Calcium imaging: a technique to monitor calcium dynamics in biological systems

Abstract

Calcium ion (Ca²⁺) is a multifaceted signaling molecule that acts as an important second messenger. During the course of evolution, plants and animals have developed Ca²⁺ signaling in order to respond against diverse stimuli, to regulate a large number of physiological and developmental pathways. Our understanding of Ca²⁺ signaling and its components in physiological phenomena ranging from lower to higher organisms, and from single cell to multiple tissues has grown exponentially. The generation of Ca²⁺ transients or signatures for various stress factor is a well-known mechanism adopted in plant and animal systems. However, the decoding of such remarkable signatures is an uphill task and is always an interesting goal for the scientific community. In the past few decades, studies on the concentration and dynamics of intracellular Ca²⁺ are significantly increasing and have become a trend in modern biology. The advancement in approaches from Ca²⁺ binding dyes to in vivo Ca²⁺ imaging through the use of Ca²⁺ biosensors to achieve spatio-temporal resolution in micro and milliseconds range, provide us phenomenal opportunities to study live cell Ca²⁺ imaging or dynamics. Here, we describe the usage, improvement and advancement of Ca²⁺ based dyes, genetically encoded probes and sensors to achieve extraordinary Ca²⁺ imaging in plants and animals.

Graphical abstract

Introduction

Ca²⁺-mediated signaling, i.e., the signal-specific transient changes in the cytosolic Ca²⁺, [Ca²⁺]_cyt concentration to mediate downstream signaling, has remained constant and universal for all life forms in spite of several differences (Luan and Wang 2021). The transient and spatio-temporal variations in Ca²⁺ concentrations generated in response to any stimulus are also known as “Ca²⁺ signatures” (Qudeimat and Frank 2009). The Ca²⁺ signatures in intracellular compartments and cytosol are very much specific for a particular stimuli or developmental event, which further regulate downstream Ca²⁺ signaling events. The downstream signaling components include Ca²⁺ sensors such as calmodulins (CaMs), calmodulin like proteins (CMLs), calcineurin-B like proteins (CBLs) and Ca²⁺ dependent protein kinases (CDPKs), which sense the modulations in Ca²⁺ concentrations. Ca²⁺ sensors bind to the Ca²⁺ and subsequently transduce the signal downstream to a set of responder proteins such as CBL-interacting protein kinases (CIPKs) and phosphatases in the designated signaling pathway (Luan et al. 2001, 2002; Cheng et al. 2002; Yang and Poovaiah 2003; Kolukisaoglu et al. 2004; Batistič et al. 2010; Hashimoto and Kudla 2011; Bender and Snedden 2013; Shankar et al. 2015). Based on the type of the stimulus, the sensor-kinase complex further binds and phosphorylate their respective targets such as transcription factors, transporter or channels and enzymes, which brings about an adaptive response. The Ca²⁺ transporters such as Ca²⁺ pumps or exchangers and channels are mainly responsible for generating as well as maintaining Ca²⁺ signatures and hence, are crucial components of Ca²⁺ signaling and homeostasis tool-kit (Berridge et al. 2003; Demidchik et al. 2018). Their synergistic transport activity is solely responsible for the maintenance of Ca²⁺ concentrations in the cytosol as well as internal compartments. Channels such as cyclic nucleotide gated channels (CNGC), glutamate receptor-like (GLRs) channels/Glutamate-like receptor channels, two pore channels (TPCs), annexins, mechanosensitive channels (MSLs) constitute the influx system which brings Ca²⁺ into the cytosol whereas Ca²⁺-ATPases, Ca²⁺/H⁺ exchangers (CAXs), Ca²⁺/cation exchangers (CCXs) act as an efflux system to remove Ca²⁺ out of the cytosol for restoring the resting [Ca²⁺]_cyt². This intricate functioning of Ca²⁺ influx-efflux system regulates Ca²⁺ levels under normal and adverse conditions (Ghosh et al. 2022).

Plants are rooted and continuously challenged with a large number of stimuli including the adverse cues in their growth habitat. In response to this, evolutionarily they have developed diverse intrinsic and sophisticated mechanisms to endure against these adverse conditions or stresses. Ca²⁺ signaling is involved in various physiological, biochemical, reproductive and developmental pathways (Pandey 2008; Ghosh et al. 2021, 2022). Perhaps this could be the only reason why plant based Ca²⁺ signaling received much attention and is well studied. Plants perceive stimuli via receptors which are considered as the first line of sensing and signaling events. Secondly, the resulting cellular events are dependent on  spatial and temporal variations in apoplastic, cytoplasmic and organellar Ca²⁺ concentrations (Batistič and Kudla 2012; Thor 2019). In animals, Ca²⁺ signaling machinery is comprised of a large number of components, similar to that in plants and microbial world. Still, there remains a large distinction in the presence and governance of the Ca²⁺ signaling pathway. Some of the peculiar differences in animals and plants are the presence or absence of these Ca²⁺ signaling components such as Calcineurin (a ubiquitous Ca²⁺ sensor of animal world), which is absent in plants (Kudla et al. 1999; Nagata et al. 2004). However, animals also possess CaM, Ca²⁺ sensor that is highly conserved throughout the eukaryotes. To transduce the signal downstream in the signaling pathway, CaM binds the Ca²⁺ via its EF-hands in the presence of Mg²⁺ (Fang et al. 2002; La Verde et al. 2018; Luan and Wang 2021). The selectivity of cation Ca²⁺ over Mg²⁺ is achieved by the structural folding of the binding loop of CaM. The Ca²⁺/CaM complex targets a large array of functional proteins including kinases, phosphatases, channels, transporters, and cytoskeletal components (Floyd and Wray 2007; Luan and Wang 2021). Ca²⁺ levels in animals are also regulated by a diverse array of channels such as voltage-gated Ca²⁺ channels (VGCCs), CNGCs, ionotropic glutamate receptors (iGluRs), Ca²⁺ -permeable mechanosensitive channels like PIEZOs and Hyperosmolality-gated calcium-permeable channels (OSCAs), TPCs and transporters like exchangers and Ca²⁺-ATPases. In adverse conditions, the animal [Ca²⁺]_cyt levels are elevated with the opening of Ca²⁺ channels localized on plasma membrane (PM)/or endomembrane followed by removal of Ca²⁺ by activation of PM Ca²⁺-ATPase (PMCAs), sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs) and Ca²⁺ exchangers like NHXs. The synergistic activity of channels and transporters results in Ca²⁺ entry and release which maintains Ca²⁺ levels responsible for excitation and contraction in animal muscle cells (Floyd and Wray 2007; Luan and Wang 2021).

Growing knowledge of Ca²⁺ signaling has improved our understanding of various physiological and biochemical processes occuring in a cell. It also encourages researchers to develop tools and techniques to quantify and monitor Ca²⁺ levels or signatures in nano or micromolar (nM or µM) ranges during stressed or normal conditions. The optical quantification of Ca²⁺ dynamics or signatures in the isolated cell, tissue or medium with the utilization of microscope is known as “Ca²⁺ imaging” (Reis et al. 2020). In the early 1960s and 1970s, the dye-based indicators such as murexide, azo dyes, and chlortetracycline were used. The major drawback of using these dyes are low sensitivity, difficulty in performing live cell imaging, poor accuracy and their hazardous nature (Kanchiswamy et al. 2014). With time, the advancement in Ca²⁺ imaging with advanced Ca²⁺-based indicators came into existence. Among them, green fluorescent protein (GFP), Aequorin (AEQ) and Fluorescence resonance energy transfer (FRET) based Ca²⁺ imaging are very popular. These Ca²⁺ indicators have enabled us to understand the dynamics of Ca²⁺ at cellular level (Zhou et al. 1980; Kanchiswamy et al. 2014). The present study is focused on a detailed study of Ca²⁺ imaging aspects in plants and animals describing the ancient probes and dyes, their merits and demerits as well as evolution. We have also covered the utilization and application of these techniques in modern plant and animal sciences.

Live cell [Ca²⁺]_cyt imaging: advancement in tools and techniques

Ca²⁺ imaging has benefitted from the advancements in technology based on the principle of Ca²⁺-based indicators. Although the Ca²⁺ concentration in plants is in the nM range, it can reach mM concentration in specific organelles. Ca²⁺ indicators can visually monitor Ca²⁺ concentrations in the cytoplasm because it is difficult to accurately quantify such small concentrations. Many Ca²⁺ indicators have been developed over time, but each one has its own advantages and disadvantages. In the beginning, intracellular Ca²⁺ variations were studied using the Ca²⁺-sensitive bioluminescent protein aequorin. However, it has certain drawbacks as well. To address these drawbacks, new indicators were created, and this series of advancements over the earlier dyes resulted in the production of a large number of new dyes. Also, the goal of acquiring high-resolution images underlies the modernity of the microscopy world. The discovery of light microscope by Anton van Leeuwenhoek (1632–1723) enabled the study of samples as uniformly as possible through the illuminating light. However, it was not sensitive enough for thicker samples where an advanced objective lens was required which could penetrate to a sufficient depth and focus in the region of interest (Evennett and Hammond 2004). Genetically encoded probes can also be covered in widefield microscope where the whole sample is illuminated by light source and can be viewed via eyepiece or displayed on monitor. The widefield microscopes are way less complexed as compared to the confocal microscope but path of light as well as out-of-focus light are a concern (Köhler and Blatt 2002; Kanchiswamy et al. 2014). In 1845, fluorescence microscope was discovered by Fredrick W. Herschel, which utilized fluorescent-based dyes. However, it could stimulate out-of-plane dye molecules as well, with out-of-focus light producing blurred images (Renz 2013). Later on, in mid-1950s, the confocal microscope was invented by Marvin Minsky to overcome the limitations of fluorescence microscopy (Merchant et al. 2005). Optical sectioning-based confocal microscope was able to reduce blurring of images by physically blocking out-of-focus light and enabling the three-dimensional reconstruction of samples. Over the years, several other confocal microscopes were invented (Merchant et al. 2005). One such example is confocal laser scanning microscopy (CLSM) which is primarily utilized for in situ 3D modelling of tissues (Stricker and Whitaker 1999; Elliott 2020). It is a fluorophore-based Ca²⁺ imaging method with spatial resolution that is unmatched by other Ca²⁺ imaging devices. CLSM visualizes Fluo-3 fluorophore emitted fluorescence and majorly transmits the powerful laser beam onto the sample (Mithöfer et al. 2009). Interestingly, CLSM has been shown to be a good tool to visualize live samples. It has been primarily used to measure spatial intracellular Ca²⁺ signal in eukaryotes ranging from protists, fungi, plants and animals (Mithöfer et al. 2009; Kanchiswamy et al. 2014). Confocal microscope such as Nipkow-disk-types rely on arc lamp for its illumination to acquire transient intracellular Ca²⁺ images (Williams 1990; Takahashi et al. 2011; Elliott 2020). The single plane illumination microscopy (SPIM) is used for 3D imaging of multicellular specimens with utmost high resolution. This was invented in the early 2000s in Ernst Stelzer’s laboratory (Gomez-Cruz et al. 2022). Alex Costa and co-workers utilized this microscopy to study the dynamics of Ca²⁺ in transgenic Arabidopsis expressing genetically encoded Ca²⁺ probe i.e., NES-YC3.6 which is based on the phenomenon of Förster Resonance Energy Transfer (FRET); (Costa et al. 2013). Deconvolution microscopy has also been used to study high resolution Ca²⁺ imaging. Pnevmatikakis et al., (2015) reported the position of neurons from the slow dynamics of Ca²⁺ indicators (Kanchiswamy et al. 2014; Pnevmatikakis et al. 2016). Allen and co-workers also used a deconvolution microscope to measure the fluorescence of Yellow Cameleon YC2.1 (Allen et al. 1999).

The advancement of Ca²⁺ imaging surely has paved the path of modernity of microscopy with high resolution, excellent accuracy and live cell imaging (Fig. 1). The improvement in technology will definitely enlighten the phenomenon of Ca²⁺ imaging in a more advanced manner.

Fig. 1.

The quantification of cellular Ca²⁺ dynamics measured using confocal microscope. The analysis of Ca²⁺ dynamics in living organisms encompasses various components. The entire plant sample is placed onto a specifically designed plate, which facilitates continuous flow within a chamber via a pump. The incident beam traverses the excitation filter and undergoes reflection upon interaction with the sample. The emitted fluorescence is collected and directed towards the detector by the return beam, which passes through the filter wheel before being visualized on the system [adapted from (Behera and Kudla 2013a)]

Measurement of Ca²⁺ using Ca²⁺ based indicators

This section is dedicated to the examination of different dyes, sensors, probes, and indicators that rely on the presence of Ca²⁺ ions and have been employed in various instances. The indicators are categorized on the basis of their chemical nature and usage (Table 1; Fig. 2). This section provides a description of the underlying principle on which these indicators operate to analyze their respective advantages and disadvantages for their utilization in plant and animal sciences.

Table 1.

Properties of various Ca²⁺ dyes, probes and indicators and their special features.

Indicator dyes	Properties		Special Features	References
AEQUORIN	Kd	2600 nM	Based on biolouminescence, which has restricted utility for rapid kinetics and allowing detection between 0.1 and 100 µM	Kaestner et al. (2014)
	Excitation	–
	Emission	465 nm
QUIN-2	Kd	115 nM	Preferred for studying resting Ca2+ concentration at intracellular level due to its high affinity for Ca2+	Grynkiewicz et al. (1985), Giacomello and Campeol (2013), Matuz-Mares et al. (2022)
	Excitation	339 nm
	Emission	495 nm
FURA-2	Kd	224 nM	Bigger range of Ca2+-free and bound forms higher photobleaching activity resistance	Eerbeek et al. (2004), O’Connor and Silver (2013), Tinning et al. (2018)
	Excitation	380–340 nm
	Emission	510 nm
INDO-1	Kd	822 nM	Increased speed of measurement absence of special quartz optics	Bassani et al. (1995), Eerbeek et al. (2004), Bannwarth et al. (2009)
	Excitation	351–364 nm
	Emission	405 and 485 nm
FLUO-3	Kd	390 nM	Good spectroscopic qualities and  better photostability	Castell et al. (1997), Takahashi et al. (1999), Gee et al. (2000b), Contreras et al. (2010), Blass (2015)
	Excitation	506 nm
	Emission	526 nm
FLUO-4	Kd	345 nM	Fast and high affinity Ca2+ dye used in live imaging	Wallace et al. (2008), Blass (2015), Di Virgilio et al. (2019), Liao et al. (2021)
	Excitation	480 nm
	Emission	525 nm
CALIBRYTE 520 AM	Kd	320 nM	Do not need probenecid improve dye retention	Liao et al. (2021) https://www.aatbio.com/products/cal-520-am
	Excitation	493 nm
	Emission	515 nm
CAL-590	Kd	561 nM	Useful in deep imaging experiments fall in near infrared range good signal to noise ratio	Tischbirek et al. (2015)
	Excitation	570 nm
	Emission	590 nm
RHOD-2	Kd	570 nM	Low affinity Ca2+ indicator among all fluorescent Ca2+ probes it has the highest dissociation constant and possess the strongest signal intensity	Hurley et al. (1992), MacGowan et al. (2001b), Liao et al. (2021)
	Excitation	524 nm
	Emission	589 nm
CALCIUM ORANGE AM	Kd	7.40 nM	signal to noise ratio is high reduce autofluorescence related issues	Eberhard and Erne (1991), Lam et al. (2005) https://pubchem.ncbi.nlm.nih.gov/compound/16186222
	Excitation	549 nm
	Emission	576 nm
CALCIUM GREEN-1	Kd	190 nM	Excited by visible light reducecellular photo-damage	Eberhard and Erne (1991), Hurley et al. (1992) https://pubchem.ncbi.nlm.nih.gov/compound/16186222
	Excitation	490 nm
	Emission	531 nm
YC2.1	Kd	100 nM	Improved pH sensitivity	Allen et al. (1999)
	Excitation	488 nm
	Emission	522 nm
YC3.6	Kd	250 nM	Provides a high signal-to-noise ratio has acid stability	Miyawaki et al. (1999), Nagai et al. (2004), Saito et al. (2010), Bischof et al. (2019)
	Excitation	430 nm
	Emission	475/525 nm
D3cpv	Kd	600 nM	Good Rmax/Rmin sensitivity to Ca2+ fluctuation	Rochefort and Konnerth (2008), Wallace et al. (2008), Giacomello et al. (2010)
	Excitation	488 nm
	Emission
N33D1cpv	Kd	800 nM and 60 µM	Higher affinity for Ca2+	Bischof et al. (2019), Gouriou et al. (2023b)
	Excitation	430 nm
	Emission	475/525 nm
N33D3cpv	Kd	10 M	Higher Ca2+ sensitivity greater F/F0 ratio peak provides accurate and reproducible Ca2+ measurements	Gouriou et al. (2023b)
	Excitation
	Emission
GCaMP3	Kd	660 nm	Possibility of cell specific expression ease of use with microscopy photostable	Akerboom et al. (2013), Chen et al. (2013b), Cho et al. (2017), Defalco et al. (2017), Zhong and Schleifenbaum (2019), Shemetov et al. (2021)
	Excitation	485 nm
	Emission	513 nm
GCaMP6	Kd	375 nM` nM	Consists of 3 sensors i.e., GCaMP6s,6 m,and 6f which exhibits slow, medium and fast kinetics respectively	Chen et al. (2013a), Lohr et al. (2021)
	Excitation	496 nm
	Emission	513 nm
GCaMP-HS	Kd	102 nM	More stable and stronger fluorescence higher affinity for calcium higher cooperativity	Muto et al. (2011), Muto and Kawakami (2013)
	Excitation	488 nm
	Emission	509 nm
G-GECO	Kd	Kd1 = 15 nM	Longer wavelength, less photoxicity deeper penetration in tissue	Geiger et al. (2012), Shen et al. (2018), Bi et al. (2021)
		Kd2 = 890 nM
	Excitation	390 nm
	Emission	640 nm and 700 nm
i-GECI	Kd	Kd1 = 15 nM	High photostability high brightness Approximately 60% fluorescence increase upon Ca2+ binding	Matlashov et al. (2022)
		Kd2 = 890 nM
	Excitation	390 nm
	Emission	670 nm and 700 nm
Twitch	Kd		A smaller size, a broad range of calcium affinities, superior photostability, a wider dynamic range, and faster kinetics compared to the TN-XXL	Thestrup et al. (2014b), Cho et al. (2017)
	Excitation
	Emission
CaRuby-Nano	Kd	295 nM	Highly sensitive Versatile indicator	Otsu et al. (2015), Collot et al. (2015)
	Excitation	575 nm
	Emission	610 nm
TN-XXL	Kd	800 nM	shows increased fluorescence change allows repetition of images	Mank et al. (2008), Dedecker et al. (2013)
	Excitation	435 or 515 nm
	Emission	480 or 535 nm

Fig. 2.

The categorization of various Ca²⁺ indicators utilized for studying Ca²⁺-dynamics is depicted. There are two main classes of Ca²⁺ indicators: Chemical-based and Genetically encoded Ca²⁺indicators (GECI). Chemical-based indicators are small molecules that can be loaded into cells, while GECIs are proteins that are expressed within the cell. Chemical-based indicators are easy to use and provide a quick visual indication of the presence or absence of a specific substance whereas GECIs are genetically modified proteins that fluoresce in the presence of Ca²⁺ ions

Chemical-based Ca²⁺ indicators

Chemical indicators have been used for intracellular Ca²⁺ detection over a wide range from < 50 nM to > 50 µM. High affinity chemical indicators have been optimized for [Ca²⁺]_cyt whereas, low affinity indicators have been refined for Ca²⁺ of intracellular compartments (Paredes et al. 2008).

AEQUORIN, QUIN-2, FURA-2, INDO-1

Aequorin, a chemical based photoprotein obtained from the jellyfish Aequorea victoria, has attracted considerable scientific interest for its distinctive ability to demonstrate Ca²⁺ dynamics within living cells (Shimomura 2005). The bioluminescent protein functions as a highly sensitive Ca²⁺ sensor through its high-affinity binding to Ca²⁺ (Shimomura 2005). When aequorin and Ca²⁺ come into contact, the protein changes its shape, causing its coelenterazine chromophore to become oxidised. This process ultimately leads to the emission of light within the visible spectrum (Shimomura 2005; Fig. 3). The bioluminescent reaction has a linear relationship with the amount of Ca²⁺ in the cellular environment. This makes it easy to keep track of changes in Ca²⁺ levels inside cells without hurting them. Through the process of genetic modification, cells can be manipulated in order to express aequorin (Yoshimoto and Hiramoto 1991). Alternatively, targeted aequorin probes can be used to examine a wide range of Ca²⁺-dependent cellular processes. These processes include neurotransmission, muscle contraction, cellular signaling, and apoptosis (Wier et al. 1983; McConkey and Nutt 2004; Ryu et al. 2010).The major disadvantage of using aequorin is having low light emission which actually affects the spatio-temporal resolution of Ca²⁺ imaging monitored from whole seedlings or tissue. Also, mathematical simulations have proved that single-cell oscillatory Ca²⁺ signals cannot be detected by aequorin by using conventional microscopes (Grynkiewicz et al. 1985; Krebs et al. 2012). A number of Ca²⁺ imaging studies have been undertaken in plants as well as in animals with the utilization of aequorin, to elucidate the potential role of Ca²⁺ under stress physiology and developmental pathways. With the help of aequorin transformed tobacco cells, the involvement of Ca²⁺ signaling was studied during oxidative burst condition (Chandra and Low 1997). Because of the technical difficulties, mitochondrial Ca²⁺ concentration measurement is a little tricky. But in the year 2003, Logan et al. (2003) were able to study mitochondrial Ca²⁺ dynamics by generating stable Arabidopsis aequorin lines under various stresses such as cold, osmotic, mechanical and oxidative stress  (Logan and Knight 2003). Aequorin has also been used in order to study [Ca²⁺]_cyt changes in presence of flagellin (flg22), Pep1 (a plant derived DAMP; damage associated molecular pattern), and NaCl (Xie et al. 2017). Aequorin has been also utilized to visualize in vivo Ca²⁺ signaling in different sub-cellular compartments and comparative analysis of the same has been done (Mehlmer et al. 2012). A hybrid Ca²⁺ sensor which has a fusion of green fluorescent protein (GFP) and aequorin has been engineered as a chimeric protein, termed as G5A. The chimeric protein sensor G5A was based on the principle of bioluminescence resonance energy transfer (BRET). G5A was potentially used in animal systems for over ten years. Interestingly, G5A has been used in intact Arabidopsis to study the Ca²⁺ dynamics in whole seedling. G5A can be an influential tool to study Ca²⁺ dynamics with enhanced fluorescence property (Xiong et al. 2014). Kiegle et al. (2000) have studied the transient changes in Ca²⁺ levels under cold, osmotic and salt stress conditions in Arabidopsis (Kiegle et al. 2000). The rice harbouring aequorin was used to monitor the differences in peak durations of Ca²⁺ spikes under salt and oxidative stresses (Zhang et al. 2015). Ca²⁺ signaling in biotic stress conditions has also been studied in plants. The SlCNGC1 and SlCNGC14 silenced tomato lines expressing aequorin showed reduced Ca²⁺ spikes upon flg22-treatment (Zhang et al. 2018). A very interesting study by Moreno et al. (2017), showed the change in Ca²⁺ level in Arabidopsis root upon exposure to sound wave (200 Hz) for around 2 weeks (Moreno et al. 2017). Ca²⁺ imaging has also been undertaken in rice where recombinant aequorin was introduced and Ca²⁺ spikes were recorded upon salt stress (Zhang et al. 2015). The roots of transgenic rice showed strong luminescence during excess exogenous Ca²⁺ (Zhang et al. 2015). Actually, recording of [Ca²⁺]_cyt dynamics in monocot plants is way more difficult as compared to dicots. However, Volkmann et al. (2009) were able to generate transgenic winter wheat lines which showed stable constitutive expression of aequorin in the cytosol. With the help of luminometry device, temperature-induced Ca²⁺ dynamics were recorded. The Ca²⁺ dynamics was easily detectable under cold stress implying the significance of Ca²⁺ signaling under stress in wheat (Volkmann et al. 2009). Aequorin-based luminescence has also been utilized to analyse the specific amino acid responsible for evoking Ca²⁺ signaling in rice roots. Glutamate (Glu) was found to participate in triggering Ca²⁺ flux in the root system (Ni et al. 2016).

Fig. 3.

The pictorial representation of aequorin bioluminescence based Ca²⁺ imaging. The cells that make apo-aequorin are first subjected to an incubation process with the coelenterazine compound, which gets into the cells. This incubation leads to the generation of functional aequorin within the cells. The binding of Ca²⁺ to aequorin induces a conformational alteration in the protein, resulting in the destabilisation of the peroxide group (-O-O-). This interaction facilitates the attachment of apoaequorin to coelenterazine, causing its decomposition into coelenteramide and CO₂. The excited state of coelenteramide emits blue light with a maximum wavelength of 469 nm. When cells are subjected to an environmental stress, the second messenger, Ca²⁺ permeates the cytoplasm and interacts with aequorin, resulting in the emission of light by the cell. The luminescence traces provide information on both the magnitude and the duration of the Ca²⁺ concentration wave in the cytoplasm (Shimomura 1995; Webb et al. 2015)

Quin-2 is used in case of intact cells along with membrane permanent ester derivatives. Quin-2 works under an interesting principle. The ester group is separated by the cytosolic esterase, trapping the quin-2 and tetra anion in the cytosol. Quin-2 is excited at 339 nm that excites majorly autofluorescence, thus, causes damage to the cells. To overcome this auto-fluorescence, Quin-2 must be used in a very low concentration, for instance, one tenth of a milli-mole (mM). Upon binding Ca²⁺, Quin-2 does not show much shift in emission or excitation wavelength. However, fluorescence intensity is dependent on various factors such as intensity of illumination, efficiency in emission collection, concentration of dye and most importantly, on the emission-excitation shift. Therefore, it is always recommended to select a dye that shows better emission-excitation shifts (Grynkiewicz et al. 1985). Whereas, Quin-2 can be used for the Ca²⁺ at resting state, which is approximately 10^–7 M. However, at  µM concentrations, the dye loses its resolution and reaches its saturation point. Therefore, to compensate this loss of efficacy, low affinity dyes are preferably used. Quin-2 also shows affinity for Mg²⁺ ions, without affecting fluorescence intensity. However, variation in Mg²⁺ concentration could be a possible reason for effective binding of Quin-2 with Ca²⁺ and thus, the fluorescence intensity (Grynkiewicz et al. 1985). Quin-2 has been used to investigate unknown biological functions related to Ca²⁺ signaling in plants. Plasma membrane associated cation-binding protein (PCaP1) is involved in a number of developmental functions and Ca²⁺ signaling in plants. PCaP1 is attached to PM via N-myristoylation and a polybasic cluster. It also binds Ca²⁺-CaM moiety via N-terminal. Impact of myristoylation on the complex PCaP1-Ca²⁺-CaM and the modulation in Ca²⁺ sensitivity property of CaM in the complex remains unelucidated. The kinetic study of the complex in association with myristoylation and Ca²⁺ was studied with the help of Quin-2 and the functional role of PCaP1 and its  structural aspect was demonstrated (Pedretti et al. 2023). With the utilization of Quin-2, Walton et al. (2017) reported that the divergent soybean CaMs (CaM1 and CaM4) behaved similarly on Ca²⁺ transient (Walton et al. 2017). Quin-2 has also been used to study the role of IQ motif in regulating function of CaM (Putkey et al. 2003; Shen et al. 2016).

The ratiometric fluorescent Ca²⁺ indicator was developed as an enhanced alternative to Quin-2 (Tinning et al. 2018).

FURA-2, a derivative of salicylaldehyde, represents a member of the initial cohort of Ca²⁺ fluorescent indicator (Liao et al. 2021). Its excitation and emission wavelengths depends on Ca²⁺ levels (Eerbeek et al. 2004). There is a shift in the excitation peak wavelength from 380 to 340 nm upon binding of free [Ca²⁺]_cyt to Fura-2. However, there is no alteration in the peak emission at 510 nm. The fluorescence intensity is stimulated between the wavelengths of 340 and 380 nm with subsequent emission occurring at a wavelength of 540 nm (Tinning et al. 2018). The amount of Ca²⁺ required, needs to  be standardized with the utilisation of a Ca²⁺ ionophore, such as ionomycin, to establish a stable Ca²⁺ detection via Fura-2 (Di Virgilio et al. 2019). The suggested optimal concentration of Fura-2 AM is 1 mM. Actually, a lower concentration of 250 nM can also be used much accurately . The FURA-2 dye is less likely to bleach under light and has a wider range of Ca²⁺-free and Ca²⁺-bound conformations (Tinning et al. 2018). Fura-2 wide-field excitation technique employs an arc lamp equipped with a monochromator as a light source. The requirement of rapid alterations of the wavelength within milliseconds is a significant constraint (Tinning et al. 2018). Additionally, the arc lamp light source exhibits a 5% amplitude instability, thereby diminishing the efficacy of precise Ca²⁺ concentration measurements. The use of light-emitting diodes (LEDs) presents a potential solution to address certain drawbacks associated with Arc lamps. LEDs offer advantages such as enhanced stability during rapid switching at the millisecond level and the ability to provide precise control over output. (Tinning et al. 2018). A number of studies  have been reported  in animals as well as in plants with the utilization of Fura-2. It has been used to study the distribution of Ca²⁺ levels in vegetable tissues in plants (Bonomelli et al. 2010). It has also been used as an intracellular Ca²⁺ indicator to study the role of chitinases in rice. Chitinase protein is involved in defense, growth and development processes in plants. Wu et al. (2017) functionally characterized one of the potential genes, OsCLP (encodes chitinase-like protein), involved in growth and developmental processes in rice. It has been observed that T-DNA insertion in OsCLP resulted in retarded root and shoot growth phenotype in rice. Additionally, Fura-2 staining and inductively coupled plasma-mass spectrometry (ICP-MS) results demonstrated the low level of Ca²⁺ in osclp mutants as compared to the wild type plants (Wu et al. 2017). With the utilization of Fura-2, Ca²⁺ oscillations have been measured in plant-bacterial/fungal symbiosis (Harper and Harmon 2005). The Ca²⁺ binding property and dynamics of another Ca²⁺ binding protein, OsCCD1, was studied via Fura-2 under osmotic and salt stress conditions in rice (Jing et al. 2016). Chitosan elicitation mediates synthesis of anthraquinone (Aq) in Rubia tinctorum L. However, stimulation of Aq can be blocked by BAPTA-AM but not by EGTA and VDCC-antagonists, verapamil and nifedipine. Interestingly, it has been observed that upon chitosan treatment, Fura-2 loaded R. tinctprum cells showed enhanced Ca²⁺ concentration in Ca²⁺ devoid media (Vasconsuelo et al. 2005). Vafadar et al., (2021) has also demonstrated the role of melatonin, which improves the rate of photosynthesis in Dracocephalum kotschyi. Also, the Ca²⁺ dynamic was studied with the help of Fura-2 in the presence and absence of melatonin and its respective effect on photosynthesis (Vafadar et al. 2021). With the use of Fura-2, Ca²⁺ signaling was studied in order to understand the somatic embryogenesis of pro-embryonic cells of Santalum album (Anil and Rao 2000). A study utilized Fura-2 and Fluo-3 to observe the Ca²⁺ transient in root cells under salt and osmotic stress conditions in Arabidopsis (DeWald et al. 2001). The role of Ca²⁺ signaling in the formation of important secondary metabolites has also been seen. Resveratrol is known to modulate cellular Ca²⁺ responses (Kopp et al. 2014). Interestingly, only specific Ca²⁺ indicators such as Fura-2 could monitor the change in [Ca²⁺]_cyt upon resveratrol application (Kopp et al. 2014). However, advanced indicators like Fura-4 and YC3.60 could not indicate any response (Kopp et al. 2014).

Indo-1 is a Ca²⁺ fluorescent indicator of the first generation (Liao et al. 2021). Ultraviolet (UV) light can excite Indo-1 as it exhibits absorption within the wavelength of 351–364 nm, and its emission peaks occur at 405 nm and 485 nm (Eerbeek et al. 2004; Betzenhauser 2011). The emission ratio at 485 nm is actually influenced by the incomplete hydrolysis of Indo-1 AM (Eerbeek et al. 2004). It is well-known that a single valid calibration is very challenging task to achieve. Still, Indo-1 AM is found to be a more effective approach for analysing alterations in Ca²⁺ levels in heart tissues within a semi-quantitative framework. Another important fact is the Ca²⁺ buffering capacity of Indo-1, which is relatively low and is essential to exhibit rapid kinetic properties (Eerbeek et al. 2004). All these characteristics render Indo-1, a suitable tool for evaluating Ca²⁺ transients in myocytes. Most importantly, Indo-1 transients remain unaffected by the background fluorescence of NADH which makes it more prominent to use among other chemical based Ca²⁺ indicators (Eerbeek et al. 2004). Indo-1 has been used to study Ca²⁺ oscillations in many studies. Auxin-induced [Ca²⁺]_cyt was measured in root hairs of tomato. Plant–pathogen interactions is an interesting area of research where Ca²⁺ signaling play essential role. In Rhizobium–legume interaction, Nodulation (NOD) factors are the essential components which are synthesized by Rhizobium and are required for interaction with plants. It has been observed that NOD factors induce intracellular Ca²⁺ oscillations, which have been detected by Indo-1 (Downie and Walker 1999). With the use of Indo-1, Ca²⁺ dynamics were studied during microspore development and embryogenesis in Brassica napus and Solanum melongena (Sendra et al. 2017). Indo-1 has been utilized to study the Ca²⁺ dynamics in guard cells (Gilroy et al. 1991; Israelsson et al. 2006). Studies showinghow hormones coordinate with [Ca²⁺]_cyt and the level of [Ca²⁺]_cyt changes have been well documented via Indo-1 in barley (Gilroy and Jones 1992).

FLUO

The Ca²⁺ indicator of Fluo series emits the basal level of fluorescence at resting Ca²⁺ concentration which gets increased more than hundreds folds with increase in the Ca²⁺ concentration (Paredes et al. 2008). A number of Fluo series have been discovered time to time with advancement over the previous ones.

Fluo-3 is a long-wavelength fluorescent Ca²⁺ indicator whose excitation and emission wavelength lies in a range of 506 nm and 526 nm, respectively. Fluo-3 can be used in flow cytometry, microplate screening assays, confocal laser scanning microscopy, or light microscopy with standard sets of fluorescence filters (Gee et al. 2000a; Liao et al. 2021). The K_d value is 0.40 µM. It is easy to use, stable, and can be loaded in cells in  different forms i.e., membrane soluble, acetoxymethyl (AM) ester form, or salt form. Most of the chemical indicators impose physiological impacts in a cell. However, Fluo-3 does not impart a negative impact on normal physiological processes, which makes it suitable to use (Gee et al. 2000a). On binding Ca²⁺, Fluo-3 fluorescence increases up to 40 folds, thus, it does not require ratiometric recording. Researchers have utilized Fluo-3 to study the qualitative Ca²⁺ concentration in mitochondria under various experimental conditions. Hence, it is way more helpful in studying the kinetic changes of mitochondrial Ca²⁺ concentration (Saavedra-Molina et al. 1990). Also, it is used in conjugation with BAPTA-like Ca²⁺ chelators, which are covalently linked with fluorogenic components (Gee et al. 2000a). It has been reported that Fluo-3 is predominantly localized in the cytoplasm but in the case of fibroblast cell, it is profoundly present in cell organelles as well (Kao et al. 1989). It has also been observed that the concentration of Fluo-3 is two-fold greater in the nucleoplasm than that present in the cytoplasm under normal conditions.

Contrary to Fluo-3, Fluo-4 is a single excitation wavelength indicator, but it shares some common features with Fluo-3 such as spectral value, K_d value, stability, ease in loading the dye, or Ca²⁺ dependent fluorescent increase (Tinning et al. 2018). Also, it is a second generation Ca²⁺ fluorescent indicator and is commonly used in high throughput screening (HTS), confocal, and flow cytometry imaging techniques (Liao et al. 2021). It gets excited at 480 nm, and emitted at 525 nm, which can be used for single-cell microscopy to observe Ca²⁺ concentration dynamics in numerous individual cells in one setting (Di Virgilio et al. 2019). Like Fluo-3, Fluo-4 also responds to Ca²⁺ binding by increasing the intensity of fluorescence, but not the spectral shift (Gee et al. 2000a). However, Fluo-4 has slightly higher Ca²⁺ binding affinity than Fluo-3. The structural composition of Fluo-4 is somewhat similar to Fluo-3. The only difference is the substitution of two chlorines in place of two fluorines in the fluorophore. This substitution is actually a major modification which is responsible for increasing the absorbance near 488 nm, thereby, imparting a greater advantage to Fluo-4 with less photo bleaching and photo-toxicity compared to Fluo-3 (Gee et al. 2000a). Therefore, a low amount of Fluo-4 dye can generate the same intensity of fluorescence as  Fluo-3 . Moreover, the lower usage of dye reduces the buffering effect of Ca²⁺ and lowers the production of toxic by-product such as acetoxymethyl ester which is another advantage of Fluo-4 (Gee et al. 2000a). Fluo-4 shows more intense fluorescence when it is used with an argon ion source or with a standard set of fluorescence filters (Gee et al. 2000a). It is one of the most commonly used dyes for the study of pancreatic acinar cells and imparts minimal physiological disturbances in the cell (Betzenhauser 2011).

MAG FLUO-4 is a lesser-known, low-affinity Ca²⁺ dye which is used in cultured cells for the quantification of endoplasmic reticulum (ER) stress. Interestingly this particular dye is sensitive for Ca²⁺ as well as Mg²⁺ ions. Actually, it has high affinity for Mg²⁺ than Ca²⁺. Its Ca²⁺ quantification results provide an accurate rate and time. It is also very cost-effective for ER stress examination (Lebeau et al. 2021). Fluo-5 exhibits fluorescence properties, characterised by an excitation peak at a wavelength of 494 nm and an emission peak at 516 nm. The excitation process can be achieved by utilising a laser with a wavelength of 488 nm in conjunction with a bandpass filter of 530/30 nm. Fluo-5 exhibits spectral similarities with Fluo-8, matrix metalloproteinase (MMP), Ca²⁺-Green, Cal-520, and Calbryte 520 (Kabbara and Allen 2001).

Fluo-8 is an innovative Ca²⁺ indicator that employs the fluorescence core, also present in Fluo-3 and Fluo-4, for the purpose of monitoring Ca²⁺ concentration and flux within cells. This allows Fluo-8 to maintain spectral characteristics that are indistinguishable from Fluo-4. However, it addresses the limitations of Fluo-3 and Fluo-4 (Rietdorf et al. 2014). The introduction of minor structural modifications has contributed to the development of Fluo-8, resulting in various improvements in its application. One notable improvement is the optimised loading conditions of Fluo-8. Similar to other fluorescent dyes, Fluo-8 lacks fluorescence in its Ca²⁺-free state. When it binds Ca²⁺, it exhibits a significant increase in fluorescence intensity, approximately 200 times greater than its initial level. This exhibits a twofold increase in brightness compared to Fluo-4 and a fourfold increase in brightness compared to Fluo-3. The temperature dependence of Fluo-8 is comparatively lower in comparison to other probes, thereby, generating more consistent and reproducible outcomes. It is frequently employed to ascertain the dynamic alterations in Ca²⁺ levels within the guard cells (Lock et al. 2015).

There are a number of studies where researchers have employed various versions of Fluo in plant research. [Ca²⁺]_cyt level is of utmost importance in various developmental pathways in plants including pollen tube (PT) growth, fertilization and developmental processes in angiosperms. A very interesting study came into picture in 1997, when transient elevation of free [Ca²⁺]_cyt was monitored during fusion of egg and sperm cells in maize during in vitro fertilization (Digonnet et al. 1997). The study was undertaken by the use of Fluo-3 dye under controlled physiological conditions to study the importance of Ca²⁺ transients during egg activation and early zygote development in plants (Digonnet et al. 1997). Fluo-3 was used in studying Ca²⁺ changes due to the infection caused by Spodoptera littoralis (Kanchiswamy et al. 2010). Ca²⁺ signaling plays a significant role in developing female sex organs in higher plants. In one of the reports, Fluo-3 was utilized to report Ca²⁺ in isolated female sex cells; egg cells and central cells of tobacco,  thus, contributing to study of developmental aspects in plants (Peng et al. 2009). Glycine betaine (GB) is an important component which is responsible to enhance heat tolerance via accumulation of heat shock proteins (HSPs) in plants. But, how GB stimulates HSPs under salt stress, is not known. Li et al. (2013) observed Ca²⁺ transient in tobacco seedling under salt stress.

Fluo-3 has been utilized to monitor [Ca²⁺]_cyt which contributes to anthocyanin biosynthesis in hypocotyl of radish sprouts under UV-A irradiation (Li et al. 2014). Ceramide sphingolipids are important components of cell membrane and promote programmed cell death (PCD) in plants as well as in animals. The mechanism by which ceramide induce PCD in rice is not well known. It has been speculated that Ca²⁺ levels go high just after ceramide application in rice. With the utilization of Fluo-4, researchers monitored Ca²⁺ levels on the application of ceramide in rice (Zhang et al. 2020). Fluo-4 was also used in measuring Ca²⁺ which worked parallelly with electric, ROS and hydraulic signaling in Arabidopsis (Fichman and Mittler 2021). Interestingly, jujube growth and development rely on Ca²⁺ which has been monitored by Fluo-4-AM in one of the studies (Wang et al. 2023). Apple pulp was stained by Fluo-4 to calculate the [Ca²⁺]_cyt (Qiu et al. 2021). MAG-Fluo-4 and Fluo-5 have been used majorly in animal sciences. MAG-Fluo-4 has been used to study the involvement of Mg²⁺ in the development of flowers in shoot apex of Pharbitis nil (L.). The elemental distribution has been studied via Fluo-3 AM and Mag-Fluo-4 AM. The former is solely for Ca²⁺ detection whereas the latter can be used to detect both Ca²⁺ along with Mg²⁺ (Kobayashi et al. 2006). With the help of Fluo-8, Ca²⁺ change was measured in stomatal closure via ABA modulated NADK2 (NAD kinase 2) pathway in Arabidopsis guard cell (Sun et al. 2017). Protoplasts are a useful toolbox to detect plant responses under stress conditions. Fluo-8 has also been used to monitor Ca²⁺ level under various stress in protoplast of Arabidopsis (Gilliard et al. 2021). Submergence effect induces hypoxia condition in plants. Hypoxia signaling is evolved in submerged species in order to protect the plants from hypoxia stress. Nitric oxide (NO) plays a significant role during hypoxia stress conditions. NO signaling cross-talk with Ca²⁺ signaling plays an essential role in plant defense mechanism under stress conditions. With the utilization of Fluo-8, Ca²⁺ levels were reported under NO-induced tolerance against hypoxia stress response in maize (Li et al. 2022b).

CALIBRYTE 520 AM and RHOD-2

Calibryte 520 AM is a chemical based Ca²⁺ indicator, that was developed in order to get rid of the limitation of Fluo-4 AM that requires probenecid. Probenecid is an organic ion transporter inhibitor, which basically prevents leakage of dye. But, it also negatively affects the efficiency of the indicator (Liao et al. 2021). Interestingly, Calibryte 520 AM does not require probenecid, and hence, limits the harmful effects of the probenecid, or improves dye retention. It is a Ca²⁺ indicator with similar fluorescence and spectral properties that of Fluo-4 AM. Calibryte 520 AM shows passive diffusion across the cell membrane (Liao et al. 2021). After entry into the cell, the intracellular esterase cleaves the lipophilic blocking group of Calibryte 520 AM which subsequently, results in a negatively charged dye and hence, restricts the exit of Calibryte 520 AM from the cell. Upon binding Ca²⁺, its fluorescence intensity is enhanced by more than 100 folds. This dye can be used for the sample which requires a longer time for signal recording and processing and has been extensively utilized in animal research (Liao et al. 2021).

RHOD-2 is likewise one of the first-generation Ca²⁺ fluorescent indicators (Liao et al. 2021). It shows excitation at 524 nm and emission at 589 nm (MacGowan et al. 2001a). It has greater tissue penetration ability because of the longer excitation and emission wavelengths and has lower interference resulting from the natural occurring fluorescent compounds, e.g., NAD(P)H. The binding of the Ca²⁺ with the Rhod-2 increases the fluorescence by 100 folds. The major drawback of using Rhod-2 is that ratio techniques for fluorescence quantification cannot be used. It is because of the fact that this dye does not display a shift in emission or excitation spectra. For the measurement of maximum fluorescence of Rhod-2, it is saturated with Ca²⁺ using cyclopiazonic acid, which inhibits the ATPase and leads to the blockage of Ca²⁺ uptake by the sarcoplasmic reticulum (MacGowan et al. 2001a). Earlier, for the measurement of maximum fluorescence, digitonin was used. However, it causes leakage of Rhod-2 and cytosolic proteins from the cell thus, affecting maximal fluorescence value (MacGowan et al. 2001a). Rhod-2 is also extensively utilized in animal sciences, however plant based Ca²⁺ signaling has also been undertaken with the help of this indicator in a few studies. With the help of Rhod-2, [Ca²⁺]_cyt was measured during mitochondrial Ca²⁺ release because of the disruption of actin filaments (Wang et al. 2010). Salt stress induced Ca²⁺ spikes were measured by Rhod-1 in the perennial herb, Glycyrrhiza uralensis (Lang et al. 2017).

Ca²⁺ ORANGE-AM, Ca²⁺ Green, Ca²⁺ green dextran, Ca²⁺ Ruby

Ca²⁺ Orange-AM has excitation wavelengths of 549 nm and 576 nm. The ratio of signal to noise is the highest. The fluorescence of Ca²⁺ Orange-AM exponentially increases with an increase in cell density up to 10⁶ per well (Lam et al. 2005). Usually 0.04% pluronic F-127 (a non-ionic surfactant polyol) is used with Ca²⁺ Orange to increase its efficiency. Pluronic F-127 is a non-ionic, polyglot surfactant that helps in Ca²⁺ indicator AM ester dispersion. It has been observed that when 0.04% of pluronic F-127 is increased to 0.08%, there is a 60% increase in background fluorescence (Lam et al. 2005). However in 0.04% pluronic F-127, there is almost negligible or no background fluorescence (Lam et al. 2005). When the dye is stained/loaded with ionomycin (Ca²⁺ chelator), there is an enhancement of fluorescence intensity, whereas the fluorescence gets diminished when treated with BAPTA-AM just after 20 min of incubation. Therefore, it can be concluded that the Ca²⁺ Orange exhibits a change in Ca²⁺ concentration in presence of Ca²⁺ chelators and ionophores in the cell. Due to its longer excitation wavelength, it can reduce the autofluorescence-related problem, and can be visualized under confocal microscope and spectrofluorometer (Lam et al. 2005). Again, Ca²⁺ Orange has been utilized less in plant based research. With the help of this Ca²⁺-orange AM, it has been proven that intracellular Ca²⁺ is required for the development and pathogenesis of the fungus, Magnaporthe oryae (Rho et al. 2009). It has also been used to study Ca²⁺ signaling in the green alga, Ulva linza, wherein Ca²⁺ spiking has been recorded during settlement and adhesion of zoospores (Thompson et al. 2007).

Ca²⁺ Green-1 is a fluorescent chemical indicator which gets energised at visible light wavelengths. Like other indicators, it gets intensified when it binds Ca²⁺. The excitation and emission wavelengths are 506 nm and 531 nm, respectively. The usage of Ca²⁺ Green-1 in [Ca²⁺]_cyt was compared with two regularly used dyes, i.e., Fura-2 and Fluo-3. It has been observed that Fura-2 which requires UV for its excitation, showed a quenching effect in platelet cells upon treating with menadione (UV absorbent and a quinone derivative agent). Also, Fluo-3 leaked from platelets very quickly. Additionally, Fluo-3 requires anion channel blockers (as mentioned earlier) which are known to alter platelet physiological responses (Lee et al. 1999), whereas, Ca²⁺ Green-1 can be used to detect the changes in [Ca²⁺]_cyt levels without such issues (Lee et al. 1999; Kuchitsu et al. 2002). Ca²⁺ Green-2 is a non-ratiometric fluorescent Ca²⁺ indicator having lower affinity for Ca²⁺ (K_d = 3 µM) than more regularly used indicators such as Fura-2 and Fluo-3 etc. Because of the low Ca²⁺ affinity and elevated quantum yield, cells can be supplied with low concentrations of Ca²⁺ Green dye, hence, [Ca²⁺]_cyt buffering can be overcome. Ca²⁺ Green-2, the advanced version, is even more advantageous in terms of low signal-to-noise ratio (Spencer and Berlin 1995).

Ca²⁺ Green Dextran (CGD) was originally created to address issues with Ca²⁺ -sensitive dyes in their free form, such as intracellular dye compartmentalization and dye leaking from cells. CGD stays sensitive to changes in intracellular Ca²⁺ after being taken up by the cell for at least 4 days, which is the longest life duration. If Ca²⁺ sensitivity is found to persist over longer survival durations, cells may be labelled more intensively and over a greater distance (Kreitzer et al. 2000). Ca²⁺ diffusion from the site of release was reduced using a high-molecular-weight Ca²⁺ buffer (Ca²⁺ green-1 dextran), which also served as a Ca²⁺ indicator (Lee et al. 1999). Ca²⁺ Green-1 has been used to study Ca²⁺ oscillation during touch in Arabidopsis (Legué et al. 1997). It has also been utilized to study the dynamics of Ca²⁺ during self-incompatibility response in Papaver rhoeas (Wang et al. 2016). Ca²⁺ mediated CO₂ signaling pathways has been studied with the help of Ca²⁺ Green-1 in Chlamydomonas reinhardtii (Franklin-Tong et al. 1993). It is reported that the stress stimulus induced Ca²⁺ oscillation, which was monitored by Ca²⁺ Green-1 in guard cells of Commelina communis (McAinsh et al. 1995). Ca²⁺ Green-1 was also used in reporting [Ca²⁺]_cyt change in pollen tube with the regulation of phosphoinositides and phosphatidic acid in Agapanthus umbellatus (Monteiro et al. 2005). The pollen tube orientation due to [Ca²⁺]_cyt change was also studied by the use of Ca²⁺ Green-1 in A. umbellatus (Malhó and Trewavas 1996). Ca²⁺ green dextran has also been utilized in studying elemental propagation of Ca²⁺, cell-to-cell communication induced by Ca²⁺ fluxes, mitochondrial Ca²⁺ release due to disruption in actin filament, and Ca²⁺ wave propagation in Fucus rhizoid cells in plants (Tucker and Boss 1996; Goddard et al. 2000; Coelho et al. 2002; Wang et al. 2010).

Furthermore, the continuous need of high affinity and signal to noise ratio of red-emitting Ca²⁺ probes led to the creation of Ca²⁺ Ruby Nano, a novel functional red Ca²⁺ indicator with a nanomolar sensitivity (Collot et al. 2015). To get the high affinity CaRuby, a few modifications were made in the already existing CaRuby of first generation. The major change was the introduction of oxygen group on BAPTA cycle in order to make it electronically powerful. The next modification was to transfer the fluorophore moiety from the para- to the meta-position. This moiety is a positively charged rhodamine, that has an electron-depleting effect. These modifications lead to the production of a new CaRuby i.e., CaRuby-Nano (Mallet and Collot 2015). It exhibits absorption wavelength at 575 nm and emission wavelength at 610 nm with a K_d value of 295 nM (Otsu et al. 2015). Hence, it gives a bit higher affinity compared to the Fluo-4 (K_d value of 335 nM). It exhibits a 50-fold increase in fluorescence upon binding Ca²⁺. Therefore, it is considered as the best candidate for measuring small intracellular changes when it binds Ca²⁺. The quantum efficiency of CaRuby-Nano is 0.45 which is lower than another chemical Ca²⁺ indicator i.e., Oregon Green-488b BAPTA-1 AM (OGB-1) having quantum efficiency approx. 0.7, but greater than Fluo-4 i.e. approx. 0.14. CaRuby-Nano is suited for both in vivo and in vitro Ca²⁺ imaging studies. It can be used along with other probes to obtain a dual colour image of brain (Collot et al. 2015).

Genetically encoded Ca²⁺ indicators (GECI)

The GECI binds to Ca²⁺ and detect intracellular Ca²⁺ via emitting fluorescence and monitor in vivo cell activity. It is characterized into two basic classes i.e., single fluorophores based and fluorescence resonance energy transfer (FRET) based indicators.

Single fluorophores

Camgaroos

The Camgaroo indicator is the first discovered single fluorescent GECIs (Griesbeck et al. 2001). Camgaroos are intrinsically sensitive to the pH, thus, it is possible that the change in the fluorescence is more likely to be caused by changes in proton ([H⁺]) than by Ca²⁺ (Hasan et al. 2004). Camgaroos do not comprise of CaM binding peptide and thus, results in low level of Ca²⁺ binding affinity compared to the G-CaMP or Pericam (Griesbeck et al. 2001). Camgaroo-1 is a chimeric protein, yellow in color and contain CaM in its pouch with high bouncing capability in signal and hence justified the name. It has been observed that when histamine is added to the Camgaroos-1 expressing HeLa cells, there is a modest increase of fluorescence intensity i.e., 40% which results in diminished Ca²⁺ spiking activity, in such case Camgaroos-1 is not as bouncy as its name depicts while sensing [Ca²⁺]_cyt. Whereas, with the usage of ionomycin, the intensity goes 7 folds high (Ai 2015; Yu et al. 2002). Camgaroo-1 has a large absorption peak at about 400 nm in the absence of Ca²⁺, and a dominating peak at around 490 nm under Ca²⁺ saturation. The deprotonation form which absorbs at 490 nm is extremely fluorescent in contrast to the protonated form, which shows absorbance at the same wavelength. An emission maximum occurs at about 515 nm after a single peak in the fluorescence excitation spectrum at 490 nm. Thus, Camgaroo-1 is a fluorescent intensometric probe (Ai 2015). However, it shows poor expression at 37 °C and is not targeted to organelles such as mitochondria which limit its usage (Griesbeck et al. 2001). Camgaroo-2 had a similar structure as Camgaroo-1since it is generated from random mutagenesis of Camgaroo-1. It also exhibits 6-to-sevenfold increase in fluorescence intensity upon Ca²⁺ binding as that of the Camgaroo-1 and has the same K_d value (Ai 2015; Yu et al. 2002). There is an increase in basal fluorescence and this is due to the replacement of 69th amino acid residue i.e., glutamine to methionine in Camgaroo-2 (Q69M) (Yu et al. 2002). Camgaroo-2 shows better expression at 37 °C and can be targeted to organelles such as mitochondria (Griesbeck et al. 2001).

Pericam

Pericam line of sensors were created by the Miyawaki group (Whitaker 2010). Enhanced yellow fluorescent protein (EYFP)-Val68Leu/Gln69Lys was circularly permuted to develop a novel protein which consists of Y145 and N144 as N terminal and C terminal (cpEYFP), respectively along with M13 and CaM (Ai 2015; Miyawaki et al. 1997; Whitaker 2010). It has been observed that when CaM fuses with N-terminus, the resulting construct (CaM-cpEYFP-M13) does not exhibit any Ca²⁺-dependent properties (Nakai et al. 2001). But in reverse conditions (M13-cpEYFP-CaM), there is about 3 fold increase in fluorescence at 520 nm under high Ca²⁺ levels as compared to 485 nm in Ca²⁺ free media (Shimozono et al. 2002). Additionally, three point mutations in Pericam resulted in the production of another Pericam, known as Flash Pericam. This point mutation resulted in the increase in fluorescence upto 8 folds at 520 nm. It is a non-ratiometric single wavelength indicator with a 0.7 µM K_d value (Whitaker 2010). Its absorbance spectrum is comparable to cpEYFP (V68L/Q69K) in the absence of Ca²⁺. On Ca²⁺ saturation, the pH titration curve shifts towards left due to the chromophore ionization which is actually caused by Ca²⁺ association with CaM and M13. At an optimum pH (9.0) for the ionization of chromophore, the Flash Pericam bound to Ca²⁺ exhibits twofold higher brightness than the free Flash Pericam. When Flash Pericam carries a substitution of phenylalanine at 203 residue within YFP, this resulted in the fluorescence on the protonated form and generation of a new construct i.e., Ratiometric Pericam, which is basically a sensor having an absorbance at 494 or 415 nm and emission at 520 nm. The functional properties of Ratiometric Pericam is analogous to Fura-2. It varies tenfold between Ca²⁺ free and Ca²⁺ saturated condition with a K_d of 1.7 µM (Whitaker 2010). Similar to Flash Pericam, it shows Ca²⁺-dependent changes in its absorbance spectra, and shifts the pH titration curve to the left upon binding Ca²⁺ (Pologruto et al. 2004). Additionally, semi-random mutagenesis in Ratiometric Pericam results in a construct named Inverse Pericam with having single wavelength and decreased intensity of fluorescence at 513–515 nm on binding with Ca²⁺ (Whitaker 2010). Interestingly, it can detect even low Ca²⁺ concentration in mitochondria and is a better option than Rhod-2 (Korzeniowski et al. 2009). The two important advantages of Inverse Pericam are the powerful brightness and the similarity of excitation/emission properties to fluorescein which is also a characteristic feature of Flash Pericam. Flash and Ratiometric Pericam are the YFP-based indicators which share common features with single wavelength Ca²⁺ sensors i.e., Fluo-3 and Fluo-4 (Pologruto et al. 2004). The difference between Ratiometric Pericam and Inverse Pericam is that Ratiometric Pericam shows slower decay time whereas Inverse Pericam shows constant decay time (Kaestner et al. 2014). Both Camgaroo and Pericam are used extensively in animal research.

GCaMP

GCaMP3 is a monochromatic sensor primarily used for observing live cells and performing in vivoCa²⁺ imaging. It relies on components such as calmodulin (CaM), the CaM binding domain RS20, circularly permuted green fluorescent protein (cpGFP), and the Ca²⁺/CaM-binding "M13" peptide. When the cellular Ca²⁺ concentration changes and Ca²⁺ binds to CaM, it leads to the protection of the cpGFP chromophore from the surrounding aqueous environment. Consequently, this results in an increase in fluorescence emission intensity. In mice, GCaMP3 finds utility in detecting neuronal activity in large populations within the motor cortex, barrel cortex, and hippocampus. These indicators are not suitable for quantitative ratiometric measurements because they exhibit changes in intensity without spectral shifts. Moreover, the maximum fluorescence intensity also varies with the level of expression. GEX-GECO1 (Fura-2-like) and GEM-GECO1 (Indo-1-like) were developed through random mutagenesis of GCaMP3 (Cho et al. 2017). Furthermore, each line expressing GCaMP3 was tested to observe how it responds to a non-injurious application of ice-cold water. The study aimed to understand the wound signaling in plants by using GCaMP3 as an indicator. They tested whether the mid-vein alone could transmit the wound signals to a distant leaf by removing extracellular tissue surrounding the primary vasculature of the leaf. The researchers also examined the spatial distribution of wound-stimulated GCaMP3 fluorescence (Nguyen et al. 2018). In another aspect of the study, the focus was on determining how aphid saliva affects changes in [Ca²⁺]_cyt levels in plants. They used CaMV35S: GCaMP3 transgenic N. benthamiana and showed that saliva obtained from both Serratia-free and Serratia-infected aphids induced a powerful Ca²⁺ signal within the first 90 s of a 300-s observation period (Wang et al. 2020). The role of Ca²⁺ in biotic stress using GCaMP3 was shown through live biotic interactions between the green peach aphid (Myzus persicae) and Arabidopsis (Vincent et al. 2017). In addition, the study sought to uncover how different cell types in plant roots react to fluctuations in [Ca²⁺]_cyt triggered by both internal and external signals. To investigate this, scientists engineered Arabidopsis plants with GCaMP3 expression in five distinct root cell types: columella, endodermis, cortex, epidermis, and trichoblasts. They observed both commonalities and differences in how [Ca²⁺]_cyt levels changed in response to substances like adenosine tri-phosphate (ATP), glutamate, aluminium, and salt, all of which are known to induce [Ca²⁺]_cyt increases in root cells. These specialized GCaMP3-modified plant lines provide a valuable resource for conducting more in-depth studies aimed at understanding the connections between specific environmental cues and distinct root development pathways mediated by [Ca²⁺]_cyt signaling (Krogman et al. 2020). In another study, the altered root growth due to impaired PEIZO1 channel activity in root tips exposed to mechanical stress was studied. To investigate this, the Ca²⁺ response to mechanical stimulation in living plants was examined using a transgenic line that expressed GCaMP3 (Mousavi et al. 2021). Additionally, how leaves and roots react to extracellular ATP and its potential association with Ca²⁺ signaling and DORN1/P2K1 was examined. The alterations in [Ca²⁺]_cyt levels in response to extracellular ATP were observed by employing wild-type roots that expressed GCaMP3 (Matthus et al. 2019).

GCaMP5s are derived from the GCCaMP3 framework through a specific library screening process that targets the interface between cpGFP/CaM and two interdomain linkers. Several GCaMP5 variants exhibit a remarkable fluorescence increase of over 150-fold upon binding Ca²⁺. Notably, in cultured neurons, GCaMP5G demonstrated a higher response to maximal stimulation (Akerboom et al. 2013). As RHO OF PLANTS (ROPs) are known to play key roles in controlling Ca²⁺ gradients, post-Golgi secretion, ROS generation, and the dynamic arrangement of actin microfilaments (MFs) during pollen tube growth, researchers were curious if these intracellular processes undergo dynamic changes during pollen germination in a BDR dependent manner. To investigate this, researchers introduced specific fluorescence probes into bdr8/bdr9 plants. These probes included GCaMP5 for monitoring Ca²⁺ concentrations, mRFP-RabA4b to track post-Golgi secretory vesicles, H2DCF-DA for visualizing ROS, and LifeAct-GFP to observe the dynamics of actin microfilaments (MFs;Xiang et al. 2023). G-CaMP5 was found to be an effective Ca²⁺ indicator for imaging Ca²⁺ levels in Arabidopsis pollen cells. The research utilized G-CaMP5 to reveal that Ca²⁺ forms a gradient focused on the tip of the pollen tube and exhibits oscillations during pollen tube growth. Additionally, a significant and temporary increase in cytosolic Ca²⁺ concentration was observed during pollen tube emergence. G-CaMP5 was also used to monitor changes in cytosolic Ca²⁺ levels in response to various treatments (Diao et al. 2018).

GCaMP6 variants are characterized by their high brightness and rapid detection of changes in [Ca²⁺]_cyt levels, often triggered by a single action potential. These variants were developed by introducing specific point mutations into the GCaMP5G framework, resulting in the creation of the GCaMP6 series. Within this series, the selected members, GCaMP6f, GCaMP6m, and GCaMP6s, differ in their response kinetics to fluctuations in Ca²⁺ levels. Among them, GCaMP6s stands out as the most sensitive to Ca²⁺ changes.

Recently, using GCaMP6f-mCherry under the control of UBQ10 promoter, Fu et al. (2022) demonstrated the role of Ca²⁺ signaling in maintaining manganese uptake and homeostasis in Arabidopsis (Fu et al. 2022). Based on genetically encoded Ca²⁺ sensor and advanced microscopy, Vigani and Costa (2019) revealed the potential of Ca²⁺ signaling in nutrient homeostasis in plant (Vigani and Costa 2019). Liu et al. (2017) showed that nitrate signaling coordinates with Ca²⁺ signaling via live imaging. Nitrate signaling involves NRT1.1/NPF6.3 transporters; transcription factors (TFs), CIPKs, phosphatases 2C and nitrate cis-regulatory elements (NRE). NLP6 and NLP7 are the major TFs which participate in nitrate responses. Nitrate-induced Ca²⁺ signaling was detected using transgenic aequorin seedlings and found to be more prominent as compared to flg22-derived Ca²⁺ signaling. This result was further validated by using Ca²⁺ biosensor, GCaMP6, co-expressing with nuclear mCherry and found that unique Ca²⁺ spikes were enhanced in nucleus and cytosol upon supplementation with nitrate. Interestingly, the signal vanished away when EGTA (Ca²⁺ chelating agent) was applied (Liu et al. 2017). Ca²⁺ signaling plays a crucial role in plant-pathogen and plant–insect interactions. Recently, Parmagnani et al. (2022) demonstrated the evocation of Ca²⁺ and ROS during herbivore-associated molecular patterns (HAMPs). The group has utilized various Ca²⁺ dyes, modern genetic probes and indicators in plant-pathogen interactions to study Ca²⁺ oscillation under such conditions (Parmagnani and Maffei 2022). To study local and distal Ca²⁺ signaling upon herbivory attack, GCamP6s was used, which is more advanced and sensitive than GCaMP1 to GCamP5 and demonstrated the role of GLR3.3 and GLR3.6 channels in systemic Ca²⁺ signaling in Arabidopsis (Xue et al. 2022).

Efforts in the past to enhance GCaMP kinetics has seen limited success. However, the development of jGCaMP8 sensors marks a significant breakthrough. These sensors comprise three distinct variants: jGCaMP8s, known for its rapid rise and slow decay, combined with high sensitivity; jGCaMP8f, characterized by fast rise and fast decay; and jGCaMP8m, featuring swift rise and medium decay rates. Remarkably, all jGCaMP8 sensors exhibit nearly tenfold faster fluorescence rise times when compared to earlier GCaMP iterations. What sets the jGCaMP8 sensors apart is their improved linearity, which is a significant advantage over previous GCaMP variants. This enhanced linearity facilitates accurate spike estimation through advanced deconvolution techniques. Notably, these sensors excel in addressing key limitations seen in earlier GECIs. They demonstrate rapid kinetics in response to fluctuations in Ca²⁺ levels. Moreover, the jGCaMP8 sensors maintain several essential characteristics shared with other GCaMP sensors, including exclusion from the nucleus, robust fluorescence, as well as similar excitation and emission spectra. Specifically, jGCaMP8s stand out as they exhibit the most significant fluorescence change in response to a single spike compared to all other Ca²⁺ indicators. It also boasts a moderate half-decay time of 200 ms in the mouse brain. However, there's a trade-off to its heightened sensitivity. jGCaMP8s can saturate at a lower spike count, resulting in a reduced dynamic range. Nonetheless, this limitation is partially mitigated by its improved linearity and kinetics (Zhang et al. 2023).

The genetically encoded Ca²⁺ indicator, GCaMP-HS (GCaMP hyper-sensitive), represents an upgraded version of the original GCaMP, offering heightened sensitivity to changes in cellular Ca²⁺ ion (Ca²⁺) concentrations (Muto et al. 2011). This enhanced sensitivity is achieved through the incorporation of superfolder GFP mutations which is formed by amino acid substitution. The resultant construct increases folding activity but also enhances fluorescence output (Akerboom et al. 2012). Muto et al. (2011) introduced four specific superfolder mutations—S30R, Y39N, N105T in the N-terminus region of GFP, and A206V in the C-terminus region of GFP—into the previous iteration of GCaMP, known as GCaMP2 (Muto et al. 2011). This genetic modification led to a novel variant termed GCaMP-HS. GCaMP-HS exhibited an absorbance wavelength at 488 nm and emission at 509 nm, along with a K_d value of 105 nM in the presence of 300 nM Ca²⁺. One notable finding was that the levels of the GCaMP-HS protein consistently exceeded those of the GCaMP2 protein by a factor of 1.7. This suggests that the heightened folding efficiency induced by the superfolder mutations led to increased cellular expression levels. Consequently, this elevation in cellular expression contributed to the baseline fluorescence enhancement observed in GCaMP-HS (Muto et al. 2011).

FRET-based sensors

Genetically encoded Ca²⁺ biosensors are valuable tools in cell biology and neurology, but areas like signal strength and response kinetics need improvement. Forster resonance energy transfer (FRET) biosensors have revolutionized live cell imaging by allowing real-time chemical processes to be observed with high temporal resolution (Yoon et al. 2020). EGFP and Fusion-Red fluorophores were used to make a new Ca²⁺ biosensor called FRET-GFP-Red. This biosensor allows precise targeting of subcellular areas and repeated stimulation for longitudinal studies. The study supports a consistent 10–15% FRET response of a FRET-GFP-Red construct (Yoon et al. 2020). A new Ca²⁺ ion probe, H2BD3cpv, based on FRET, targets the nucleoplasm and measures Ca²⁺ in a precise way. The probe, adapted to mCerulean3, showed increased brightness and photostability and validated the similarity of cytoplasmic and nucleoplasmic Ca²⁺ concentrations in both basal and stimulated cellular states (Galla et al. 2021).

Yellow Cameleon

Yellow Cameleon indicators are majorly expressed in the cytosol and also expressed in guard cells (Allen et al. 1999). YC2.1 has been used successfully in the study of Arabidopsis guard cell Ca²⁺ dynamics (Allen et al. 1999). In Arabidopsis, ratiometric imaging of YC2.1 allowed Ca²⁺ imaging in a time-dependent manner (Grynkiewicz et al. 1985). In pollen tube growth, YC2.1 is used as an invasive method. In root hairs, the use of YC2.1 and a range of probes have limited the usefulness of YC2.1 (Lam et al. 2005). In most of the transgenic studies, increasing the expression level of YC2.1, CaMV 35S promoter driven expression is preferred. But the CaMV 35S promoter is associated with events like gene silencing and co-suppression. So, UBQ10 promoter can be used in the place of CaMV 35S as it is stable in plants, but has moderate target gene expression (Krebs et al. 2012). YC2.1 was first used for Ca²⁺ imaging in pollen tubes. The results showed that both Lilium longiflorum and Nicotiana tabacum had a Ca²⁺ gradient and tip-focused oscillations (Parton et al. 2003). Time-to-time a number of other modifications have been done to improvise the efficacy of these techniques.

Yellow Cameleon 3.6 was developed in Roger Tsien's laboratory. It is composed of CaM, donor chromophore (CFP), glycine linker, CaM-binding peptide, myosin light-chain kinase (M13), and acceptor chromophore (YFP). When Ca²⁺ binds to the CaM, an intramolecular interaction occurs between CaM and M13 that results in a change in the protein to a more compact form from an extended form, which increases the efficiency of FRET between CFP and YFP (Krebs et al. 2012; Behera et al. 2015). These dyes are FRET-based ratiometric Ca²⁺ indicators used (Fig. 4). They do not include the artifacts, which are the results of fluctuations in the Ca²⁺ indicator expression levels (Behera et al. 2015). It consists of a circularly permuted Venus (cpVenus). It is brighter, and on maturation, it is more efficient and has acid stability when compared with enhanced yellow fluorescent protein (EYFP) used in YC2.1 (Krebs et al. 2012). Thus, YC3.6 provides a high signal-to-noise ratio, which is why it can be used for various Ca²⁺ imaging experiments that were previously not possible with the other YCs (Nagai et al. 2004). Various YCs are targeted to the endoplasmic reticulum, peroxisome, and mitochondria and are thus successfully used to study the organellar Ca²⁺ dynamics and homeostasis (Behera et al. 2015). In guard and root cells, YC3.6 probes give efficient results for mitochondrial Ca²⁺ changes in response to different types of stimulus. Its K_d value for Ca²⁺ is 0.25 µM. The Ca²⁺ gradient  during growth of the tip can be monitored using YC3.6 (Lam et al. 2005).

Fig. 4.

The principle model presenting YC-based Ca²⁺ imaging. In the absence of Ca²⁺ binding to the Cameleon protein, the excitation of the CFP by light with a wavelength of 488 nm leads to the emission of light with wavelengths approximately centered around 480 nm, which falls within the blue light spectrum. The process of Ca²⁺ binding to the M13 domain induces a conformational change in the protein, resulting in the proximity of the CFP and YFP domains. The proximity of the domains enables FRET to occur, facilitating the transfer of energy from the CFP to the YFP. Consequently, light with a wavelength of around 535 nm (yellow) is emitted. The relationship between the fluorescence intensities of YFP and CFP is directly proportional to the concentration of Ca²⁺ within the cell (Yellow-to-Cyan FRET Ratio = I_Yellow/I_Cyan) (adapted from Monshausen et al. 2008)

Ca²⁺ levels in cells are crucial signals for various biological activities. Using ratiometric Ca²⁺ reporter proteins like Yellow Cameleon (YC) has enabled us  to understand more about the dynamic behavious of Ca²⁺ transient/signature in the living cells. This pioneering development in plant research has expanded to include single-cell models like pollen tubes and root hairs. However, the use of YC reporter proteins in plants has mainly focused on (Ca²⁺)_cyt levels. YCs is composed of two forms- Green Fluorescent Protein (GFP): CFP and YFP, representing cyan and yellow, respectively (Behera et al. 2015). The M13 calcium-binding domain, derived from calmodulin, links the two forms. Under low Ca²⁺, the cyan fluorescent protein (CFP) emits blue light, which is induced by presence of higher Ca²⁺ and hence binding CaM which interact with the M13 domain and causes conformational changes. This conformational change causes the CFP and YFP domains to be near to each other, allowing FRET to occur, resulting in emission of yellow light (Fig. 4) (Behera et al. 2015). The fluorescence intensities of YFP and CFP are directly proportional to the concentration of Ca²⁺ in the cell or at a specific location. This ratio is advantageous as it remains unaffected by the quantity of YC3.6 protein in the cellular environment. Periodic measurements can be conducted to monitor changes in Ca²⁺ concentration. During development, reproduction, biotic and abiotic stress, plants generate a unique Ca²⁺ signatures. YC is a popular option for scholars looking to explore these characteristics. Some of the recent studies related to this sensor are briefly described here. Chlamydomonas reinhardtii, a single-cell microalgae, responds to light and the role of intracellular Ca²⁺ signals in these responses  has been studied  by using YC3.6 (Pivato et al. 2023). The ratiometric Ca²⁺ indicator YC3.6 was used, with a focus on the cytosol, chloroplasts, and mitochondria. In vivo single-cell confocal microscope imaging was used to examine light-induced Ca²⁺ signaling under various situations and genotypes, including the photoreceptor mutants, phot and acry. A genetically encoded H₂O₂ sensor was used to explore the potential involvement of hydrogen peroxide production in light-induced Ca²⁺ signaling (Pivato et al. 2023). The study found that Chlamydomonas reinhardtii cells exhibited a Ca²⁺ ion response reliant on light, found only inside the chloroplast. The intensity of light and photosynthetic electron transfer impacted this response. The study found a link between elevated H₂O₂ gradients in chloroplasts and transient increases in Ca²⁺ levels, confirming the role of light-induced chloroplast Ca²⁺ signaling (Pivato et al. 2023). Ca²⁺ signatures are crucial in Ca²⁺ signaling pathways and require high spatial and temporal resolution. Vectors and transgenic lines were developed to visualize intracellular Ca²⁺ dynamics in live plant cells using YC. The ubiquitin-10 (UBQ10) promoter from Arabidopsis is used to ensure consistent expression of YCs in transgenic plants. The vector repertoire includes multiple iterations directed towards specific areas. This provides an opportunity to examine Ca²⁺ dynamics in various cellular compartments and plant species, facilitating the development of innovative methodologies (Krebs et al. 2012). Behera et al. (2015) compared how the 35S promoter and the UBQ10 promoter works in the plants Arabidopsis thaliana and Oryza sativa. The study focused on the expression of the Ca²⁺ indicator YC3.6, controlled by either the UBQ10 or 35S promoters (Behera et al. 2015). The UBQ10 promoter performed better in terms of expression pattern, levels, and stability in both species. However, significant differences were observed in the spatiotemporal characteristics of the detected Ca²⁺ signals between the two species (Behera et al. 2015). Rice showed a decreased peak signal amplitude but a significantly prolonged signal duration compared to Arabidopsis. The study highlights the importance of comparative research for a comprehensive understanding of Ca²⁺ signaling in plant (Behera et al. 2015).

In eukaryotes, subcellular compartments like mitochondria, endoplasmic reticulum, lysosomes, and vacuoles can transport Ca²⁺ across their membranes to modulate enzyme activity or convey specific cellular signaling events. In plants, chloroplasts also display Ca²⁺ regulation. However, monitoring stromal Ca²⁺ dynamics in vivo has been limited by the use of the luminescent Ca²⁺ probe aequorin. A toolkit of Arabidopsis Ca²⁺ sensor lines expressing plastid-targeted FRET-based YC sensors has been presented. The probes reliably reportin vivo Ca²⁺ dynamics in the stroma of root plastids in response to extracellular ATP and leaf mesophyll and guard cell chloroplasts during light-to-low-intensity blue light illumination transitions. Applying YC sensing to single chloroplasts, the findings confirmed gradual, sustained stromal Ca²⁺ increases at the tissue level after light-to-low-intensity blue light illumination transitions. The toolkit identifies novel facets of chloroplast Ca²⁺ dynamics and refines the understanding of plastid Ca²⁺ regulation (Loro et al. 2013).Cells that grow from the tip are thought to have a tip-high Ca²⁺ gradient that controls parts of the growth machinery, such as the cytoskeleton, Ca²⁺-dependent regulatory proteins, and the secretory apparatus. In pollen tubes, both the Ca²⁺ gradient and cell elongation show oscillatory behaviour, reinforcing the link between the two (Monshausen et al. 2008). In Arabidopsis root hairs, a YC3.6 FRET-based Ca²⁺ sensor was used to monitor the oscillating tip-focused Ca²⁺ gradient (Monshausen et al. 2008). Both the elongation of root hairs and the associated tip-focused Ca²⁺ gradient show a similar dynamic character, oscillating with a frequency of 2 to 4 min⁻¹. Cross-correlation analysis indicates that Ca²⁺ oscillations lag growth oscillations by 5.3 ± 0.3 s. However, growth never completely stops, and the concomitant tip Ca²⁺ level is always slightly elevated compared to the resting Ca²⁺ concentration along the distal shaft. Artificially increasing Ca²⁺ leads to an immediate cessation of elongation and thickening of the apical cell wall (Monshausen et al. 2008). Cross talk between Ca²⁺ and ROS signaling has been reported during root developmental processes in plants (Monshausen et al. 2007). Actually, [Ca²⁺]_cyt is responsible for ROS generation which further stimulates downstream signaling pathway (Monshausen et al. 2007). The modulation in Ca²⁺, ROS and pH levels during root hair cell growth in Arabidopsis was monitored by YC3.6 Ca²⁺ biosensor, Oxyburst (ROS-sensitive dye), and GFP-H148D, a pH- sensitive probe, respectively (Monshausen et al. 2007). External and internal stimuli can temporarily increase Ca²⁺ in the cytoplasm, triggering a cascade of reactions that govern plant growth. A Ca²⁺oscillatory gradient at the pollen tube tip is essential for polarised growth. Disrupting this gradient can inhibit pollen tube development. Researchers have created genetically engineered Ca²⁺ YCs to assess the physiological function of (Ca²⁺)_cyt in biological systems. The Cameleon uses FRET to measure changes in (Ca²⁺)_cyt levels in developing pollen tubes (Barberini and Muschietti 2015). The transgenic plants that express the YC3.6 under the pollen-specific promoter LAT52 were employed to monitor how Ca²⁺ moves in the pollen tubes of tomato plants (Barberini and Muschietti 2015). The study aimed to understand the processes responsible for the generation and maintenance of the intracellular Ca²⁺ gradient, which is crucial for exocytosis and the transportation of fresh membrane and cell wall materials to the apex of the pollen tube (Barberini et al. 2018). Ratiometric fluorescent imaging techniques, specifically the YC3.6, have been used to investigate and interpret the Ca²⁺ signature generated by flg22 in individual guard cells of Arabidopsis thaliana. The determination of Ca²⁺ reserves and the kinds of channels involved in their creation was conducted using a pharmacological technique (Thor and Peiter 2014). The fluorescence-based Ca²⁺ sensors R-GECO1 and NES-YC3.6, are based on the intensity of the light. The results showed that R-GECO1 had a larger signal shift in response to different stimuli, such as the fungal elicitor chitin, which caused a strong [Ca²⁺]_cyt oscillation in epidermal and guard cells. The elongation zone was found to be the starting point for both flg22 and chitin-induced Ca²⁺ signals in the root (Keinath et al. 2015). CCX2 is one such Ca²⁺ exchanger, localized on endoplasmic reticulum (ER), strongly expressed under salt and osmotic stress conditions (Corso et al. 2018). Cameleon Ca²⁺ biosensors revealed the importance of AtCCX2 gene, absence of which resulted in impaired Ca²⁺ dynamics in the ER and cytosol as compared to wild type (WT) and over-expression lines in Arabidopsis (Corso et al. 2018). Ca²⁺ imaging with fluorescent sensor, YC3.6, helped in the study of Ca²⁺ dynamics under dehydration and rehydration treatment in moss (Physcomitrella patens) (Storti et al. 2018).

D3cpv, N33D1cpv and N33D3cpv

D3cpv consists of a circular permutation of Venus (cpVenus). It is brighter, and on maturation, is more efficient and has acid stability compared with enhanced yellow fluorescent protein (EYFP) used in YC2.1 (Krebs et al. 2012). Since D3cpv comprises a dual fluorophore, the fluorescence ratio has the potential to be adopted to monitor Ca²⁺, overcoming movement aberrations (Kuhn et al. 2012). D3cpv is especially useful for researchers who are looking for minor changes in Ca²⁺ beyond basal levels because its Ca²⁺ K_d is precisely between YC2.1 and YC3.3, but it is no longer affected by excess CaM and has a fivefold increase in dynamic range. D3cpv might get localized to the plasma membrane of hippocampal neurons, where it is sensitive to both spontaneous and proven Ca²⁺ influx KCl-induced depolarization, exogenous glutamate addition, and electrical simulation (Palmer et al. 2006). This modified Cameleon overcomes the issue of prior Cameleons that were unable to measure Ca²⁺ influx when focused on the plasma membrane, enabling us to view Ca²⁺ influx without any artifacts. Tandem CytC signal sequence repetitions were employed to D2cpv, D3cpv, and D4cpv (Palmer et al. 2006). D3cpv, like other GECIs, has restrictions when it comes to displaying spike rates and timeframes. Because each of the currently used GECIs has positive cooperativity, the reportable Ca²⁺ range of concentrations for any given GECI is constrained, and Ca²⁺-binding saturation is quickly achieved. D3cpv also has slow response dynamics, with off-responses having a time constant of nearly two seconds (Kuhn et al. 2012). D3 Cameleon variants (D3cpv) are distinguished by a single and appropriate affinity for Ca²⁺ (K_d) and a good dynamic range (R_max/R_min) sensitivity to Ca²⁺ fluctuations (Greotti et al. 2019). Certain variants of the original YC protein exhibited a failure to accurately detect and report fluctuations in Ca²⁺ levels when localised to the plasma membrane, a cellular compartment where CaM may accumulate in mM quantities. As a result of this, Palmer et al. (2006) introduced a new lineage of Cameleons, referred to as Dcpv. In summary, the Dcpv family originates from the classical Cameleons, wherein two variants of GFP, namely CFP and cpVenus (cpv, a variant of yellow fluorescent protein (YFP) that is circularly permuted), are connected via a mutated CaM and M13 peptide (Palmer et al. 2006). This linkage serves to eliminate or significantly diminish interference from endogenous CaM. Similar to the conventional Cameleon, within the Dcpv family, the binding of Ca²⁺ triggers a conformational change in CaM, leading to its subsequent interaction with M13. As a consequence of the decreased distance between the CFP and the chromophore-binding protein (CPV), there is an observed augmentation in the efficiency of FRET. The rise in FRET and thus, the increase in Ca²⁺ may be simply quantified by measuring the increase in the ratio of emission intensities between cpv and CFP, respectively, after excitation of CFP (Palmer et al. 2006).

Mitochondria are essential organelles in the eukaryotic cell, generating unique Ca²⁺ patterns in response to abiotic and biotic stimuli. The discovery of the mammalian mitochondrial Ca²⁺ uniporter has opened up new research in plant biology. A transgenic Arabidopsis line expressing the genetically encoded Cameleon D3cpv probe, specifically targeting mitochondria,  showed more fluorescence as compared to the established Cameleon YC3.6 (Loro and Costa 2013).

Giacomello et al. (2010) developed a GFP-based Ca²⁺ probe i.e. N33D1cpv targeted on the outer mitochondrial membrane (OMM) suitable for tracking Ca²⁺ hotspots (high Ca²⁺ levels ranging from 1 to 200–300 M) in a limited cellular area. The Ca²⁺ biosensor N33D1cpv comprises a structure made up of a N33, that codes for an outer mitochondrial membrane (OMM) peptide, a FRET donor (CFP-Cyan Fluorescent Protein), a Ca²⁺ binding domain D1, and a FRET acceptor (ccpv173-circularly permutated Venus protein); (Gouriou et al. 2023a).The Dcpv protein, often known as Cameleons, has undergone evolutionary changes resulting in the emergence of many variations, namely D1, D2, D3, and D4. These variants exhibit distinct affinities for Ca²⁺ ions and possess diverse cellular localization signals. Using the N33D3cpv biosensor, both genetic suppression and pharmacological inhibition of endogenous IP₃R activity led to a decrease in the rate of Ca²⁺ leakage from the endoplasmic reticulum (ER) through the OMM when the oxygen level was low (Gouriou et al. 2023a).

Gouriou et al. (2023a, b) developed the N33D3cpv indicator by replacing the N33D1cpv D1 Ca²⁺ binding domain with the D3 Ca²⁺ binding domain of the cytosolic biosensor D3cpv, which has a greater Ca²⁺ affinity (Gouriou et al. 2023a). N33D3cpv having a D3 ligand domain with a dissociation constant (K_d) of 0.6 µM, is well matched to the range of low intensity Ca²⁺ fluctuations in the cytosol (from 0.1 to 10 µM). First, Ca²⁺ response amplitude with an ATP stimulus showed that the relative fluorescence intensity (F/f₀) ratio peak average is higher with N33D3 than with N33D1cpv, which is 1.573 and 1.107, respectively. The overall F/F₀ ratio was higher with N33D3cpv than with N33D1cpv, which is 1.426 and 1.123, and there also existed a distinction in the rate of decay of Ca²⁺ levels. N33D3cpv enabled precise and reliable readings of Ca²⁺ dynamic at OMM. Overall, these findings clearly indicate the improved sensitivity of the new N33D3cpv sensor and its capacity to recognise lower fluctuations in Ca²⁺ dynamic at OMM (Gouriou et al. 2023a). D3cpv, N33D1cpv, and N33D3cpv have extensively been used in recording Ca²⁺ in animal system.

iGECI, TN-XXL and TnC (Troponin C)-based FRET sensor (Twitch)

The near infrared Ca²⁺ sensor (NIR) called iGECI, is based on FRET. Mammalian cells benefit from its comparatively high effective brightness and photostability, which enables mouse cortex deep layer imaging. Although iGECI has a huge molecular weight (86 kDa) and slower signal fading, these factors may impair the effectiveness of targeting and packaging of proteins in Adeno associated virus (AAV); (Matlashov et al. 2022). iGECI exhibited a modest absorption peak at 390 nm as well as two large peak values of 640 nm and 700 nm for absorption. The fluorescence of iGECI peaked at 670 and 720 nm, respectively. iGECI showed two sensitivity constants, K_d1 = 15 nM and K_d2 = 890 nM, as a consequence of four Ca²⁺ binding sites (Shemetov et al. 2021).

TN-XXL is a FRET based Ca²⁺ biosensor and is based on Troponin C (Tian et al. 2009; Geiger et al. 2012). It is mainly used for long term Ca²⁺ imaging (Mank et al. 2008). TN-XXL consists of four EF-Hands, which can be divided into two classes. In one, EF-hand 3.1 and 3.2 are involved in FRET output, and in the second, 4.1 and 4.2 are involved, which are responsible for stabilising function, but not FRET output (Geiger et al. 2012). It has been detected that the change in the FRET level in TN-XXL is not only due to the various changes in the conformation but also due to the changes in fluorophore orientation. In a Ca²⁺-free form, it consists of fluorescent donor and acceptor proteins at opposite ends, which should not be in a close proximity with each other. However, in Ca²⁺-bound form, they are always in close proximity (Geiger et al. 2012). The signal amplitude of the TN-XXL is lower than that of the OGB-1 (Mank et al. 2008). In most of the studies, Ca²⁺ oscillations have been detected more strongly and more consistently using TN-XXL in Drosophila neurons (Whitaker 2010).

TnC (Troponin C)-based FRET sensor (Twitch) offers a linear response, a smaller size, a broad range of Ca²⁺ affinities, superior photostability, a wider dynamic range, and faster kinetics compared to the TN-XXL. Even with all these improvements, FRET sensors experience a low signal-to-noise ratio (SNR). In comparison to GCaMPs, the kinetics of Twitch with in on/ and off conditions is still slower (Cho et al. 2017). Twitch-5's K_d value of 9.25 µM suggests rapid kinetics, while Twitch-3's K_d value of 250 nM shows noticeably delayed kinetics. Slow kinetics produces more photons, which increases the signal and makes indicators more sensitive (Thestrup et al. 2014a). The indictors have been named because of their sensor parts are derived from Troponin C which is a Ca²⁺-binding protein present in the skeletal and cardiac muscle that governs muscle twitching. A single Troponin C molecule, like most Ca²⁺-binding proteins, binds up to four Ca²⁺ ions using four specialised protein domains known as EF- hands (Wilms and Häusser 2014). In addition, for measuring the accurate kinetics and function of Ca²⁺ binding structure, resolved spectroscopy is used due to its simple and compact structure. By decreasing the number of binding Ca²⁺ ions, the biocompatibility of the sensor can be improved as it reduces buffering while using it for long term expression. The structure of the Ca²⁺ binding domain and whole structure of Twitch-1 was determined by using nuclear magnetic resonance (NMR) and the whole prototype indicator by small angle X-ray scattering spectroscopy (Wilms and Häusser 2014). As it is a FRET indicator, it shows ratiometric detection. However it relies on change in volume and movement of Ca²⁺ but not the change in intensity of Ca²⁺. Hence, it is a reliable Ca²⁺ imaging indicator for motile cells. When comparing this with the previous FRET-based GECIs, which are brighter at resting condition of the cell, the Twitch has been found to be more brighter. Therefore, it allows imaging of cell structure via increasing signal-to-noise ratio and makes it suitable for doing functional imaging (Wilms and Häusser 2014). iGECI, TN-XXL and Twitch have been used in animal research extensively.

Ca²⁺ imaging: applications in animal sciences

Animal systems are more complex than plant and therefore, it is very important to visualize the changes occurring at cellular and sub-cellular levels. Several fields have been revolutionized from the advances in Ca²⁺ imaging, particularly pathology and neurosciences. Quin-2 was the first member of the first generation of fluorescent Ca²⁺ indicators to be utilised in biological research. Pozzan et al. (1982) used the Quin-2 indicator to determine the influence of mouse spleen lymphocyte anti-immunoglobulin on cytoplasmic Ca²⁺ concentration (Pozzan et al. 1982). Because Quin-2 is not an excellent Ca²⁺ indicator, it must be used with elevated intracellular concentrations to prevent cellular autofluorescence. Another first-generation indicator dye Fura-2 has been used by many neuroscientists since it outperforms Quin-2 in many ways (Grienberger and Konnerth 2012). Phillips et al. (2019) measured the neuronal Ca²⁺ levels in DFP (OP-diisopropyl fluorophosphate)-based rat model for GWI (Gulf War Illness). GWI is a chronic multi-symptom disorder that affects veterans of the First Gulf War. It includes depression and memory loss as neurological symptoms (Phillips et al. 2019). It’s utilisation is not only limited to the neurology but also includes other fields of biology. One of the common kind of muscular dystrophy is Duchenne muscular dystrophy (DMD). The severity of the illness in DMD is considered to be influenced by changes which was measured by Fura-2 (Mázala et al. 2015). Numerous other Ca²⁺ indicators have been developed over time, with a variety of excitation spectra and affinities for Ca²⁺. These include, Fluo-4 dye families, Oregon Green BAPTA families and many more. The Fluo-3AM has been used to study the intracellular Ca²⁺ concentration and other ROS assays to uncover whether the blood–brain barriers (BBB) in cerebrovascular endothelial cells destruction after ischemic stroke was caused by the inactivation of Wnt7/β-catenin signaling (Haiping et al. 2023). Fluo-4 is another widely used Ca²⁺-sensitive florescent dye. The development of Fluo-4 has had a significant impact on the study of Ca²⁺ signaling in biological systems, enabling researchers to gain deeper insight into intracellular Ca²⁺ dynamics and its role in various physiological processes. Bajnok et al. (2023) have used the Fluo-4AM to study the Ca²⁺ flux which plays a key role in B cell signaling pathway and might act as an important tool for drug discovery towards the development of autoimmune disease caused by B-cells. In this study, they standardised a method based on flow cytometry to study the patterns of Ca²⁺ flux of Peripheral Human B-cells. This approach can be used in clinical research, multi-centric studies and also facilitate the assessment of rare conditions (Bajnok et al. 2023). With time, Ca²⁺ indicators have been developed with the goal of enhancing the performance of Ca²⁺ imaging experiments. Following the Fluo family, a new synthetic Ca²⁺ indicator Cal-520, Cal-590, and so on have been reported. Daily et al. (2017) have shown that when iPSC-derived (induced Pluripotent Stem Cells) human cardiomyocytes for drug screening were employed, FLIPR Calcium 6 and Cal-520 were found to be appropriate dyes (Daily et al. 2017). It has been seen that Cal-590 applied ultra-short laser pulse to reduce photoxicity, showcasing the potential for deep two-photon imaging in vivo with single-cell resolution, allowing for the examination of neuronal population in all six layers of the mouse brain cortex (Birkner et al. 2017). Cal-520, a comparable alternative, offers several advantages over visible-light dyes such as Fluo-4 and Oregon Green 488 BAPTA-1 (OGB-1). It enhances intracellular retention and signal-to-noise ratio (SNR). In a study conducted by (Tada et al. 2014), Cal-520 proved to be highly effective for in vivo detection of dendritic Ca²⁺ transients in Purkinje cells of anesthetized mice, displaying both high SNR and rapid decay time (Tada et al. 2014).

The dysregulation of neural circuit function leads to development of neuropsychiatric disorders. Monitoring neural population and its activity under drug supplementation is still challenging for the scientific world. The regulation of behavioural addiction by neural circuits makes the monitoring of cellular granularity in brain region highly important. With the utilization of genetically encoded Ca²⁺ indicator (GECI), Siciliano and Tye (2019) were able to target the cells of interest and study in vivo Ca²⁺ dynamics to access neural activity. The researchers have developed miniaturized two-photo microscope (MINI2P) to visualize high resolution, fast and multiplane Ca²⁺ imaging in moving mice to study almost thousands of neurons at a time. The group were able to observe cells of medial entorhinal cortex, hippocampus and visual cortex (Zong et al. 2022). With the utilization of transgenic Pirt-GCaMP6s mice, the gauge responses of DRG (dorsal root ganglion) neurons were studied under in vivo visceral and somatic stimulation to understand the phenomenon of visceral pain sensitization and the concept of somatic hypersensitivity (Gao et al. 2021).

Ca²⁺ currents are very essential for proper functioning of neuron cells. To image a native neuron, Ca²⁺ current in brain slices, a combined approach was adopted by using low affinity Ca²⁺ indicators along with voltage sensitive dyes. Using this technique, Ca²⁺ kinetics was estimated via modulations in Ca²⁺ fluorescence activity which was simultaneously correlated with change in surface potential through change of voltage fluorescence (Ait Ouares et al. 2020). Ca²⁺ imaging in higher organisms such as Rhesus macaque is also being studied via GCaMP Ca²⁺ indicator, which will help in understanding the human brain function as well as neurological diseases (Bollimunta et al. 2021). With the help of genetically encoded Ca²⁺ indicators, Ca²⁺ oscillations were measured in neural cells of mammals (Jercog et al. 2016). Also, with the utilization of genetically encoded Ca²⁺ sensor, a protocol of brain surgeries for deep brain Ca²⁺ imaging via miniature fluorescence microscope (miniscope) was developed (Thapa et al. 2021).

Guillain-Barré syndrome is the a type of syndrome which causes numbness, weakness and pain in feet, hands and limbs of human body. This syndrome is majorly associated with activation of complement on the axonal PM by the attack of motor axons. In animal systems, Ca²⁺ influx is triggered by axonal damage which is caused by protease calpain. Cunningham et al. (2022), were able to monitor ex vivo intra-axonal Ca²⁺ changes in mouse affected with axonal Guillain-Barré syndrome and demonstrated the role of Ca²⁺ during axonal injury and explained the effects of calpain inhibition (Cunningham et al. 2022). Ca²⁺ act as an important messenger in neuronal system however, its function in regeneration of axon is not fully understood. A non-invasive in vivo Ca²⁺ imaging using GCaMP6f for studying Ca²⁺ oscillations was carried out during regeneration of axon in zebrafish Mauthner cells. An inward rectifying K⁺ channel protein i.e., Kir2.1a is solely responsible for decrease in the activity of Mauthner neural activity. The Kir2.1a overexpression resulted in the reduced prohibition of Ca²⁺ induced axonal regeneration (Chen et al. 2019).

Caenorhabditis elegans which is considered as an excellent model system to study Ca²⁺ imaging in animals was utilized to study in vivo Ca²⁺ dynamics in the cells of body wall muscles. It was observed that with the co-expression of presynaptic channel rhodopsin, there was a provocation of acetylcholine from excitatory motor neurons accompanied by blue light pulses, resulting in the depolarization of muscle cells and ultimately leading to transient changes in [Ca²⁺]_cyt levels. The change in Ca²⁺ was monitored by genetically encoded Ca²⁺ sensors (Martin et al. 2019). With the utilization of GECI, the amygdala in deeper brain region of mouse was captured via Ca²⁺ imaging with head-mount miniaturized microscope (Lee and Han 2020).

Pancreatic islet hormones are important for the maintenance of glucose homeostasis. Interestingly, it was found that any change in blood glucose level induces change in [Ca²⁺]_cyt in pancreatic islet cells. The excess of Ca²⁺ in these cells promote the secretion of three major hormones such as insulin (reside in beta-cells), glucagon (alpha-cells), and somatostatin (delta-cells). Beta cells are connected to each other to form one single entity. These are stimulated on glucose supplementation and are a major constituent of islet cells. Therefore, the excitability of others such as alpha and delta cells could show interesting variations and are of special interest. With the help of Ca²⁺ imaging, it has been analysed that the minor cell responses on the application of adrenaline and ghrelin which induces transient change in [Ca²⁺]_cyt in specific populations of islets cells of the mouse (Hamilton et al. 2019).

Brugia malayi is a filarial nematode which causes lymphatic filariasis/ elephantiasis disease in human. Unlike C. elegans, Ca²⁺ dynamics in B. malayi is less studied (Williams et al. 2020). Williams et al. (2020) has developed Ca²⁺ imaging method for the first time to study the cellular Ca²⁺ dynamics in muscle of B. malayi. Ca²⁺ imaging is found to be a suitable technique to uncover the mode of action of anthelmintic drugs and their effect on the parasite. Two methods were generated to study Ca²⁺ dynamics: muscles of B. malayi were soaked with Fluo-3AM and the other, where Fluo-3 was directly micro injected. By using these approaches, Ca²⁺ dynamics as well as electrophysiological recordings were measured (Williams et al. 2020). The transgenic mouse expressing genetically encoded Ca²⁺ indicator, RCaMP1.07, under alpha-smooth muscle actin promoter was used to study Ca²⁺ fluctuations in brain cells and correlate it with hemodynamics in mice (Meza-Resillas et al. 2021).

The voltage gated calcium channel (VGCC) of the retinal ganglion mediate Ca²⁺ signaling which was monitored via Ca²⁺ imaging tool in rat (Sargoy et al. 2014). Also, the long-term memory formation is correlated with Ca²⁺ dynamics in animals. It is a well know mechanism that G-protein coupled receptors (GPCR) activation leads to an increase in intracellular Ca²⁺ concentration. Simultaneously, the activation of neuron also causes increase in intracellular Ca²⁺ level. It has been observed that GCaMP-expressing Drosophila larval brain cell was able to enhance Ca²⁺ level upon test peptide and the fluorescent signal was captured via spinning disc confocal microscope associated with CCD camera (Ishimoto and Sano 2018). With the utilization of in vivo functional and Ca²⁺ imaging, Rigosi et al. (2015a) has demonstrated that the right antenna of bees were better in distinguishing a target odour than left antenna (Rigosi et al. 2015b).

Single photon Ca²⁺ imaging via real-time processing correction (RTMC) method in animals has also been reported (Li et al. 2022a). The whole brain Ca²⁺ imaging with good cellular resolution was reported in freely swimming larval zebrafish by using HiLo microscopy (Kim et al. 2017). Jacob et al. (2018) described the usage of low-cost compact head-mounted endoscope (CHEndoscope) to monitor Ca²⁺ imaging (Jacob et al. 2018). Many such studies have brought us a clear understanding of the cellular pathways and have helped in identifying highly important mechanisms of Ca²⁺ dynamics and signaling in regulating diverse cellular processes.

Unveiling intricacies: important contributors in the field of Ca²⁺ imaging techniques

The field of Ca²⁺ imaging has evolved from recording Ca²⁺ activity in individual cells to visualising and analysing Ca²⁺ signals within complex tissues and organs. This has significantly improved our understanding of cellular signaling and physiological mechanisms in plants and animals. In plant biology, Ca²⁺ imaging is used to examine Ca²⁺ transitions during growth and development processes and to study plant responses to biotic and abiotic stresses. Several scientists have worked to improve Ca²⁺ imaging techniques in plants. This has made it possible to see and understand how Ca²⁺ signals work in different parts of plant physiology, growth, and responses toward diverse stimuli. In animal systems, Ca²⁺ imaging has enabled the observation of neuronal activity patterns, synaptic transmission, and Ca²⁺ signaling in various tissues and organs, including the brain, heart, and immune system. Recent advancements in imaging technology have further propelled the field of Ca²⁺ imaging (Kanchiswamy et al. 2014). Advancements in Ca²⁺ imaging have enabled high precision in observing Ca²⁺ dynamics in both spatial and temporal domains (Russell 2011; Nietz et al. 2022). These advancements and improvements in multiple imaging systems have improved the accuracy and sensitivity of visualising Ca²⁺ ion dynamics, with notable contributions from researchers over a period of time (Fig. 5).

Fig. 5.

The present roadmap aims to provide an overview of the milestones in the field of in vivo studies pertaining to Ca²⁺ biosensing in plants

The discovery of aequorin (1963) by Shimomura was of significant importance in understanding bioluminescence. Aequorin is a Ca²⁺-sensitive protein found in jellyfish that emits blue light when it binds Ca²⁺ ions (Prasher et al. 1985). This discovery helped scientists gain insights into the biochemical processes involved in bioluminescence and paved the way for further research on the mechanisms and applications of this phenomenon. The use of synthetic Ca²⁺ indicators has played a crucial and influential role in the field of Ca²⁺ imaging. During the early 1980s, Charles F. Stevens (1983) introduced the initial synthetic Ca²⁺ indicator known as Fura-2 (Neher 1992). In the 1990s, Roger Y. Tsien performed significant research on Ca²⁺ Green-1, a highly adaptable Ca²⁺ indicator that facilitated the assessment of intracellular Ca²⁺ levels in both plants and animals (Tsien 1998). These indicators established the fundamental methods for Ca²⁺ imaging. A new set of fluorescent markers have been developed to study the physiological significance of cytosolic free Ca²⁺. These markers have an 8-coordinate tetracarboxylate chelating site and stilbene chromophores, which improve quantum efficiency and photochemical stability. They exhibit fluorescence up to 30 times more intense than the widely used "Quin-2" dye. They also show significant wavelength changes upon binding with Ca²⁺, with slightly reduced affinities and longer excitation wavelengths. These features make them ideal for intracellular applications (Grynkiewicz et al. 1985). The introduction of GECIs brought about a significant transformation in the field of Ca²⁺ imaging (Tsien 1998). GECIs are a class of proteins that demonstrate alterations in fluorescence upon binding with Ca²⁺. These indicators have the ability to be expressed in particular cell types or tissues, thereby facilitating more precise and enduring investigations of Ca²⁺ imaging in plants and animals (Kotlikoff 2007). The initial GECI, known as “Cameleon” was developed by the research team led by Roger Y. Tsien. The development of Cameleon allowed the specific visualisation of Ca²⁺ through imaging techniques (Miyawaki et al. 1997). In the realm of animal research, initial investigations into Ca²⁺ imaging primarily centred on single-cell recognition, employing methodologies such as microinjection of Ca²⁺ indicators or the introduction of indicator dyes into cells (Li and Saha 2021). These techniques enabled researchers to observe the temporal dynamics of Ca²⁺ in individual cells with a relatively high level of precision. The initial microinjection technique for the introduction of Ca²⁺ indicators into individual cells was pioneered by David W. Tank in 1985 (Tank et al. 1988).

GECIs, such as Cameleon, GCaMP, and R-GECO, were devised as a means to address certain constraints associated with chemical indicators. Nakai et al. (2001) optimised GECIs, specifically GCaMP, to enhance their ability to sense changes in Ca²⁺ levels. Loren L. Looger and Mark H. Ellisman (2003) further developed enhanced GECIs, including various variants of GCaMP. These variants demonstrated improved sensitivity to Ca²⁺ and exhibited notable changes in fluorescence levels (Terai and Nagano 2008). The research conducted by Simon Gilroy has significantly contributed to the field of plant biology by developing GECIs that are specific to plants, such as GCaMP and R-GECOs variants. These indicators have enabled accurate and detailed imaging of Ca²⁺ in plant cells and tissue (Monshausen et al. 2008; 2011). Ca²⁺ ions, pH levels, and reactive oxygen species (ROS) are crucial cellular regulators in plant physiological and developmental processes. Understanding their spatial and temporal dynamics across various scales is essential to comprehending their extensive influence. Fluorescent sensors, when used with appropriate controls, provide a reliable method for quantifying dynamic changes in these entities in real-time and within living organisms. Fluorescent cellular probes can be classified into two main classes: dyes and genetically encoded sensors based on GFPs. GFP probes can be selectively directed towards specific subcellular regions, allowing for detailed mapping of these signals within the cellular environment at a high level of precision (Swanson et al. 2011).

José A. Feijó primarily centred on the utilisation of Ca²⁺ imaging techniques to investigate the process of polarised growth. This work provided valuable insights into the intricate dynamics of Ca²⁺ during the growth of pollen tubes and the subsequent fertilisation process (Feijó and Moreno 2004; Feijó and Wudick 2018). Fluorescent genetic probes and ion-sensitive vibrating probes are used to find out where and when H⁺ and Ca²⁺ are present in tobacco pollen tubes. The results showed a discernible acidity gradient, with oscillations lasting 1–4 min. The (Ca²⁺)_cyt was visualised using confocal microscopy, revealing a V-shaped gradient spanning 40 μm from the tip. Oscillatory patterns of extracellular Ca²⁺ fluxes were observed in the majority of pollen tubes, ranging from 2 to 50 pmol cm⁻² min⁻¹. The study also found distinct spatial distributions and morphologies of H⁺ and Ca²⁺ inside the cellular environment, indicating a potential synergistic relationship between these two second messengers in facilitating cellular development. The findings suggest that the fluxes occurring at the apex of the pollen tube have a direct role in both the development and maintenance of the gradient (Michard et al. 2008). Another interesting study uses a genetically encoded FRET-based reporter to visualise conformational changes in Ca²⁺⁻dependent protein kinases (CDPKs and CPKs) in Arabidopsis thaliana. The researchers found that CPK21-FRET showed oscillatory changes in emission ratios, reflecting changes in (Ca²⁺)_cyt levels. CPK23-FRET did not show similar changes, indicating different Ca²⁺-sensitivity and a lack of reversibility. The conformational dynamics of CPK21 in Arabidopsis guard cells indicate it interprets signal-specific Ca²⁺ patterns in response to abscisic acid and the flagellin peptide flg22 (Liese et al. 2023). CapHensor, a dual-reporter, was used to study signaling processes in pollen tubes, guard cells, and mesophyll cells. The study revealed previously unreported linkages between membrane voltage, Ca²⁺ dynamics, and pH dynamics, revealing spatio-temporal interactions (Li et al. 2021).

Zhenbiao Yang and collaborators have successfully developed enhanced variants of Ca²⁺ indicators, such as YC, which have been specifically optimised for utilisation in plant cells. Ca²⁺ is a universal second messenger that transmits unique cellular signals through a spatiotemporal signature derived from both its extracellular source and internal reserves (Endo 2006). But we don't know much about how a Ca²⁺ signature is made because we don't have better ways to track the changing levels of Ca²⁺ in different subcellular compartments at the same time. To deal with this, molecular tools called CamelliA lines have been built in Arabidopsis to make it possible to watch the movement of Ca²⁺ in different subcellular compartments at the same time and with high resolution. Ca²⁺ signatures were identified in three types of Arabidopsis cells, relating to both internal and external stimuli. Some of these signs were fast changes in the amount of Ca²⁺ in the cytosol and the flow of Ca²⁺ into the plasma membrane at the tip of fast-growing pollen tubes. Also, when root epidermal cells were put under salt stress, the spatiotemporal correlation of Ca²⁺ dynamics was observed in four subcellular compartments (Guo et al. 2022).

The work of Frantisek Baluska and his collaborators involved the utilisation of Ca²⁺ imaging techniques to investigate the occurrence of Ca²⁺ waves and oscillations in plant root hairs. In Arabidopsis root hairs, the study showed how actin filaments affect mitochondrial Ca²⁺, how much Ca²⁺ is in the cytoplasm, and how these two phenomena are affecting each other. The researchers used fluorescent mitochondrial dyes, MitoTracker and Rhod-2, to visualise and quantify mitochondrial Ca²⁺ levels (Wang et al. 2010). Another study focused on the transition zone in the root apex, which plays a crucial role in determining cellular fate and facilitating root development. This zone integrates various inputs, including hormones and sensory stimuli, into signaling and motor outputs, leading to adaptive differential growth responses. Ca²⁺ green-1-dextran was microinjected into the root hair and  the amounts of Ca²⁺ in the cytoplasm were assigned pseudo-colours based on the initial intensity of green fluorescence. This understanding is essential for understanding root-apex tropisms and various aspects of adaptive root activity, as it is crucial for understanding root-apex tropisms and adaptive root activity (Baluška et al. 2010). The luminescent Ca²⁺ indicators, including aequorin, were developed by Marc Robert Knight and his research team. These indicators enabled the quantification of Ca²⁺ concentrations in plants through the use of bioluminescence methodologies. This method was a good alternative to fluorescent indicators because it made easier to spot changes in the amount of Ca²⁺ in different plant tissues (Gao et al. 2004; Liu et al. 2020). Julian Schroeder's study aimed to accurately measure the amount of Ca²⁺ in the cytosol by introducing Ca²⁺ Green-1 AM into guard cells in epidermal strips. The strategy involves introducing the dye and measuring the guard cells' response to abscisic acid. Their team used Arabidopsis thaliana to create Yellow Cameleon 2.1, a Ca²⁺ indicator based on a green fluorescent protein, to measure [Ca²⁺]_cyt in guard cells (Allen et al. 1999). Fluorescence ratio imaging allowed time-dependent measurements of [Ca²⁺]_cyt in guard cells, showing repetitive transients with peak values of up to 1.5 μM. Technical advancement by using combined cell biological and molecular genetic approaches will pave the path for understanding of mechanistic role of Ca²⁺ dynamics in plant signaling pathways (Israelsson et al. 2006).

Alex Costa, a renowned scholar in Ca²⁺ imaging in plant sciences, has made significant contributions to understanding the role of Ca²⁺ signaling in plant growth, development, and responses to environmental stimuli using advanced Ca²⁺ imaging methodologies. In vivo imaging of Arabidopsis cells shows that Ca²⁺ transients are linked to pH changes in the cytosol. This suggests that Ca²⁺ movement in plant cells and pH changes are linked in a fundamental way (Behera et al. 2018). Selective Plane Illumination Microscopy (SPIM) is a type of biological imaging that is often used to study transparent specimens for long periods of time in vivo. It is especially useful for studying small organs and organisms (Costa et al. 2013). A line of genetically encoded Ca²⁺ indicators was developed to simultaneously image Ca²⁺ dynamics in both the ER and cytosol. Live cell imaging revealed a wounding-induced Ca²⁺ wave in mature plants, enhancing high-resolution analyses of intracellular Ca²⁺ dynamics and paving the path for a new methodology (Resentini et al. 2021). They developed a protocol for imaging the R-GECO1 Ca²⁺ sensor in the roots of Arabidopsis, which can be used with other intensiometric Ca²⁺ sensors, as well as a method for analysing images (Himschoot et al. 2018). Loro and Costa have developed a new method to study mitochondrial Ca²⁺ in plant root cells using the Cameleon sensor 4mt-YC3.6. This method allows simultaneous monitoring of both nuclear and mitochondrial Ca²⁺ within a single cell using both nuclear and mitochondrial-targeted Cameleons by using high-resolution confocal laser scanning microscopy (Loro and Costa 2013).

Jörg Kudla, a plant biologist, has made significant contributions toward the understanding of Ca²⁺ signaling in plants. Kudla and team found that ratiometric fluorescence imaging works better with transgenic Arabidopsis plants that express the FRET-based Ca²⁺ probe Cameleon. This allows for better analysis of in vivo Ca²⁺ dynamics, focusing on (Ca²⁺)_cyt concentrations (Behera and Kudla 2013a). Excessive Na⁺ in soils inhibits plant growth by triggering primary Ca²⁺ signals within the root differentiation zone. A Ca²⁺-sensing mechanism measures stress intensity, allowing salt detoxification responses and optimizing Na⁺ extrusion capacity (Steinhorst et al. 2022). Behera and Kudla developed a technique to visualize (Ca²⁺)_cyt dynamics in roots using confocal laser scanning microscopy (CLSM) using the FRET technique, specifically using the genetically engineered Ca²⁺ indicator YC3.6. This method allows for precise imaging of seedlings aged 5–7 days using high-magnification objectives (Behera and Kudla 2013b). Behera and Kudla also developed a method to measure the movement of Ca²⁺ in the cytoplasm of Arabidopsis thaliana guard cells using the FRET method and the genetically modified Ca²⁺ indicator YC3.6 (Behera and Kudla 2013b; Himschoot et al. 2018).

The modulation of cellular Ca²⁺ concentrations is crucial for signal transduction in plant cellular processes. Carvalho-Niebel and team showed how Ca²⁺ oscillations in the nucleus and around the nucleus are important signs of symbiotic signaling responses in legumes. FRET yellow-based Ca²⁺ Cameleon probes have been effective in quantifying spatial and temporal patterns of symbiotic Ca²⁺ signaling in legumes. However, these sensors were limited in their capacity to measure changes in Ca²⁺, specifically inside individual subcellular compartments. This study examined the potential of GECOs, single fluorescent protein-based Ca²⁺ sensors, for simultaneous and multicolor imaging of cytoplasmic and nuclear Ca²⁺ signaling dynamics in root cells. Transgenic Medicago truncatula roots and Arabidopsis thaliana were used to investigate Ca²⁺ responses to microbial biotic and abiotic elicitors. The results showed that GECOs exhibit greater sensitivity in detecting differences in symbiosis-related Ca²⁺ spiking in M. truncatula compared to yellow FRET-based sensors. The dual sensor accurately analyses synchronised spatial and temporal patterns of nuclear and (Ca²⁺)_cyt signaling inside a single root cell while the organism is alive (Kelner et al. 2018).

 Wang et al. studied the pollen-tube pathway method of transformation to see how it is affected by the amount of plasmid DNA, the type of plant tissue/organ i.e., tomato used in the study, and the transformation solution. The YC3.6 vector was used as a transformation indicator. The study found a significant correlation between plasmid DNA density and transformation solution components and fruit set ratio. The optimum combination for the transformation solution was 600 ng μL⁻¹, sucrose, and 0.05% Silwet-L-77. This technique efficiently and precisely screens transgenic plants using in vivo imaging and fluorescence microscopy, making the pollen-tube route a cost-effective approach for introducing foreign genes into tomato plants (Wang et al. 2017). [Ca²⁺]_cyt is crucial for cellular signal transduction in plants and controls plant development. Pollen tubes establish an oscillatory Ca²⁺ gradient, which is essential for polarised growth. Genetically encoded Ca²⁺ indicators like YC quantify its role. A method for imaging (Ca²⁺)_cyt dynamics in growing pollen tubes using laser-scanning confocal microscopy is described by Barberini and Muschietti (2015).

Advancements in the enhancement of Ca²⁺ imaging have been achieved not just within the discipline of plant science but also in other disciplines. Numerous scientists and researchers have made noteworthy contributions to the advancement of Ca²⁺ imaging resolution. Ca²⁺ imaging has significantly enhanced our understanding of plant and animal physiological and developmental processes. Hillman and Chédotal (2011) work enabled high-resolution images of Ca²⁺ movement in intact animal tissues and organs (Santi 2011). In the laboratory of Mark Schnitzer, the work focused on miniaturised microscopes and fibre-optic-based systems for Ca²⁺ dynamics visualisation in live animals (Jercog et al. 2016). This allowed researchers to investigate Ca²⁺ dynamics in various behavioural contexts (Helmchen 2002; Russell 2011; Helmchen et al. 2013). GECIs have facilitated targeted manipulation and observation of Ca²⁺ dynamics within neurons and brain circuits (Oh et al. 2019; Lohr et al. 2021; Zhang et al. 2023). Carlos Pantoja and Takashi Nakamura's non-invasive imaging techniques have expanded the scope of studying neural activity in live animal subjects (Albers et al. 2018; Streich et al. 2021). These advancements have contributed immensely to our understanding of Ca²⁺ signaling and its role in various physiological processes, including neuronal activity, synaptic transmission, and organ function.

The significant contributions made by researchers have strongly influenced the development of Ca²⁺ imaging in both plants and animals. The collective endeavours have established a strong basis for forthcoming breakthroughs, facilitating novel understandings of the intricate realm of cellular signaling and physiological mechanisms in both plants and animals.

Conclusion and future prospects

Ca²⁺ is a macronutrient which also acts as a second messenger. It participates in a number of physiological activities such as cell wall structure and integrity maintenance, nutrient uptake, stress response, blood clotting, nerve function etc., developmental (cell differentiation, seed germination, root development, meristem activity) and biochemical pathways (enzyme activity, protein regulation, cell signaling, apoptosis etc.). Plants and animals perceive stress stimuli via receptors which results in the generation of Ca²⁺ transient in cytosol. This change in Ca²⁺ is solely responsible for downstream signaling events. Interestingly, the modulation in Ca²⁺ concentration can be quantified by Ca²⁺ imaging. Time-to-time various indicators have been discovered and utilized for cellular Ca²⁺ quantification. With the utilization of various Ca²⁺ dyes, probes and indicators researchers have been able to successfully attempt in vivo live cell Ca²⁺ imaging in higher organisms. Ca²⁺ imaging unveils the mechanism of Ca²⁺ signaling at the cellular level and unravels the minor changes that could not have been identified accurately. It also encourages development of various tools and techniques which help researchers to perform live cell Ca²⁺ imaging. The association of Ca²⁺ imaging with the machine learning and artificial intelligence algorithms can reduce the researcher's burden of complex Ca²⁺ imaging dataset analysis and also improves the accuracy.

The development of miniaturized and portable super resolution Ca²⁺ imaging system can make the technology more accessible for field studies and for visualizing the Ca²⁺ dynamics with nanometre scale precision, revealing intricate sub-cellular structure and interactions. The Ca²⁺ indicators advancement is a crucial aspect of advancing the field of molecular biology, biochemistry and cell physiology. Some of the advancements that can be done are calibration free indicators, multiparametric indicators (indicators that can measure multiple parameters like Ca²⁺, pH etc.), quantum dots integration (to improve brightness, photostability and multiplexing capabilities), use of bio-orthogonal chemistry (activate or deactivate selectively in response to specific cellular events or signals), biocompatibility and many more. The modernization of the Ca²⁺ indicators can improvise the accuracy and quality of Ca²⁺ imaging, thus, helping us to picture the vital changes occurring at cellular level. Many more advancements can be done in Ca²⁺ imaging to deepen our knowledge regarding plant biology, stress responses and adaptations to environmental changing conditions. This is a huge support for the scientific community that is always keen to identify and make better the existing means to derive accurate representation of the responses in living systems. It has the potential to solve issues in biotechnology, environmental science and agriculture, ultimately promoting food security and plant based solutions that can adapt to a changing environment.

Authors contribution

GKP: Conceptualization, Supervision, Funding acquisition, Writing—review and editing. SG: Investigation, Methodology, Writing—original draft, Writing—review and editing. MD: Investigation, Methodology, Writing—review and editing. AK: Investigation, Methodology, Writing—review and editing. MB: Writing—review and editing.

Funding

Department of Biotechnology (DBT), Govt. of India; Science and Engineering Research Board (SERB), Govt. of India; Council for Scientific and Industrial Research (CSIR), Govt. of India; Delhi University (IoE/FRP grant), India.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Consent for publication

All authors give their consent to publish this article.