Imaging Ca²⁺ with a fluorescent rhodol

Abstract

Ca²⁺ mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca²⁺-sensitive fluorophores has played an instrumental role in understanding of the temporal and spatial dynamics of Ca²⁺. Coupling Ca²⁺-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca²⁺ affinities suited to a variety of cellular contexts. One under-developed area is the use of hybrid rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca²⁺ sensing. Rhodols are bright, photostable, and have good two-photon absorption cross-sections (σ_TPA), making them excellent candidates for incorporation into Ca²⁺-sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca²⁺ sensor- 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca²⁺ binding constant of 240 nM and a 10-fold turn-response to Ca²⁺. RCS-1 effectively absorbs infrared light and has a σ_TPA of 76 GM at 840 nm, 3-fold greater than its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca²⁺ fluctuations in cell lines and cultured neurons using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca²⁺ sensing using two-photon illumination in neurons.

Graphical Abstract

The development of synthetic fluorescent indicators for Ca²⁺ ions is one of the success stories of organic chemistry and molecular recognition. The majority of fluorescent Ca²⁺ indicators utilize BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) as the key Ca²⁺-recognizing domain and couple Ca²⁺ binding to changes in fluorescence intensity or wavelength.¹ First reported in 1980 by Roger Tsien,² BAPTA solved dual problems of slow Ca²⁺-binding kinetics and pH dependence associated with the state of the art Ca²⁺-selective chelator of the day, EGTA (ethyleneglycol- N,N,N′,N′-tetraacetic acid). Importantly BAPTA maintains selectivity for Ca²⁺ over Mg²⁺, which is present in concentrations exceeding cytosolic Ca²⁺ by 4 to 5 orders of magnitude. Widely used Ca²⁺ indicators include fura-2,³ which provides a ratiometric read-out of Ca²⁺ concentration using UV excitation; the fluo- and rhod-family of indicators that report on fluctuations in Ca²⁺ concentration via changes in fluorescence intensity at around 520 and 580 nm, respectively;⁴ and OregonGreen-BAPTA (OGB)⁵ and CalciumGreen-BAPTA (CGB)⁶ derivatives that employ fluorinated and chlorinated fluorescein as an optical reporter. More recently, the use of modern xanthene scaffolds has enabled access to Ca²⁺ sensing in the orange (Cal-590,⁷ CaRuby-Nano⁸) to far-red (CaTM2⁹ or Cal-630) region of the electromagnetic spectrum by employing halogenated carbofluorescein¹⁰ or silicon-fluorescein, respectively.

Ca²⁺ indicators are widely used in neurobiology, where fluorescent indicators provide a convenient method to measure the relative activity of neurons. OGB, in particular, continues to be used for in vivo applications,¹¹ despite the recent emergence of excellent genetically-encoded Ca²⁺-sensitive fluorescent proteins,¹² on account of its well-tuned K_d (170 nM) and fast Ca²⁺-binding kinetics. ¹³ The use of fluorescent Ca²⁺ indicators, whether synthetically- derived or genetically-encoded, is frequently paired with two-photon microscopy, which allows greater tissue penetration on account of the use of high energy, pulsed lasers in the infrared regime. Despite the critical dependence on the use of two-photon illumination to image in thick tissues, most widely-used Ca²⁺ indicators show only modest ability to effectively absorb two-photon (2P) illumination.¹⁴ CGB has a two photon excitation cross section of approximately 30 GM (when saturated with Ca²⁺).¹⁵ Alternatively, indicators based on rhodamines, with larger 2P cross sections in the 50 to 100 GM range (for example, Calcium Orange or Calcium Crimson),¹⁵ often localize to cellular compartments rather than cytosol.¹⁶ To surmount the problems of low 2P cross section while maintaining good Ca²⁺ affinity, sensitivity, and cellular localization, we hypothesized that rhodol-based fluorophores¹⁷ would enable Ca²⁺ sensing under 2P illumination in live cells. Rhodols have intermediate to strong 2P cross sections, at around 120 GM,^{18, 19} and show good cellular localization. ^{20, 21} Despite these promising characteristics, rhodols have never been combined with BAPTA to create a Ca²⁺ sensor. We here report the design, synthesis, characterization and application of rhodol Ca²⁺-sensor 1, a fluorescent Ca²⁺ indicator with good 2P excitation cross section, sub-micromolar dissociation constants for Ca²⁺, and the ability to report on [Ca²⁺] fluxes in live cells and neurons under 2P illumination.

Rhodol Ca²⁺ sensor 1 is synthesized in a single step from the corresponding 6′-carboxy-dichloropyrrolidylrhodol (1) and the AM-ester of 5-aminoBAPTA (2) (Scheme 1). Synthesis of 1 proceeds in 4 steps from carboxy-dichlorofluorescein. Construction of isomerically pure 6′-carboxy-dichlorofluorescein is achieved via recrystallization from acetic anhydride/pyridine to give the 6′ isomer in gram quantities.²² Protection of the pendant carboxylate as the t-butyl ester²³ enables selective protection of a single phenolic oxygen as the methyoxymethyl (MOM) ether²⁴ and subsequent conversion of the remaining oxygen to the triflate. Conversion of the fluorescein to rhodol proceeds via Pd-catalyzed C-N bond formation with pyrrolidine followed by removal of the t-butyl ester to give compound 1 in 30% yield over two steps. Formation of amide bond between rhodol 1 and the tetra-acetoxymethyl ester of 5′-amino-BAPTA 2⁹ provides the acetoxy methyl ester of rhodol Ca²⁺ sensor 1 (RCS-1 AM) in 15% overall yield following purification. Saponification of this material provides the hexapotassium salt of RCS (RCS-1 K) for in vitro spectroscopic characterization.

Scheme 1.

Synthesis of rhodol Ca²⁺ sensor 1.

RCS-1 shows excitation and emission profiles characteristic of substituted rhodols, with a λ_max absorption at 515 nm and emission maximum centered at 551 nm (Figure 1a). Addition of Ca²⁺ results in an increase in fluorescence intensity (Figure 1a). The quantum yield for RCS-1 is Ca²⁺-dependent. In the absence of Ca²⁺, Φ₅₅₁ is 0.073; in the presence of saturating levels of Ca²⁺ (39 μM), Φ₅₅₁ is 0.73. There is no Ca²⁺-dependent change in absorption (Figure 1a). Titration with Ca²⁺ reveals a dissociation constant of 240 nM (±6 nM, S.E.M., n = 3 separate determinations), which is well-matched to the K_d reported for CGB-1 (190 nM)^{6, 13} (Figure 1b, Figure S1). In our hands, we determined a K_d of 180 nM for CGB-1 (±5 nM, S.E.M., n = 3 separate determinations), indicating a good match between RCS-1 and CGB-1. RCS-1 possesses a large dynamic range in response to Ca²⁺, exhibiting a 10- fold increase (±1, S.E.M., n = 3) in fluorescence intensity upon Ca²⁺ binding (Figure 1b), again, well-matched to the dynamic range for CGB-1 (which we measured as a 13-fold increase).⁶ We measured the two-photon excitation spectrum of RCS-1. Using rhodamine B as a standard, we determined the two-photon absorption cross-section (σ_TPA) of RCS-1.^{15, 19} RCS-1 displays a σ_TPA maximum at 840 nm, with a value of 72 GM. This is 3-fold larger than the 24 GM σ_TPA value we determined for CGB-1 (approximately 24 GM at 840 nm).¹⁵

Figure 1.

Spectroscopy characterization of rhodol Ca²⁺ sensor 1. a) Absorbance spectrum of rhodol Ca²⁺ sensor 1 (10 μM) in the absence (black dotted line) and presence (black solid line) of 39 μM Ca²⁺. Fluorescence emission spectra of rhodol Ca²⁺ sensor 1 (10 μM) in the absence (green dotted line) and presence (green solid line) of 39 μM Ca²⁺. Excitation provided at 515 nm. b) Ca²⁺ titration of rhodol Ca²⁺ sensor 1. The fluorescence emission spectra of rhodol Ca²⁺ sensor 1 was recorded at increasing concentrations of Ca²⁺ from 0 μM to 39 μM, with intermediate concentrations of 17 nM, 38 nM, 65 nM, 100 nM, 150 nM, 225 nM, 351 nM, 602 nM, and 1.35 μM. Excitation provided at 515 nm. c) Two-photon absorption spectra of rhodol Ca²⁺ sensor 1 with Ca²⁺ (39 μM). Error bars are ± standard deviation for n = 3 separate determinations.

The combination of large turn-on response to Ca²⁺ binding, nanomolar dissociation constant, and strong two-photon absorption cross-section suggests RCS-1 will be useful for monitoring Ca²⁺ transients in living cells. To investigate the ability of RCS-1 to report on Ca²⁺ fluxes in live cells, we utilized the tetra-AM ester version of RCS-1. Masking polar carboxylates with AM esters enable uptake anionic BAPTA-containing indicators into cells.²⁵ Cellular esterases remove the AM esters, liberating the Ca²⁺-responsive fluorophore. Bath application of RCS-1 to HeLa cells (1.7 μM, with Pluronic F-127, 0.01%) resulted in bright intracellular fluorescence, as determined by laser scanning confocal microscopy (Figure 2a). Stimulation of RCS-1-loaded cells with histamine (5 μM) resulted in the induction of periodic Ca²⁺ oscillations that were readily observable with RCS-1 fluorescence (Figure 2b). Gratifyingly, Ca²⁺ transients could be monitored using infrared, two-photon microscopy, with excitation provided at 840 nm (Figure 2c and d). Under both one- and two-photon illumination, RCS-1 displays photostability comparable to CGB-1.

Figure 2.

Live-cell imaging of histamine-evoked Ca²⁺ fluctuations with rhodol Ca²⁺ sensor 1. a) Confocal fluorescence microscopy images (one-photon) of HeLa cells incubated with rhodol Ca²⁺ sensor tetra-AM (1.7 μM). Scale bar is 20 μm. b) Quantification of intracellular [Ca²⁺] fluctuations measured in response to stimulation with histamine (5 μM). c) Two photon laser scanning fluorescence microscopy images of HeLa cells incubated with rhodol Ca²⁺ sensor tetra-AM (1.7 μM). Scale bar is 20 μm. d) Quantification of intracellular [Ca²⁺] fluctuations measured in response to stimulation with histamine (5 μM).

Building on these observations, we next determined whether RCS-1 could report on neuronal activity in cultured mammalian hippocampal neurons. Incubation of RCS-1 (1.7 μM, with Pluronic F-127, 0.01%) with cultured rat hippocampal neurons resulted in bright cellular fluorescence, as determined by widefield epifluorescence microscopy (Figure 3a). RCS-1 can report on spontaneous activity in hippocampal cultures (Figure 3b). Action potentials evoked by field stimulation give clear increases in fluorescence (Figure S2). Importantly, RCS-1 can also report on spontaneous activity in cultured neurons when imaged under 2P illumination, using 840 nm excitation light (Figure 3c and d).

Figure 3.

Visualization of Ca²⁺ transients in rat hippocampal neurons using rhodol Ca²⁺ sensor 1. a) Widefield fluorescence micrograph of cultured rat hippocampal neurons incubated with rhodol Ca²⁺ sensor 1 tetra AM ester (1.7 μM). Scale bar is 20 μm. b) Relative changes rhodol Ca²⁺ sensor 1 fluorescence vs. time for neurons in panel a. c) Two-photon laser scanning microscopy image of rat hippocampal neuron stained with rhodol Ca²⁺ sensor 1 tetra AM ester (1.7 μM). Scale bar is 20 μM. d) Intracellular Ca²⁺ transients recorded as relative changes in rhodol Ca²⁺ sensor 1 fluorescence vs. time for the neuron in panel c.

In summary, we present the design, synthesis, and application of RCS-1, a rhodol-based fluorescent indicator for Ca²⁺. The large turn-on response to Ca²⁺, high Ca²⁺ affinity, and good two-photon cross-section of RCS-1, coupled with the ability to monitor spontaneous activity in cultured neurons, make it a promising candidate for monitoring [Ca²⁺] transients in the context of neurobiology and beyond.