Photoswitchable Fluorophores Research Articles

Photoswitchable AIE‐Active Supramolecular Self‐Assemblies for Biosensing and Bioimaging

Photoswitchable fluorescent self‐assemblies exhibiting aggregation‐induced emission (AIE) properties have garnered widespread attention. Due to their great adaptiveness and responsiveness towards different wavelengths required by photoreaction, photo‐isomerization, and photoluminescence excitation, these fluorescent self‐assemblies show diverse emission behavior in a spatial, temporal, and polychromatic manner. Integrating organic photoswitches and AIE fluorophores, these photoluminescent nano‐assemblies has been proven to be an effective approach, allowing the development of sophisticated systems for drug release and cell regulation that can be precisely manipulated through light or other stimuli. This review systematically encapsulates recent advances in photoswitchable fluorescent self‐assemblies under multi‐scenario biological applications. The construction of photoswitchable AIE fluorophores through covalent and non‐covalent interactions and their multimodal regulation are detailed, along with their biological applications in biosensing, bioimaging, and drug therapy.

A Visible-Light-Responsive Fluorescent Diarylethene Having a Betaine Structure.

As a new molecular scaffold of photoswitchable fluorophore, we developed a photochromic diarylethene containing a betaine structure based on pyridinium N-enolate. A facile reaction of a pyridyl-substituted dithienylperfluorocyclopentene derivative with octafluorocyclopentene constructed the betaine structure. The introduction of the betaine moiety provided the diarylethene molecule with bathochromically shifted optical absorption and fluorescing ability, thus enabling the molecule to function as a visible-light-sensitive turn-off mode photoswitchable fluorophore. The molecule in the open-ring form emitted bright blueish green fluorescence. Upon irradiation with 405 nm light, the molecule underwent cyclization isomerization to form the closed-ring isomer and the fluorescence intensity significantly decreased. The turn-off mode fluorescence photoswitching was observed not only in solution but also in polymer films.

Cover Feature: A Photoswitchable Solvatochromic Dye for Probing Membrane Ordering by RESOLFT Super‐resolution Microscopy (ChemPhysChem 12/2023)

The Cover Feature illustrates how photoswitchable solvatochromic fluorophores can be used to distinguish lipid membrane domains with different lipid ordering using super-resolution microscopy. More ordered membranes have lower water content than disordered membranes so by detecting solvatochromic fluorescence in multiple spectral regions, the lipid ordering in different parts of the membrane can be mapped. Cover design by Kathryn G. Leslie. More information can be found in the Research Article by Andrew T. Frawley, Christian Eggeling, Harry L. Anderson and co-workers.

Designs and Applications of Multi-stimuli Responsive FRET Processes in AIEgen-Functionalized and Bi-fluorophoric Supramolecular Materials

Materials capable of displaying strong ratiometric fluorescence with Förster resonance energy transfer (FRET) processes have attracted much research interest because of various chemosensor and biomedical applications. This review highlights several popular strategies in designing FRET-OFF/ON mechanisms of ratiometric fluorescence systems. In particular, the developments of organic and polymeric FRET materials featuring aggregation-induced emission-based luminogens (AIEgens), supramolecular assemblies, photochromic molecular switches and surfactant-induced AIE/FRET mechanisms are presented. AIEgens have been frequently employed as FRET donor and/or acceptor fluorophores to obtain enhanced ratiometric fluorescences in solution and solid states. Since AIE effects and FRET processes rely on controllable distances between fluorophores, many interesting fluorescent properties can be designed by regulating aggregation states in polymers and supramolecular systems. Photo-switchable fluorophores, such as spiropyran and diarylethene, provide drastic changes in fluorescence spectra upon photo-induced isomerizations, leading to photo-switching mechanisms to activate/deactivate FRET processes. Supramolecular assemblies offer versatile platforms to regulate responsive FRET processes effectively. In rotaxane structures, the donor-acceptor distance and FRET efficiency can be tuned by acid/base-controlled shuttling of the macrocycle component. The tunable supramolecular interactions are strongly influenced by external factors (such as pH values, temperatures, analytes, surfactants, UV-visible lights, etc.), which induce the assembly and disassembly of host-guest systems and thus their FRET-ON/FRET-OFF behavior. In addition, the changes in donor or acceptor fluorescence profiles upon detections of analytes can also sufficiently alter the FRET behavior and result in different ratiometric fluorescence outputs. The strategies and examples provided in this review offer the insights and toolkits for future FRET-based material developments.

Hemipiperazines as peptide-derived molecular photoswitches with low-nanomolar cytotoxicity

Molecular photoswitches transform light energy into reversible structural changes. Their combination with known pharmacophores often allows for photomodulation of the biological activity. The effort to apply such compounds in photopharmacology as light-activated pro-drugs is, however, hampered by serious activity reduction upon pharmacophore modifications, or limited biostability. Here we report that a potent antimitotic agent plinabulin and its derivatives demonstrate up to 56-fold reversible activity photomodulation. Alternatively, irreversible photoactivation with cyan light can enhance the cytotoxicity up to three orders of magnitude—all without compromising the original activity level, as the original pharmacophore structure is unchanged. This occurs due to the presence of a peptide-derived photoswitchable motif hemipiperazine inside the plinabulin scaffold. Furthermore, we systematically describe photochromism of these thermally stable and biocompatible hemipiperazines, as well as a photoswitchable fluorophore derived from plinabulin. The latter may further expand the applicability of hemipiperazine photochromism towards super-resolution microscopy.

Minimizing Molecular Misidentification in Imaging Low-Abundance Protein Interactions Using Spectroscopic Single-Molecule Localization Microscopy.

Super-resolution microscopy can capture spatiotemporal organizations of protein interactions with resolution down to 10 nm; however, the analyses of more than two proteins involving low-abundance protein are challenging because spectral crosstalk and heterogeneities of individual fluorescent labels result in molecular misidentification. Here we developed a deep learning-based imaging analysis method for spectroscopic single-molecule localization microscopy to minimize molecular misidentification in three-color super-resolution imaging. We characterized the 3-fold reduction of molecular misidentification in the new imaging method using pure samples of different photoswitchable fluorophores and visualized three distinct subcellular proteins in U2-OS cell lines. We further validated the protein counts and interactions of TOMM20, DRP1, and SUMO1 in a well-studied biological process, Staurosporine-induced apoptosis, by comparing the imaging results with Western-blot analyses of different subcellular portions.

Supramolecular Complex of Photochromic Diarylethene and Cucurbit[7]uril: Fluorescent Photoswitching System for Biolabeling and Imaging.

Photoswitchable fluorophores—proteins and synthetic dyes—whose emission is reversibly switched on and off upon illumination, are powerful probes for bioimaging, protein tracking, and super-resolution microscopy. Compared to proteins, synthetic dyes are smaller and brighter, but their photostability and the number of achievable switching cycles in aqueous solutions are lower. Inspired by the robust photoswitching system of natural proteins, we designed a supramolecular system based on a fluorescent diarylethene (DAE) and cucurbit[7]uril (CB7) (denoted as DAE@CB7). In this assembly, the photoswitchable DAE molecule is encapsulated by CB7 according to the host–guest principle, so that DAE is protected from the environment and its fluorescence brightness and fatigue resistance in pure water improved. The fluorescence quantum yield (Φfl) increased from 0.40 to 0.63 upon CB7 complexation. The photoswitching of the DAE@CB7 complex, upon alternating UV and visible light irradiations, can be repeated 2560 times in aqueous solution before half-bleaching occurs (comparable to fatigue resistance of the reversibly photoswitchable proteins), while free DAE can be switched on and off only 80 times. By incorporation of reactive groups [maleimide and N-hydroxysuccinimidyl (NHS) ester], we prepared bioconjugates of DAE@CB7 with antibodies and demonstrated both specific labeling of intracellular proteins in cells and the reversible on/off switching of the probes in cellular environments under irradiations with 355 nm/485 nm light. The bright emission and robust photoswitching of DAE-Male3@CB7 and DAE-NHS@CB7 complexes (without exclusion of air oxygen and addition of any stabilizing/antifading reagents) enabled confocal and super-resolution RESOLFT (reversible saturable optical fluorescence transitions) imaging with apparent 70–90 nm optical resolution.

3D Single Molecule Super-Resolution Microscopy of Whole Nuclear Lamina

Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of up to ∼1 µm per acquisition. This approach however, places high demands on fluorophore brightness and photoswitching resilience meaning that imaging entire cell volumes in 3D using SM super-resolution remains challenging. Here we employ SM astigmatism together with multiplane acquisition to visualize the whole nuclear lamina of COS-7 and T cells in 3D. Nuclear lamina provides structural support to the nuclear envelope and participates in vital nuclear functions including internuclear transport, chromatin organization and gene regulation. Its position at the periphery of the nucleus provides a visible reference of the nuclear boundary and can be used to quantify the spatial distribution of intranuclear components such as histone modifications and transcription factors. We found Alexa Fluor 647, a popular photoswitchable fluorophore, remained viable for over an hour of continuous high laser power exposure, and provided sufficient brightness detectable up to 8 µm deep into a cell, allowing us to capture the entire nuclear lamina in 3D. Our approach provides sufficient super-resolution detail of nuclear lamina morphology to enable quantification of overall nuclear dimensions and local membrane features.

Extra kinetic dimensions for label discrimination

Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.

Optically-manipulated multiaddressable all-ESIPT fluorescence nanomicelles prepared using a single fluorophore

High-performance optically-manipulated multiaddressable all-ESIPT fluorescence nanomicelles were successfully prepared from a single photoswitchable ESIPT fluorophore directly regulating fluorescence switching by photoisomerization.

Development of a New Approach for Low-Laser-Power Super-Resolution Fluorescence Imaging.

The development of super-resolution fluorescence microscopy over the past decade has drastically improved the resolution of light microscopy to ∼10 nm. Stochastic optical reconstruction microscopy (STORM) can be used to achieve subdiffraction-limit resolution by sequentially imaging and localizing individual fluorophores. In principle, the super-resolution of STORM can be obtained by high-accuracy localization of photoswitchable fluorophores, which require fast photoswitching and bright fluorescence intensity from a single emitter. It is known that the switching rate of photoswitchable fluorophores depends on the laser power─a high laser power being required for the enhancement of imaging resolution. However, high laser power is usually harmful to biological specimens and limits the imaging time because of its photobleaching effects and high phototoxicity. In this study, we attempted to overcome this problem by improving the STORM resolution at a lower laser power. Through the quantitative analysis of the photoswitching behavior of single fluorophores under different laser power conditions, we developed a new approach to achieve super-resolution fluorescence images at a laser power 10 times lower than had previously been reported. This approach is expected to play an increasingly significant role in super-resolution imaging of power-sensitive samples.

Tunable Wide-Field Illumination and Single-Molecule Photoswitching with a Single MEMS Mirror.

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. Therefore, different approaches for flat-field illumination have been introduced as alternative to the conventional Gaussian illumination. Here, we introduce a single microelectromechanical systems (MEMS) mirror as a tunable and cost-effective device for adapting wide-field illumination in SMLM. In flat-field mode the MEMS allowed for consistent SMLM metrics across the entire field of view. Employing single-molecule photoswitching, we developed a simple yet powerful routine to benchmark different illumination schemes on the basis of local emitter brightness and ON-state lifetime. Moreover, we propose that tuning the MEMS beyond optimal flat-field conditions enables to study the kinetics of photoswitchable fluorophores within a single acquisition.

An expectation-maximization approach to quantifying protein stoichiometry with single-molecule imaging.

Single-molecule localization microscopy (SMLM) is a super-resolution technique capable of rendering nanometer scale images of cellular structures. Recently, much effort has gone into developing algorithms for extracting quantitative features from SMLM datasets, such as the abundance and stoichiometry of macromolecular complexes. These algorithms often require knowledge of the complicated photophysical properties of photoswitchable fluorophores. Here, we develop a calibration-free approach to quantitative SMLM built upon the observation that most photoswitchable fluorophores emit a geometrically distributed number of blinks before photobleaching. From a statistical model of a mixture of monomers, dimers and trimers, the method employs an adapted expectation-maximization algorithm to learn the protomer fractions while simultaneously determining the single-fluorophore blinking distribution. To illustrate the utility of our approach, we benchmark it on both simulated datasets and experimental datasets assembled from SMLM images of fluorescently labeled DNA nanostructures. An implementation of our algorithm written in Python is available at: https://www.utm.utoronto.ca/milsteinlab/resources/Software/MMCode/. Supplementary data are available at Bioinformatics Advances online.

Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM).

Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary data are available at Bioinformatics online.

Fluorescence Switchable Conjugated Polymer Microdisk Arrays by Cosolvent Vapor Annealing.

Depositing minute light emitters into a regular array is a basic but essential technique in display technology. However, conventional lithographic methodologies involve multistep and energy-consuming processes. Here, we develop a facile method in which organic and polymeric fluorescent dyes spontaneously aggregate to form a patterned microarray. We find that a thin film of fluorescent π-conjugated polymer transforms into micrometer-sized aggregates when exposed to binary organic vapor at ambient temperature. The arrayed microaggregates can be formed over the whole substrate surface when using a quartz substrate that is prepatterned with regular hydrophilic boxes and hydrophobic grids. The resultant microarray is applicable to optical memories and displays when photoswitchable fluorophores are doped into the polymer matrix.

Out-of-Phase Imaging after Optical Modulation (OPIOM) for Multiplexed Fluorescence Imaging Under Adverse Optical Conditions.

Fluorescence imaging has become a powerful tool for observations in biology. Yet it has also encountered limitations to overcome optical interferences of ambient light, autofluorescence, and spectrally interfering fluorophores. In this account, we first examine the current approaches which address these limitations. Then we more specifically report on Out-of-Phase Imaging after Optical Modulation (OPIOM), which has proved attractive for highly selective multiplexed fluorescence imaging even under adverse optical conditions. After exposing the OPIOM principle, we detail the protocols for successful OPIOM implementation.

Counting Proteins and Nucleic Acids with Single-molecule Microscopy

Single-molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here, we present our progress in developing a molecular counting technique for SMLM data built upon the exponentially distributed blinking statistics of photoswitchable fluorophores. The approach was motivated by a desire to quantify the single-cell variability in plasmid copy number, but can be applied to a wide range of applications. We will present our work benchmarking the method on fluorescently labeled, surface mounted DNA origami grids and our progress in counting plasmids. The accuracy of our results illustrates SMLM's utility for optical -omics analysis.

Resolution enhancement of transmission electron microscopy by super-resolution radial fluctuations

Super-resolution fluorescence microscopy techniques have enabled dramatic development in modern biology due to their capability to discern features smaller than the diffraction limit of light. Recently, super-resolution radial fluctuations (SRRF), an analytical approach that is capable of generating super-resolution images easily without the need for specialized hardware or photoswitchable fluorophores, has been presented. While SRRF has only been demonstrated on fluorescence microscopes, in principle, this method can be used to generate super-resolution images on any imaging platforms with intrinsic radial symmetric point spread functions. In this work, we show that SRRF can be utilized to enhance the resolution and quality of transmission electron microscopy (TEM) images. By including an image registration algorithm to correct for sample drift, the SRRF-TEM approach substantially enhances the resolution of TEM images of three different samples acquired with a commercial TEM system. We quantify the resolution improvement in SRRF-TEM and evaluate how SRRF parameters affect the resolution and quality of SRRF-TEM results.

Single-frame wide-field nanoscopy based on ghost imaging via sparsity constraints

Single-molecule, localization-based, wide-field nanoscopy often suffers from low time resolution because the localization of a single molecule with high precision requires a low emitter density of fluorophores. In addition, to reconstruct a super-resolution image, hundreds or thousands of image frames are required, even when advanced algorithms, such as compressive sensing and deep learning, are applied. These factors limit the application of these nanoscopy techniques for living cell imaging. In this study, we developed a single-frame, wide-field nanoscopy system based on ghost imaging via sparsity constraints (GISC), in which a spatial random phase modulator is applied in a wide-field microscope to achieve random measurement of fluorescence signals. This method can effectively use the sparsity of fluorescence emitters to enhance the imaging resolution to 80 nm by reconstructing one raw image using compressive sensing. We achieved an ultrahigh emitter density of ${143}\;\unicode{x00B5} {{\rm m}^{ - 2}}$143µm−2 while maintaining the precision of single-molecule localization below 25 nm. We show that by employing a high-density of photo-switchable fluorophores, GISC nanoscopy can reduce the number of sampling frames by one order of magnitude compared to previous super-resolution imaging methods based on single-molecule localization. GISC nanoscopy may therefore improve the time resolution of super-resolution imaging for the study of living cells and microscopic dynamic processes.

Nanoscopic Stoichiometry and Single‐Molecule Counting

AbstractSingle‐molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here, a method for molecular counting is presented with SMLM built upon the exponentially distributed blinking statistics of photoswitchable fluorophores, with a focus on organic dyes. A guide to performing this newly developed technique is provided, highlighting many of the challenges and pitfalls, by benchmarking the method on fluorescently labeled, surface mounted DNA origami grids. The accuracy of the results illustrates SMLM's utility for optical “‐omics” analysis.