Recent Advances in Application of Circularly Polarized Luminescence Probes toward Bioimaging Technology

Abstract

Circularly polarized luminescence (CPL) refers to the differential emission of left- and right-handed circularly polarized light in chiral materials, exhibiting significant potential for applications in bioimaging. The introduction of CPL can effectively eliminate the background autofluorescence interference, thereby enhancing the signal-to-noise ratio and resolution. Furthermore, CPL probes serve as robust detection tools capable of directly identifying the unique optical fingerprint information on the chiral targets, enabling real-time dynamic tracking of species within organisms, thus facilitating higher-dimensional bioimaging. Recently, a new series of CPL probes has been developed for cellular and in vivo imaging. This review summarizes the research progress of various CPL probes designed for efficient bioimaging, including CPL-active lanthanide metal complexes, small molecules, and nanomaterials. Finally, we discuss the current challenges and future prospects of these CPL probes in the field of bioimaging.

Over a century ago, Lord Kelvin introduced the concept of chirality, which derives from the Greek word “χειρ (kheir)”, meaning hand. In his famous Baltimore Lectures of 1904, he defined chirality as a property of geometrical figures or a group of points that cannot coincide with their idealized mirror image (Figure a). Chirality is one of the most fundamental geometric features widely found throughout living organisms and nature, observable at various scales from microscopic subatomic and molecular levels to the supramolecular level, and even living systems and galaxies (Figure b). The experimental identification of molecular chirality can be dated to the separation of sodium ammonium (±)-tartrate enantiomers by Louis Pasteur in 1848, noting that the enantiopure tartaric acid salts could rotate the transmitted linearly polarized light in opposite directions. , As illustrated in Figure c, chiral molecules can be categorized into several types, such as point chirality, axial chirality, helical chirality, planar chirality, and others. The molecular machinery of life is also fundamentally based on chiral building blocks. − During the evolution of life on primitive Earth, l-amino acids were predominantly selected in proteins and enzymes, while d-sugars were favored in DNA and RNA construction. − In addition, more complex biological structures, including organelles, tissues, and organs, demonstrate chiral characteristics. , These chiral components are essential for maintaining normal metabolic activities. −

1.

(a) Schematic diagram of the chirality. (b) Chiral architectures at various scales. Reproduced with permission from ref . Copyright 2015 American Chemical Society. (c) Examples of chiral elements in molecules. Reproduced with permission from ref . Copyright 2017 Springer Nature. (d) Method of obtaining CPL. Reproduced with permission from ref . Copyright 2020 Wiley-VCH. (e) Illustration of the sensor method using the CPL signal as detection output for object identification. Reproduced with permission from ref . Copyright 2018 Wiley-VCH.

As research on chiral molecules progresses, various distinctive chiral optical properties have been discovered, including optical rotation, circular dichroism (CD), and circularly polarized luminescence (CPL). , Circularly polarized (CP) light refers to the light whose end point of the optical vector rotates in a circular path perpendicular to the direction of propagation, displaying left- and right-handedness. , In natural settings, CP light primarily originates from certain stars and nebulae, as well as through reflection and scattering from optically active components and interfaces. − In biological contexts, the lanterns of firefly larvae emit CP light with opposite handedness, and the birefringent cuticles of certain crustaceans also reflect CP light. − Typically, CP light can be obtained artificially by passing linearly polarized light through a quarter-wave plate (Figure d). However, this method results in a 50% loss of luminous energy and limits the miniaturization of CPL devices. Consequently, there is growing interest in chiral CPL systems. − The difference in emission between left- and right-handed CP light in these systems can be quantitatively evaluated by the luminescence dissymmetry factor g _lum value, defined by eq

$$g_{\mathrm{lum}}=\frac{2(I_{\mathrm{L}}-I_{\mathrm{R}})}{I_{\mathrm{L}}+I_{\mathrm{R}}}=\frac{4|\mu |·|m|\cos \theta }{{|\mu |}^2+{|m|}^2}$$ 1

where I _L and I _R represent the luminescence intensities of the left- and right-handed CP light and μ, m, and θ represent the electric transition dipole moment, magnetic transition dipole moment, and the spatial angle between them, respectively.

CP light exhibits universal sensitivity and high specificity in intricate environments, as its circularly polarized state can be maintained during light transmission. Consequently, CPL probes possess great potential in terms of bioimaging. , Unlike conventional fluorescence imaging, the CPL probes are capable of collecting CPL signals (I _L ≠ I _R) from target binding sites, effectively eliminating undesirable nonpolarized autofluorescence and light scattering (I _L = I _R) from competing biological substances, resulting in higher image contrast and spatial resolution (Figure e). , The introduction of CPL into bioimaging can further provide optical fingerprint information about local chirality within target regions, achieving higher-dimensional fluorescence imaging. The CPL signals detected from the complexes formed upon the interactions of CPL probes with their corresponding targets allow for real-time monitoring of concentration fluctuations and association states of species within biological systems. Besides, given that recognition processes in biological systems typically involve chiral species such as amino acids, proteins and sugars, it is reasonable to expect that chiral CPL probes will demonstrate enantioselective fluorescent recognition capabilities. , Currently, significant strides have been made in the development of CPL probes for bioimaging, with a variety of biocompatible CPL probes meticulously developed, including CPL-active lanthanide metal complexes, CPL-active small molecules, and CPL-active nanomaterials. To the best of our knowledge, such advancements are often presented as a subsection within broader reviews addressing the biomedical applications of chiral materials. , In 2022, Kuang et al. published an insightful review summarizing the construction strategies of chiral nanomaterials and their applications in biological imaging, sensing, and therapy. Similarly, Rogach et al. provided a comprehensive overview of the fabrication techniques, chiroptical properties, and emerging biomedical applications of chiral carbon dots. Nevertheless, reviews specifically focusing on the research progress of CPL probes in bioimaging remain scarce in the literature. At present, the research on CPL probes is just in the early stages, but is advancing rapidly. Therefore, a focused review is needed to promote further progress in this promising field. Notably, while numerous studies utilize chiral inorganic probes for bioimaging, the intrinsic CPL properties of these probes have not been precisely investigated. − Moreover, these topics have been previously addressed in several pivotal reviews and are beyond the scope of this review. , This review systematically summarizes the research advances in CPL probes used for bioimaging over the past two decades, providing an in-depth analysis of their advantages and challenges. We hope that these conclusions and perspectives will facilitate the development of more efficient CPL probes for bioimaging in the future.

Probes Based on CPL-Active Lanthanide Metal Complexes

The investigation of CPL probes for bioimaging originated from chiral lanthanide metal complexes, which possess the advantages of long luminescence lifetimes, large shift between absorption and emission, and relatively narrow emission bands. − Due to the allowed magnetic dipole but forbidden electric dipole f–f transitions in chiral lanthanide complexes, CPL signals with notably large g _lum values can be observed in these chiral luminescence systems. Among the various chiral lanthanide complexes reported, europium (Eu) complexes stand out for their extensive research and the largest recorded g _lum value to date, reaching up to 1.38. , Consequently, CPL-active lanthanide probes represent attractive candidates for detecting and imaging cellular environments, as their emissions can be readily distinguished from background signals in such environments. , A summary of reported CPL-active lanthanide probes and their key performance metrics is provided in Table .

1. Summary of Probes Based on CPL-Active Lanthanide Metal Complexes.

CPL probes	year	λ max [nm]	g lum values	quantum yield	bioimaging applications	ref
[EuL1]3+	2006	616	–	9%	selective staining, target detection
[TbL2]3+	2008	545	+0.27	–	potential real-time tracking of protein association
[EuL3]3+		589/595	+0.19/–0.16	–
[Eu/TbL4]3+	2012	632/–	vary with pH	–	signaling pH variation
[Eu/TbL5]3+		624/545	–0.057	–	signaling bicarbonate concentration
[EuL6]	2017	589	–0.20	0%/0.7%	selectively binding to HSA
[EuL7]	2017	605	–	–	enantioselective staining
	2022	615	0.30/0.25	–	EDCC imaging in cells
[EuL8]	2020	616/620	0.11/–0.05	11%	EDCC imaging
Eu{(+)–facam}3		595/613	–0.76/+0.11	–
[EuL9]	2023	593/615	+0.24/–0.043	11%	biphotonic imaging

^aMeasured with the addition of HSA.

^bMeasured upon two-photon excitation.

As early as 2000, Parker et al. first reported the use of CPL to signal the reversible anion binding to chiral lanthanide probes in the simulated extracellular anionic background. Nevertheless, the corresponding cellular or in vivo imaging experiments have not been conducted. Subsequently, they discovered a chiral Eu complex [EuL¹]³⁺ capable of selectively staining cellular nucleoli in 2006 (Figure a). As depicted in Figure b, the luminescent intensity within the nucleoli is three times higher than that of the cytoplasm and twice that of the nuclear membrane. Colocalization experiments using the commercially available nucleolar stain SYTO RNA-Select were performed to validate that the bright spots observed within the nucleus were associated with the luminescence of the complex localized at the nucleolus (Figure c). Furthermore, intracellular speciation of the complex was examined via CPL spectroscopy, revealing the potential presence of multiple Eu species bound to proteins (Figure d). In 2008, Parker’s group documented the first example of CPL signal inversion originating from noncovalent interactions between chiral lanthanide complex probes and proteins and defined them as unique chiroptical probes for albumin binding. The CPL signals of the (SSS)-Δ Eu and terbium (Tb) enantiomers [TbL²]³⁺ and [EuL³]³⁺ were evidently inverted upon the addition of bovine serum albumin (BSA), whereas the (RRR)-Λ enantiomers did not display such a response (Figure e,f). This inversion behavior aligns with the helicity inversion of the Δ-isomer complexes in their reversible protein-bound form to maximize the level of binding. This discovery potentially enables real-time tracking of protein associations both in vitro and within living cells. These advancements inspired Parker and colleagues to conceive the potential applications of these complexes as CPL microscope probes. In 2011, Geraldes et al. explained further the origin of this dynamic helicity inversion using saturation transfer difference (STD) NMR techniques and molecular docking simulations. Analysis of ¹H STD NMR spectral intensity differences and docking results indicated the stronger protein interactions of (SSS)-Δ enantiomers and the interaction regions of them.

2.

(a) Chemical structure of the chiral Eu­(III) complex [EuL¹]³⁺. (b) Luminescence images of cells loaded with [EuL¹]³⁺. (c) Colocalization of [EuL¹]³⁺ and SYTO RNA-Select dye (upper row for live-cell loading images; lower row for fixed cell loading images) and (d) CPL spectrum of [EuL¹]³⁺ in aqueous solution (black), cells (red), and aqueous l-Asn solution (green). Reproduced with permission from ref . Copyright 2006 American Chemical Society. (e) CPL spectra for (SSS)-Δ-[TbL²]³⁺ (blue) and in the presence of added BSA (red) and (f) CPL spectra for (SSS)-Δ-[EuL³]³⁺ (black) and in the presence of added BSA. Reproduced with permission from ref . Copyright 2008 Royal Society of Chemistry.

In 2012, Parker et al. , developed a series of lanthanide CPL probes, demonstrating cellular localization behavior and CPL emission in response to pH ([Eu/TbL⁴]³⁺) and bicarbonate concentrations ([Eu/TbL⁵]³⁺). Specifically, the hydration state of the lanthanide ion [EuL⁴]³⁺ undergoes reversible switching during acidification, with the sulfonamide nitrogen atom bound in this process (Figure a). This pH-dependent change in sulfonamide ligation was reflected in CPL emission variations, as the local helicity at the metal center switched. The rigidified structure of the N-bound form at higher pH values results in a larger |g _lum| value (Figure b). This is the first documented instance of reversible CPL modulation for signaling pH variations. Additionally, the CPL spectrum of [Eu/TbL⁵]³⁺ revealed a dramatic and remarkable change upon the addition of sodium bicarbonate, particularly within the magnetic dipole allowed ΔJ = 1 manifold (Figure a,b). As presented in Figures d and c, [Eu/TbL⁴]³⁺ showed localization within the lysosomes, whereas [Eu⁵]³⁺ was within the mitochondrial region. The fluctuations in lysosomal pH values and equilibrium bicarbonate concentrations in mitochondrial and human serum can be evaluated in real time through monitoring the emission intensity ratio of structurally related Eu and Tb probes (Figures c and d).

3.

(a) Reversible binding of a substituted sulfonamide moiety to the lanthanide ion [EuL⁴]³⁺. (b) Variation of |g _lum| (λ = 632 nm) with pH for [EuL⁴]³⁺. (c) Variation of lysosomal pH with time was monitored through quantitatively analyzing the [Tb/EuL⁴]³⁺ emission intensity ratio in microscopic observations and (d) confocal microscopy images showing the observation of lysosomes in cells. Reproduced with permission from ref . Copyright 2012 Royal Society of Chemistry.

4.

(a) CPL spectra of [EuL⁵]³⁺ (red) in the presence of 30 mM sodium bicarbonate (blue). (b) CPL spectra of [TbL⁵]³⁺ (green) in the presence of 30 mM sodium bicarbonate (blue) and 2 mM sodium citrate (yellow). (c) Confocal microscopy images for [EuL⁵]³⁺ and (d) plot of the emission intensity ratio for [EuL⁵]³⁺/[TbL⁵]³⁺ as a function of pCO₂ in cells. Reproduced with permission from ref . Copyright 2012 Wiley-VCH.

In 2017, Parker’s group reported a CPL-active Eu probe [EuL⁶], which demonstrated selectively binding to Sudlow’s drug site I on human serum albumin (HSA) with relatively high affinity. [EuL⁶] also demonstrated solvatochromism and pH-dependent emission characteristics (Figure a–c). Specifically, no Eu emission was observed within the pH range of 3 to 8 in aqueous conditions, and the energy of the intramolecular charge-transfer transition was found to be highly sensitive to solvent polarity, leading to the switching-on of Eu emission as the solvent polarity decreased (Figure a). Moreover, the addition of HSA to a solution of [EuL⁶] in phosphate-buffered saline also activated Eu luminescence, accompanied by a strong induced CPL signal with a |g _lum| value of 0.20 (Figure b). And the emission intensity of the complex bound to the protein displayed pH-dependent behavior with a pK _a value of 7.22 ± 0.01, associated with reversible intramolecular sulfonamide ligation (Figure c). Based on these findings, they extended this study to live-cell imaging, demonstrating significantly enhanced probe emission in lysosomes when replacing BSA with HSA in the cell-growth medium (Figure d). Subsequently, their group investigated the impact of chirality on the cellular uptake of CPL probes and explored the enantioselective imaging and the distribution of the chiral Eu­(III) complex Δ/Λ-[EuL⁷] in living cells (Figure e). As depicted in the CPL spectrum of Δ/Λ-[EuL⁷] in Figure f, the CPL intensity of Λ-[EuL⁷] increased upon BSA addition, while the response of Δ-[EuL⁷] was more complicated, exhibiting a slight decrease in the ΔJ = 4 manifold, a significant decrease in the ΔJ = 1 and 2 manifolds, and minimal alteration in the ΔJ = 3 manifold. Cell uptake experiments revealed that Λ-[EuL⁷] was internalized approximately 1.6 times more than Δ-[EuL⁷] on average in treated cells. Cells incubated with enantiopure complexes were imaged by laser scanning confocal microscopy, exhibiting distinct localization profiles with the selective mitochondrial localization for the Λ enantiomer in the appearance of tubular networks, whereas lysosomal localization for the Δ enantiomer as circular dots (Figure g). This represents an exceptional example of enantioselective staining of cellular organelles in living cells. Understanding such behavior is crucial for the application of CPL probes in living cell imaging.

5.

(a) Molecular structure of [EuL⁶] and its total emission variation with Reichardt’s normalized solvent polarity parameter. (b) Variation of emission intensity upon the addition of [EuL⁶] to HSA, with the inset showing the induced CPL spectrum of the protein-bound complex. (c) pH-dependent emission behavior of [EuL⁶] in the HSA-bound complex and (d) confocal microscopy images of cells after incubation with [EuL⁶] and LysoTracker Green. Reproduced with permission from ref . Copyright 2017 Royal Society of Chemistry. (e) Chemical structure of [EuL⁷]. (f) CPL spectra of Δ/Λ-[EuL⁷] in the presence and absence of BSA and (g) confocal microscopy images of cells treated with [EuL⁷], demonstrating good correlations with mitochondrial stain for Λ-[EuL⁷] and lysosomal stain for Δ-[EuL⁷]. Reproduced with permission from ref . Copyright 2018 Royal Society of Chemistry.

In addition, the evolution of CPL probes has also contributed to the advancements in CPL instrumentation and techniques. In 2016, Parker et al. performed a proof-of-concept study that described the time-gated enantioselective differential chiral contrast (EDCC) imaging of CPL-active Eu­(III) complexes. Racemic complexes, fluorescein, and a mixture of them were applied as methanol solution onto nonoptically brightened white paper and subsequently dried in the air. The resulting test paper exhibited three different colors (red from the Eu complexes, green from fluorescein, and yellow from the mixture). The introduction of a band-pass filter to the camera system resulted in the partial suppression of the green emission from the fluorescein spot, leaving two visible red spots (Figure a). Furthermore, EDCC imaging was achieved using enantiopure Eu complexes via a modified time-resolved epifluorescence microscope setup, which allows both continuous-wave and time-resolved operations (Figure b). This study illustrated the potential to differentiate and detect objects labeled with lanthanide CPL probes through a time-gated camera. In 2020, Pal et al. designed a rapid CPL spectroscopy through combining solid state (SS) spectrometers with a novel dual-channel optical layout, enabling the acquisition of CPL spectra in merely 10 ms. Specifically, left- and right-handed CP light were converted to orthogonal linearly polarized light using a static achromatic quarter-wave plate and subsequently split into two spatially separated detection channels via a beam splitter in the SS-CPL spectrometer. Each detection channel can independently analyze the states of both left- and right-handed CP light through automated rotation of a linear polarizer. The concurrent operation of both channels facilitated real-time acquisition of whole-spectra CPL (Figure c). The validity of this equivalent CPL measurement was confirmed by measuring the CPL spectra of two reference Eu­(III) complexes (Figure d,e). This work facilitates EDCC imaging in living cells.

6.

(a) Schematic setup and proof-of-concept for time-resolved image separation (the three spots in the paper sample correspond to racemic chiral Eu complexes at the top, a mixture of racemic chiral Eu complexes and fluorescein in the center, and fluorescein only at the bottom) and (b) schematic setup and proof-of-concept for CPL microscopy consisting of a Zeiss Axiovert 200 M epifluorescence setup, coupled to a 365 nm UV LED (Nichia, 24 V, 1.2W) and EO-1312 M (Edmund Optics) camera. Reproduced with permission from ref . Copyright 2016 Royal Society of Chemistry. (c) Optical layouts of the rapid CPL spectrometers. (d) Chemical structures of two reference Eu­(III) complexes and (e) CPL spectra of two reference samples. Reproduced with permission from ref . Copyright 2020 Springer Nature.

Besides, the limited penetration depth and cellular phototoxicity of ultraviolet light in biological tissues impede the translation of measurement technologies to subcellular CPL microscopy. Therefore, it is essential to develop CPL probes incorporating two-photon excitable ligands capable of being effectively excited by near-infrared (NIR) light. , Moreover, the intrinsic confocal character of two-photon absorption (TPA) facilitates high 3D resolution with submicrometer precision, providing considerable advantages for biological applications. In 2022, Pal’s group further extended this rapid CPL measurement technology to CPL laser scanning confocal microscopy (CPL-LSCM). This methodology allows the simultaneous acquisition of EDCC imaging of endogenous and engineered CPL-active Eu­(III) complex probes Λ/Δ-[EuL⁷] in living cells. Figure a illustrates that CPL-LSCM can simultaneously capture images and differentiate between the left- and right-handed CPL arising from the CPL probes in cells. Besides, the CPL signals from enantiopure Λ/Δ-[EuL⁷] could be efficiently excited through a two-photon process (Figure b). The CPL microscopy images in Figure c show that Λ/Δ-[EuL⁷] can selectively target different cellular organelles with well-resolved brightness differences observed within the cells, which cannot be detected by conventional unpolarized microscopy. CPL-LSCM could serve as a radical new tool in bioimaging, enabling the investigation of fundamental chiral interactions within living cells through harnessing the unique characteristics of CPL.

7.

(a) Details of the CPL-LSCM setup for EDCC imaging. (b) One-photon (main figure) and (insert) two-photon CPL spectra of Λ/Δ-[EuL⁷] and (c) enantioselective localization of EuL⁷ in live cells (lysosome for Δ-EuL⁷ and mitochondria for Λ-[EuL⁷]). Reproduced with permission from ref . Copyright 2022 Springer Nature. (d) Molecular structure and CPL spectra of (R, R)/(S, S)-[EuL⁹]Cl and (e) biphotonic-microscopy imaging of living HEK293T (up) and THP-1 (bottom) cells stained with (S, S)-[EuL⁹]Cl (left: fluorescence images, middle: overlay of the fluorescence and the transmission images, and right: fluorescence images using the spectral mode). Reproduced with permission from ref . Copyright 2023 Royal Society of Chemistry.

In 2023, Piccinelli et al. introduced a pair of cationic enantiomeric complexes (R, R)/(S, S)-[EuL⁹]­Cl, exhibiting significant CPL activity in the orange-red region of the visible spectrum with |g _lum| values of 0.24 for the magnetic dipole allowed ⁵D₀ → ⁷F₁ transitions (Figure d). Notably, their metal-centered luminescence can be efficiently sensitized by TPA excitation of the ligand around 700 nm. (R, R)/(S, S)-[EuL⁹]Cl can be readily internalized in cell lines, resulting in perinuclear diffuse localization within the cytosol and displaying intense red emission upon NIR excitation. Under physiological conditions, (S, S)-[EuL⁹]Cl was dissociated into a cationic state. Biphotonic imaging experiments conducted on the enantiopure (S, S)-[EuL⁹]Cl complex revealed at least two components contributing to the overall luminescence signal, which includes a blue component at 470 nm probably for the autofluorescence of intracellular organelles located in the cytoplasm, and a more diffuse, less intense contribution corresponding to the spectral signature of (S, S)-[EuL⁹]Cl (Figure e). These complexes represent a novel class of TPA-based CPL probes for advancing chiroptical applications in the field of bioimaging.

Probes Based on CPL-Active Small Molecules

Despite significant progress made with lanthanide-based CPL probes, their broader application is limited by the complicated synthesis with high expense. Therefore, it is essential to develop CPL probes based on chiral small molecules. Due to the fact that magnetic transition dipole moments are typically much smaller than the electric dipole terms, and the molecular sizes are considerably smaller than the helical pitch of CP light, chiral small molecules generally demonstrate relatively weak CPL signals with |g _lum| values ranging from 10^–5 to 10^–2. , Nevertheless, the |g _lum| value alone is insufficient for assessing the overall performance of a CPL emitter, as it only reflects the relative imbalance of CP light in the emission. To account for the total amount of polarized photons, Zinna et al. proposed a new quantity termed CPL brightness (B _CPL), calculated by eq

$$B_{\mathrm{CPL}}={\epsilon }_{\lambda }\times \phi \times \frac{|g_{\mathrm{lum}}|}{2}$$ 2

where ε _λ and ϕ represent the molar extinction coefficient measured at the excitation wavelength (λ) and the emission quantum yield, respectively. Although chiral small molecules exhibit relatively low |g _lum| values, their high emission yields combined with other advantageous properties such as accessible structural modifications, small size, and notable biocompatibility are still attracting great attention in bioimaging. , The general strategy for constructing CPL-active small molecules involves the incorporation of a chiral moiety to a known fluorescence dye. Currently, prominent chiral perturbation moieties employed include axially chiral binaphthol (BINOL) and helical chiral helicene, among others. − The corresponding CPL probes discussed in this section are summarized in Table .

2. Summary of Probes Based on CPL-Active Small Molecules.

CPL probes	year	λ max [nm]	g lum values	quantum yield	bioimaging applications	ref
R/S-RBOL	2015	585	–	0.47%	selective imaging of Fe3+ in living cells
R/S-P1	2021	480/520	0.70 × 10–3	9.99%	sensing endogenous cysteine
R/S-5	2021	580/460	3.6 × 10–4/6.1 × 10–4	–	detecting endogenous HClO
(R,R,R)/(S,S,S) 1	2023	610	–2.5 × 10–3	25.5%	lysosomes imaging
(R,R,R)/(S,S,S) 3		600	–	62.3%	enantioselective staining of different cell organelles
R/S-BBTI	2024	640	+0.012/–0.013	4.2%	hypoxic region imaging with chiral contrast
(R/S)-BOD1	2024	528.5	6.0 × 10–4	62%	EDCC imaging of lipid droplets
(R/S)-BOD2		529	2.0 × 10–4	87%	EDCC imaging of lysosomes
(R/S)-BOD3		653	1.4 × 10–3	77%
R/S-3sa	2025	548	+1.1 × 10–4/–5.9 × 10–5	44%	potential CPL probes for CPL-LSCM
R/S-3ta		550	–	45%	lipid droplet staining with high specificity
R/S-7		589	–	95%

^aMeasured with the addition of Fe³⁺.

^bMeasured with the addition of cysteine.

^cMeasured with the addition of ClO^–.

In 2015, Yi et al. developed a pair of novel colorimetric and fluorescent off–on enantiomers R/S-RBOL based on rhodamine derivatives for highly selective imaging of Fe³⁺ in living cells (Figure a). The sensitive detection of Fe³⁺ in aqueous and biological samples was accomplished through probing the integrated changes in fluorescence, CD, and CPL. The remarkable dual response in color and fluorescence of R/S-RBOL upon the addition of Fe³⁺ indicated its potential as an effective chemosensor for bioimaging Fe³⁺ under physiological conditions (Figure b). The ESI-MS and DFT analyses further verified the formation of the RBOL-FeCl₂ ⁺ complex with the optimized geometries presented in Figure c. The free RBOL exists in the spiro-lactam form, while the spiro-lactam ring is opened to coordinate with Fe³⁺ via the lactam nitrogen and oxygen atoms, imine nitrogen and hydroxyl oxygen atoms in the form of a ferric six-coordinate structure. Due to the chiral character of R/S-RBOL, the probes exhibited CD and CPL signals. However, the changes in CD and CPL spectra were relatively insignificant, likely attributed to the long distance between the chiral center and the fluorophore groups in the open conformation. The molecular structure of the probes requires further design and modification to induce enhanced changes in the chiral output signals.

8.

(a) Structure and response mechanism of RBOL to Fe³⁺. (b) Confocal fluorescence image of HeLa cells (a: R-RBOL, d: R-RBOL and Fe³⁺, g: merge image of R-RBOL + Fe³⁺ (red) and nucleus stained by Hoechst (blue), b, e, h: corresponding bright field images, c, f, i: corresponding merge images) and (c) optimized molecular geometries of RBOL and RBOL-FeCl²⁺. Reproduced with permission from ref . Copyright 2016 Elsevier Inc.

To further broaden the application range, Zhang’s group first developed a kind of reaction-based small molecular CPL probes R/S-P1, which can localize in the endoplasmic reticulum of living cells for the detection and imaging of endogenous cysteine. They designed R/S-P1 by introducing a 2, 4-dinitrobenzenesulfonyl (DNBS) protecting group onto a chiral borondipyrromethene (IBN-BODIPY) backbone to quench fluorescence and CPL. Remarkably, robust fluorescence and CPL signals with B _CPL of 1.86 M^–1 cm^–1 could be triggered by thiols, which can easily cleave the DNBS group (Figure a). The fluorescence intensity exhibited an excellent linear correlation with cysteine concentration, while the CPL signal displayed a sensitive “off–on” response toward cysteine with |g _lum| increasing from an undetectable value to 0.70 × 10^–3 (Figure b). In this case, CPL probes significantly enhanced the resolution and target selectivity owing to eliminating false signals from achiral interferents. Although achieving quantitative detection based on CPL signals remains challenging for R/S-P1, this simple and practical approach is valuable to develop reaction-based small molecular CPL probes. Subsequently, they reported CPL probes for detecting endogenous hypochlorous acid (HClO) in living macrophages. The chiroptical probes R/S-5 were constructed by postmodifying the phenothiazine moiety as a reactive site on the CPL-active scaffold, which comprises electron-donating carbazole units and a strong electron-accepting 2, 3-difluoroterephthalonitrile moiety (Figure d). The helically chiral probes R/S-5 showed distinct optical and chiroptical responses toward ClO^– with good linear relationships and a CPL sign blue-shifting of 120 nm, alongside |g _lum| values increasing from 3.6 × 10^–4 (≈580 nm) to 6.1 × 10^–4 (≈460 nm) in vitro (Figure e,f). Fluorescence imaging of intracellular ClO^– in living cells showed pronounced red fluorescence without external stimulation. Upon the addition of lipopolysaccharides to stimulate the increased ClO^–, both red and blue fluorescence appeared in two channels, ultimately resulting in exclusive blue emission from the cells (Figure g). This approach can access ClO^–-triggered CPL imaging and associated sign variation from the probes.

9.

(a) Molecular structures and biothiol sensing mechanism of related CPL probes. (b) g _lum curves of R-P1 (black) and R-P1 + 10 equiv. cysteine (green) and (c) confocal fluorescent images of HeLa cells (left: without R-P1, middle: in the presence of R-P1, and right: pretreated with N-methylmaleimide and then incubated with R-P1). Reproduced with permission from ref . Copyright 2021 Elsevier Inc. (d) ClO^– sensing mechanism of R/S-5. (e) CPL spectra of R-5 and R-5 + 10 equiv. ClO^–. (f) Linear relationship of R-5 between the emission intensity ratio I₄₅₈/I₅₇₆ and the dosages of ClO^– and (g) confocal fluorescence images of cells for detection of endogenous ClO^– (a: cells incubated only with R-5, b: cells pretreated with lipopolysaccharides (3 μg mL^–1) and then incubated with R-5, and c: cells pretreated with lipopolysaccharides (7 μg mL^–1) and then incubated with R-5). Reproduced with permission from ref . Copyright 2022 Elsevier Inc.

Rhodamine dyes are widely considered as prominent fluorescent dyes for staining and imaging different cell organelles due to their favorable extinction coefficients, quantum yields, photostability, and biocompatibility. , Xiang et al. utilized a straightforward way to link two rhodamine chromophores via different chiral bridges, synthesizing two pairs of conjugated birhodamine dyes (R, R, R)/(S, S, S) 1 and 2, as shown in Figure a. These dyes possess one axial chiral center and two spirocyclic chiral centers in the middle and periphery of two rhodamine chromophores, respectively. The compounds showed interesting CD and CPL switching behaviors and lysosome-targetable properties (Figure b). Additionally, they also synthesized a pair of unconjugated birhodamine dyes (R, R)/(S, S) 3 with chiral cyclohexane moieties, which did not display such chiroptical switching properties but demonstrated unexpectedly different cellular staining properties. Specifically, (R, R) 3 can stain both lysosomes and mitochondria, but (S, S) 3 exclusively stained lysosomes (Figure c). This work presents a rare example of enantiopure dyes with differential cellular staining properties, which might be attributed to the different interactions between enantiomers and chiral biomolecules, such as DNA and proteins.

10.

(a) Chemical structures of chiral rhodamine dyes (R, R, R)/(S, S, S) 1–3. (b) CPL spectra and g _lum curves of (R, R, R)/(S, S, S) 1–3 in dilute MeCN solution before and after adding HCl and (c) bright field and fluorescence images of cells costained with (R, R)/(S, S) 3, chromatin, and lysotracker and their merged fluorescence images. Reproduced with permission from ref . Copyright 2023 Wiley-VCH.

In 2024, Tang’s group reported iodine-substituted R/S-binaphthyl benzothiadiazole R/S-BBTI with highly distorted spiral ring-locked heteroaromatics and heavy iodine atoms, and investigated their near-infrared circularly polarized phosphorescence (NIR-CPP) properties (Figure a). In dimethyl sulfoxide (DMSO) solution, R/S-BBTI were found to emit NIR-CPP with an efficiency of 4.2%, a lifetime of 119 μs, and a large |g _lum| value up to 0.013 (Figure b,c). The efficient NIR-CPP observed in both solution and aggregate states was attributed to the heavy-atom effect, charge-transfer characteristics, and rigid twist heterocyclic configuration of R/S-BBTI. Moreover, the phosphorescence of R/S-BBTI was oxygen-sensitive and photoactivatable in DMSO. The application in hypoxia imaging was successfully demonstrated in cellular and tumor models. In the stained cells, phosphorescence was negligible under atmospheric conditions but obviously increased in hypoxic conditions (2% O₂) (Figure e). Furthermore, intensive phosphorescence emission was observed in isolated hypoxia tumors up to 72 h, indicating that R/S-BBTI can effectively visualize the hypoxic region of tumors in vivo (Figure f). Chiral image contrast in emission was further demonstrated by translating CPL as linearly polarized light using a birefringent quarter-wave plate and a linear polarizer, which quite depends on the specific equipment setup.

11.

(a) Molecular design strategy of R/S-BBTI. (b) PL and (c) CPL spectra of R-BBTI in DMSO solution before and after UV irradiation. (d) Photoactivated NIR-CPP mechanism of R/S-BBTI in DMSO solution. (e) HeLa cells were treated with S-BBTI in hypoxia and visualized via confocal image after laser irradiation of 390 nm and (f) in vivo hypoxia imaging of HeLa cell xenograft mouse model and separated organs after intravenous injection at the indicated time scales. Reproduced with permission from ref Copyright 2024 Springer Nature.

The CPL-LSCM developed by Pal et al. represents a crucial advancement in optical microscopy, enabling the investigation of the chiral subcellular environments using CPL probes. In 2024, Moya et al. further advanced the field by developing the first small full-organic CPL probes (R/S)-BOD1–3, which enabled CPL-based bioimaging through CPL-LSCM. As depicted in Figure a, these CPL probes are based on a readily accessible BINOL-O-BODIPY scaffold (borondipyrromethene functionalized at boron with a 1,1′-bi-2-naphthol moiety). Although the corresponding enantiomers displayed weak mirror-image CPL signals with relatively small g _lum values (ranging from 2.0 × 10^–4 to 1.4 × 10^–3) due to the chiral perturbation induced by the BINOL moiety, their high fluorescence efficiencies (from 62 to 87%) and moderate B _CPL values were successfully utilized for EDCC imaging. Using (R/S)-BOD1 as the CPL probes, left- and right-handed CPL images of lipid droplets in living cells were recorded, and the replacement between enantiomers can lead to the switching of image contrast (Figure b). Similarly, (R/S)-BOD2 and 3 can also be employed to achieve CPL-based images of lysosomes in living cells. These highly biocompatible and adaptable probes are pivotal to advance emerging CPL-LSCM for studying the biological significance of chirality. Additionally, the synthetic versatility of BODIPY scaffolds has attracted considerable interest from synthetic chemists. In 2025, Hao et al. reported a chiral phase-transfer-catalyst-enabled enantioselective α-amidation/amination reaction to synthesize a series of boron-stereogenic amido/amino BODIPY fluorophores (Figure c,d). Among them, compounds R/S-3sa were selected as representatives to investigate chiral photophysical properties, yielding g _lum values of approximately −5.9 × 10^–5 and +1.1 × 10^–4, respectively (Figure e). Compounds R/S-3ta and R/S-7 were employed to further evaluate the potential as fluorescent imaging agents in living cells (Figure f). Recently, they have also synthesized a series of boron-stereogenic formyl BODIPYs via enantioselective esterification reactions catalyzed by N-heterocyclic carbene. These CPL-active BODIPY derivatives demonstrated excellent biocompatibility and high specificity for lipid droplet staining, representing an accessible scaffold for the development of efficient CPL probes for bioimaging through CPL-LSCM.

12.

(a) Small organic molecule CPL probes based on BINOL-O-BODIPY scaffold and (b) localization of (R)-BOD3 in living cells by CPL-LSCM. Reproduced with permission from ref . Copyright 2024 American Chemical Society. (c) Approach to achieve boron-stereogenic amido/amino BODIPYs. (d) Molecular structures of R/S-3sa, R/S-3ta, and R/S-7. (e) CPL spectra of R/S-3sa in toluene and (f) confocal fluorescence images of HeLa cells stained with S-3ta and S-7. Reproduced with permission from ref . Copyright 2025 Springer Nature.

Probes Based on CPL-Active Nanomaterials

With the rapid progress in chiral nanomaterials and the nanobiomedical field, chirality-dependent interactions between chiral nanomaterials and biological systems have garnered significant attention. , Chiral nanomaterials can be obtained by incorporating chiral ligands or inducers during the processes of chemical synthesis or supramolecular assembly. − Owing to their facile preparation methods, remarkable CPL properties, and broad universality, CPL-active chiral nanomaterials have been employed as probes for biological applications (Table ). −

3. Summary of Probes Based on CPL-Active Nanomaterials.

CPL probes	year	λ max [nm]	g lum values	quantum yield	bioimaging applications	ref
R/S-Cu14	2019	726/702	3.0 × 10–3 /1.0 × 10–2	8.2%/1.6	potential biolabeling agent
l/d-AuNC	2021	538	3.0 × 10–3	70%	chirality-dependent imaging and radiosensitization
C-dot/CNC	2019	460	0.20	–	eliminating background photoluminescence signals
l/d-Cys-CdSe/CdS	2023	598.7	3.9 × 10–4	53%	high-contrast imaging and photodynamic therapy
Au(I)-MPA & R/S, R/S-1 & B-GQDs	2024	435	5.1 × 10–3	≈2.5%	potential water-based cellular CPL probe
(NEt4)2[Eu2(LR/S)4]	2025	612	+1.21/–1.21	1.69%/1.79%	cellular imaging and sensitive detection of 1O2
(NEt4)2[Eu2(EP-LR/S)4]			+1.29/–1.29	21.33%/23.42%

^aMeasured in crystalline state.

^bMeasured in aggregated state.

Chiral nanoclusters represent an emerging class of CPL materials with unique chiral nanostructures. − To date, atomically precise metal nanoclusters have been extensively employed in biological applications due to several essential advantages, such as ultrasmall size, atomic structure controllability, and biocompatibility. In 2019, Zang’s group synthesized a pair of chiral alkynyl ligands R/S-DPM to prepare atomically precise enantiomeric copper­(I) alkynyl nanoclusters R/S-Cu₁₄ with inherent chirality for the first time. These chiral nanoclusters were obtained through the reaction between R/S-DPM and [Cu­(MeCN)₄]­PF₆, with an average size of approximately 2 nm (Figure a). The R/S-Cu₁₄ nanoclusters exhibited crystallization- and aggregation-induced emission effects, which triggered bright red luminescence and CPL emission with high |g _lum| values (Figure b–e). Specifically, the dilute solution of R/S-Cu₁₄ in dichloromethane (DCM) showed no luminescence and CPL signals at room temperature, whereas the CPL response occurred in the solid state with a |g _lum| value of 3.0 × 10^–3. In the DCM/n-hexane solvent system, the CPL intensity gradually increased with an increasing volume fraction of n-hexane due to changes in emission at different aggregation degrees (Figure e). Notably, the |g _lum| value reached up to 1.0 × 10^–2 and remained relatively stable as the proportion of n-hexane increased. Moreover, the emission of R/S-Cu₁₄ in the cell medium was stable over 24 h, demonstrating excellent biocompatibility suitable for bioimaging applications (Figure f). Figure g shows that most of the copper­(I) nanoclusters were distributed throughout the cytoplasm in the cells. This work opens new horizons for studying the potential applications of CPL-active copper nanoclusters in cell imaging.

13.

(a) Chemical structure of R-DPM and the synthesis of the desired Cu₁₄ nanoclusters. (b) Normalized excitation and emission spectra of R-Cu₁₄ (left) and CPL spectra of R/S-Cu₁₄ enantiomers in the solid state (right). (c) Fluorescence photographs of R-Cu₁₄ in DCM solutions with different fractions of n-hexane. (d) Emission spectra and (e) CPL spectra of R-Cu₁₄ in DCM/n-hexane mixtures with different volume fractions. (f) Confocal images of cells incubated R/S-Cu₁₄ for 24 h: red from R/S-Cu₁₄; blue from nucleus and (g) bio-TEM images of R/S-Cu₁₄ aggregates internalized by cells. Reproduced with permission from ref . Copyright 2019 Wiley-VCH.

In 2021, Zhao et al. designed and fabricated alkynyl-protected chiral gold nanoclusters l/d-Au₁₀(C₁₃H₁₇O₅)₁₀ (l/d-AuNC) with excellent biocompatibility and radiosensitization effect for bioimaging and therapy studies. The synthesis of l/d-AuNCs was achieved through a two-step reaction using Me₂SAuCl as the gold source (Figure a). The resulting l/d-AuNCs had a core–shell structure and exhibited excellent monodispersity and uniform size distribution in ethanol, with an average diameter of approximately 2 nm (Figure b). The enantiomeric l/d-AuNCs demonstrated remarkable CPL response with a g _lum value of 3.0 × 10^–3 and strong fluorescence in cell culture systems (Figure c). Owing to stereospecific interactions between chiral AuNCs and proteins, l/d-AuNCs displayed different size distributions in cell medium. As illustrated in Figure d, the diameters of l-AuNCs ranged from 2 to 16 nm, while d-AuNCs exhibited a narrower size distribution ranging from 1 to 4.5 nm. Furthermore, l-AuNCs showed significantly higher cellular uptake, which could be assigned to stereoselective interactions between chiral AuNCs and the cell membrane (Figure e). The better intracellular dispersibility and lower cytotoxicity of d-AuNCs make it a more favorable candidate than l-AuNCs as a radiosensitizer in vivo (Figure f,g).

14.

(a) Synthesis of l/d-AuNC. (b) TEM images of d-AuNC in ethanol. (c) CPL spectra of l/d-AuNC enantiomers. (d) TEM images and corresponding size distribution histograms of l-AuNC (top) and d-AuNC (bottom) in cell medium. (e) Concentration-dependent viability of cells after being treated with l/d-ligand or l/d-AuNC for 24 h. (f) Confocal images of cells treated with l/d-AuNC and (g) hematoxylin and eosin histological staining of excised organs and tumor slices. Reproduced with permission from ref . Copyright 2021 Elsevier Inc.

Exploring the CPL probes with high biocompatibility is crucial for promoting their application in biological systems. , Kumacheva et al. reported a hydrothermal method for synthesizing nitrogen-doped fluorescent carbon dots (C-dots) using cellulose nanocrystals (CNCs) as both a carbon source and chiral substrate to generate hybrid C-dot/CNC nanoparticles (Figure a). These noncytotoxic C-dot/CNC nanoparticles were biocompatible to apply as biotags for cell labeling (Figure b,c). Notably, the hybrid nanoparticles displayed enhanced left-handed CPL emission compared to right-handed emission, with a g _lum value reaching 0.20 (Figure d,e). This property can facilitate improved imaging quality of biological species by effectively eliminating background photoluminescence signals through the use of circularly polarized fluorescence microscopy (Figure f).

15.

(a) Schematic of C-dot synthesis on the CNC surface. (b) Fluorescence microscopy images of the live cells without exposure to the C-dot/CNC nanoparticles (left) and the live cells cultured in the 0.1 wt % suspension of C-dot/CNC nanoparticles (right). (c) Normalized proliferation index of cells cultured in suspensions containing various concentrations of C-dot/CNC nanoparticles. (d) Photoluminescence spectra of the C-dot/CNC suspensions synthesized for 6 h, recorded using left- and right-handed circular polarizers. (e) Variation in the g _lum value plotted as a function of the emission wavelength and (f) confocal fluorescence microscopy images of the nanoparticles suspension under left- and right-handed circular polarizers were acquired while maintaining identical microscope settings. Reproduced with permission from ref . Copyright 2020 Wiley-VCH.

Xu et al. synthesized chiral CdSe/CdS dot-in-rods (DRs) DR-1 to 3 with different sizes by the postligand-exchange method and investigated their performance in high-resolution imaging and cytotoxicity (Figure a). The coverage with chiral cysteine (Cys) was found to reduce their cytotoxicity and improve their biocompatibility, thereby making them suitable for bioimaging and therapies. Notably, the CPL signal of DRs was ten times higher than that of spherical CdSe/CdS quantum dots (QDs), achieving the highest g _lum value of approximately 3.9 × 10^–4 for d-Cys-DR-1 due to the distinctive anisotropic morphology (Figure b). Additionally, d-Cys-CdSe/CdS DRs exhibited better fluorescence imaging performance than l-Cys-CdSe/CdS DRs (Figure c). The ultrasmall size and high quantum yield of red-emissive DR-1s enabled enhanced permeability and retention effects within brain glioma cells, facilitating high-resolution fluorescence imaging (Figure d). The possible applications of chiral DRs in bioimaging-guided tumor photodynamic therapy were also explored using CP light at 473 nm as a laser source. Chiral DRs were capable of enantioselectively stimulating the generation of reactive oxygen species and hydroxyl radicals, providing synergistic therapy in glioma cells with over three times higher efficacy than conventional photodynamic therapy without chiral selection (Figure e,f). Furthermore, d-DRs exhibited better bioimaging ability and efficient tumor ablation in vivo (Figure g).

16.

(a) Scheme of chiral CdSe/CdS involved strategy for high-resolution imaging and biological theranostics. (b) CPL spectra and the corresponding g _lum factors of l/d-Cys-CdSe/CdS DR-1. (c) Comparison of average fluorescence intensity of d- and l-Cys-CdSe/CdS DRs. (d) Representative fluorescence images of different concentrations of l/d-Cys-CdSe/CdS DRs. (e) Mechanisms and energy levels involved in CdSe/CdS DRs redox processes. (f) Statistical analysis plot represents the fluorescence intensity of reactive oxygen species (ROS) generated under various conditions and (g) ex vivo major organs and tumors imaging at the in vivo imaging experimental end point. Reproduced with permission from ref . Copyright 2023 Elsevier Inc.

In 2024, Zhang et al. designed a novel water-based CPL system with high biocompatibility through the combination of zero-dimensional QDs and two-dimensional chiral nanosheets. As depicted in Figure a and b, the chiral 2D nanoassemblies were prepared by mixing Au­(I)-MPA (MPA: 3-mercaptopropionic acid) nanosheets with the chiral compound (1R/S, 2R/S)-(−)–1,2-diamino cyclohexane (R/S, R/S-1) to be combined with blue luminescent boron-doped graphene QDs (B-GQD). The effective combination was facilitated by their hydrogen-bonding interactions as well as the spectral matching for the specific absorption, providing the generation of CPL emission with a |g _lum| value of about 5.1 × 10^–3 from the blue B-GQDs (Figure c). Additionally, this water-based system exhibited good scalability, as the CPL property can be effectively regulated by various kinds of luminescent QDs. The cell viability remained consistently above 90% across various concentrations of CPL QDs, making them suitable for cellular imaging experiments. The apparent blue fluorescence observed in cells confirmed the good imaging effects of CPL QDs and validated the potential biological applications of this CPL system as a cellular probe (Figure d).

17.

(a) Schematic diagram of blue CPL system composed of chiral Au­(I)-MPA nanoassemblies and B-GQDs. (b) TEM images of Au­(I)-MPA chiral nanosheets and Au­(I)-MPA chiral nanosheets doped with B-GQDs. (c) CPL and their corresponding DC voltage readouts of Au­(I)-MPA nanosheets doped with B-GQDs and (d) fluorescence images of CPL B-GQDs in cells after incubating for 24 h. Reproduced with permission from ref . Copyright 2024 Wiley-VCH. (e) Optimized geometries of (NEt₄)₂[Eu₂(L^S)₄] and (b) ¹O₂ detection and imaging in living cells using a CPL probe. Reproduced with permission from ref . Copyright 2025 Elsevier Inc.

In photodynamic therapy (PDT), singlet oxygen (¹O₂) generated upon the activation of photosensitizers, serves as the primary active ingredient and plays a crucial role in the destruction of malignant cells. , However, excessive production of ¹O₂ may lead to unnecessary toxicity. Recently, Li’s group reported the first example of ¹O₂ detection and imaging in living cells using a novel CPL probe based on assembled helicates. The helicates were obtained by dissolving chiral β-diketones ligands L^R/S and Eu­(OTf)₃ salts in acetonitrile, with tetraethylammonium hydroxide (NEt₄OH) serving as the base at a 2:1 stoichiometric ratio. Figure e illustrates the optimized geometry of the helicate (NEt₄)₂[Eu₂(L^S)₄], which converged into a quadruple-stranded helical structure. Each Eu­(III) ion was eight-coordinated by eight oxygen atoms from four β-diketonate units, forming a dinuclear quadruple-stranded helicate with a square antiprismatic geometry. Initially, the helicates displayed weak luminescence due to the strong quenching effect of the anthryl groups on the luminescence of the β-diketonate-Eu­(III) moiety. Upon reaction with ¹O₂ via a [4 + 2] cycloaddition reaction, the endoperoxides (NEt₄)₂[Eu₂(EP-L^R/S)₄] were formed within the anthracene framework, leading to the activation of the long-lived CPL emission. Notably, the g _lum value associated with the ⁵D₀ → ⁷F₁ transition increased obviously from −1.21 to −1.29 upon oxidation. The CPL intensity showed excellent linearity with respect to the ¹O₂ concentration. During the PDT process, (NEt₄)₂[Eu₂(L^S)₄] could readily permeate the cell membranes and demonstrated a highly selective luminescent response to the ¹O₂ generated by 5-aminolevulinic acid loaded 4T1 cells, enabling efficient cellular ¹O₂ imaging (Figure f). This study provides a practical strategy for the sensitive detection of ¹O₂ in biological systems.

Conclusions and Prospects

In recent years, CPL probes have attracted growing attention as an emerging class of bioimaging tools. The transmission characteristics of CP light enable the imaging with higher spatial resolution by eliminating the nonpolarized autofluorescence and light scattering from competing substances. Moreover, CPL from the chiral probes following formation of complexes with its target molecules encodes unique optical fingerprint information, reflecting changes in the local environment, conformational dynamics, and molecular binding states. These remarkable advantages allow for the real-time dynamic tracking of biological species. Herein, we have provided a concise summary of the recent advances in CPL probes employed for bioimaging over the past two decades, including CPL-active lanthanide metal complexes, CPL-active small molecules, and CPL-active nanomaterials.

Given that strong CPL signals with large g _lum values originate from the allowed magnetic dipole but forbidden electric dipole transitions, chiral lanthanide complexes are among the first types to be utilized for bioimaging applications. Parker’s group conducted pioneering research in this field and demonstrated the chiral selectivity of CPL probes in cellular environments. To further improve the biocompatibility of probes, subsequent efforts have focused on developing CPL probes based on small molecules and nanomaterials. Although these probes generally exhibit lower g _lum values compared to those of lanthanide-based probes, their high quantum yields allow for the required proper B _CPL values to be achieved. Moreover, their facile preparations, versatile modification, and excellent biocompatibility make them increasingly attractive for bioimaging applications.

Simultaneously, the development of CPL probes has been accompanied by innovation in CPL instrumentation. The development of CPL-LSCM by Pal et al. represents a landmark achievement, enabling the investigation of chiral interactions at the subcellular level. Corresponding valid CPL probes based on both lanthanide metal complexes and small molecules have been successfully applied to CPL-LSCM for EDCC imaging. This represents one of the most compelling demonstrations of the utility of CPL imaging and microscopy to date. Additionally, there are also several novel probes with biorelated properties that have been designed to facilitate their practical applications in bioimaging and photodynamic therapy, such as the near-infrared CPL probes, two-photon excitable probes, and water-based CPL probes.

To date, there are only a limited number of studies reporting the use of CPL probes in bioimaging. Therefore, it remains a relatively emerging field to explore. Currently, practical application of CPL probes in bioimaging still faces numerous challenges, including achieving high B _CPL values (high extinction coefficients, quantum yields, and g _lum values), ensuring excellent biocompatibility, and enabling precise in vivo targeting. The rational design of efficient CPL probes can be further advanced by computational tools, such as density functional theory (DFT) calculations and molecular dynamics (MD) simulations. Furthermore, the development of biodegradable CPL probes is essential for future in vivo applications. On the other hand, the design of more stable CPL probes capable of adapting to complex biological environments for deep-tissue imaging is equally needed, which holds promise for future applications in precision medicine through real-time diagnostic monitoring.

Glossary

Vocabulary Section

Chirality

The geometric property where an object cannot be superimposed on its mirror image.

Circularly polarized luminescence

The differential emission of left- and right-handed CP light in chiral emitters.

Dissymmetry factor

A dimensionless parameter used to quantify the extent of dissymmetry in CPL signals, defined as 2­(I _L – I _R)/(I _L + I _R).

CPL brightness (B _CPL)

A comprehensive metric for CPL performance combining quantum yield and dissymmetry factor to account for the total amount of emitted polarized photons.

EDCC imaging

A highly sensitive imaging technique capable of discriminating the spatial distribution of CPL emissions from enantiomers, enabling enhanced optical dissymmetry.

CPL laser scanning confocal microscopy (CPL-LSCM)

An advanced imaging system with high spatial resolution that can rapidly and simultaneously acquire diffraction-limited CPL-differential images of CPL probes in cells, enabling the subcellular tracking of emissive chiral subcellular probes.

D.L., Z.J. and X.L. conducted the literature search. Y.C. conceived the original idea and supervised the work. The manuscript was written by D.L. and Y.C. with assistance from all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.