Reversible Saturable Optical Fluorescence Transitions Research Articles

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so scanning the sample in order to build a full, meaningful image, takes time. Parallelization of the illumination is the only way to make these microscopy techniques more suitable for real-time live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields. Parallelized illumination with both Gaussian and doughnut beams will be then used to acquire super-resolution images.

Numerical study of transient absorption saturation in single-layer graphene for optical nanoscopy applications

Transient absorption, or pump–probe microscopy is an absorption-based technique that can explore samples ultrafast dynamic properties and provide fluorescence-free contrast mechanisms. When applied to graphene and its derivatives, this technique exploits the graphene transient response caused by the ultrafast interband transition as the imaging contrast mechanism. The saturation of this transition is fundamental to allow for super-resolution optical far-field imaging, following the reversible saturable optical fluorescence transitions (RESOLFT) concept, although not involving fluorescence. With this aim, we propose a model to numerically compute the temporal evolution under saturation conditions of the single-layer graphene molecular states, which are involved in the transient absorption. Exploiting an algorithm based on the fourth order Runge–Kutta (RK4) method, and the density matrix approach, we numerically demonstrate that the transient absorption signal of single-layer graphene varies linearly as a function of excitation intensity until it reaches saturation. We experimentally verify this model using a custom pump–probe super-resolution microscope. The results define the intensities necessary to achieve super-resolution in a pump–probe nanoscope while studying graphene-based materials and open the possibility of predicting such a saturation process in other light-matter interactions that undergo the same transition.

Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 μm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1–2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.

An open-source microscopy framework for simultaneous control of image acquisition, reconstruction, and analysis

We present a computational framework to simultaneously perform image acquisition, reconstruction, and analysis in the context of open-source microscopy automation. The setup features multiple computer units intersecting software with hardware devices and achieves automation using python scripts. In practice, script files are executed in the acquisition computer and can perform any experiment by modifying the state of the hardware devices and accessing experimental data. The presented framework achieves concurrency by using multiple instances of ImSwitch and napari working simultaneously. ImSwitch is a flexible and modular open-source software package for microscope control, and napari is a multidimensional image viewer for scientific image analysis.The presented framework implements a system based on file watching, where multiple units monitor a filesystem that acts as the synchronization primitive. The proposed solution is valid for any microscope setup, supporting various biological applications. The only necessary element is a shared filesystem, common in any standard laboratory, even in resource-constrained settings. The file watcher functionality in Python can be easily integrated into other python-based software.We demonstrate the proposed solution by performing tiling experiments using the molecular nanoscale live imaging with sectioning ability (MoNaLISA) microscope, a high-throughput super-resolution microscope based on reversible saturable optical fluorescence transitions (RESOLFT).

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so we need to raster scan the sample in order to build a full, meaningful image, which takes time. Parallelization of the illumination is the only way to make these microscopies more suitable for live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields.

Multi-foci parallelised RESOLFT nanoscopy in an extended field-of-view.

Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastic-targeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers resolution of large fields of view in a few seconds. In MoNaLISA, engineered light patterns strategically confine the fluorescence to sub-diffracted volumes in a large area and provide optical sectioning, thus enabling volumetric imaging at high speeds. The optical setup presented in this paper extends the degree of parallelisation of the MoNaLISA microscope by more than four times, reaching a field-of-view of . We set up the periodicity and the optical scheme of the illumination patterns to be power-efficient and homogeneous. In a single recording, this new configuration enables super-resolution imaging of an extended population of the post-synaptic density protein Homer1c in living hippocampal neurons.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

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.

Molecular contribution function in RESOLFT nanoscopy.

The ultimate objective of a microscope of the highest resolution is to map the molecules of interest in the sample. Traditionally, linear imaging systems are characterized by their spatial frequency transfer function, which is given, in real space, by the point spread function (PSF). By extending the concept of the PSF towards the molecular contribution function (MCF), that quantifies the average contribution of a single fluorophore to the image, a straightforward concept for counting fluorophores is obtained. Using reversible saturable optical fluorescence transitions (RESOLFT), fluorophores are effectively activated only in a small, subdiffraction-sized volume before they are read out. During readout the signal exhibits an increased variance due to the stochastic nature of prior activation, which scales quadratically with the brightness of the active fluorophores while the mean of the signal scales only linearly with it. Using a two-state Markov model for the activation, showing comparable behavior to the switching kinetics of the switchable fluorescent protein rsEGFP2, we can approximate quantitatively the MCF of RESOLFT nanoscopy allowing to count the number of fluorophores within a subdiffraction-sized region of the sample. The method is validated on measurements of tubulin structures in Drosophila melagonaster larvae. Modeling and estimation of the MCF is a promising approach to quantitative microscopy.

GMars-T Enabling Multimodal Subdiffraction Structural and Functional Fluorescence Imaging in Live Cells.

Fluorescent probes with multimodal and multilevel imaging capabilities are highly valuable as imaging with such probes not only can obtain new layers of information but also enable cross-validation of results under different experimental conditions. In recent years, the development of genetically encoded reversibly photoswitchable fluorescent proteins (RSFPs) has greatly promoted the application of various kinds of live-cell nanoscopy approaches, including reversible saturable optical fluorescence transitions (RESOLFT) and stochastic optical fluctuation imaging (SOFI). However, these two classes of live-cell nanoscopy approaches require different optical characteristics of specific RSFPs. In this work, we developed GMars-T, a monomeric bright green RSFP which can satisfy both RESOLFT and photochromic SOFI (pcSOFI) imaging in live cells. We further generated biosensor based on bimolecular fluorescence complementation (BiFC) of GMars-T which offers high specificity and sensitivity in detecting and visualizing various protein-protein interactions (PPIs) in different subcellular compartments under physiological conditions (e.g., 37 °C) in live mammalian cells. Thus, the newly developed GMars-T can serve as both structural imaging probe with multimodal super-resolution imaging capability and functional imaging probe for reporting PPIs with high specificity and sensitivity based on its derived biosensor.

GMars-Q Enables Long-Term Live-Cell Parallelized Reversible Saturable Optical Fluorescence Transitions Nanoscopy.

The recent development of reversibly switchable fluorescent proteins (RSFPs) has promoted reversible saturable optical fluorescence transitions (RESOLFT) nanoscopy as a general scheme for live-cell super-resolution imaging. However, continuous, long-term live-cell RESOLFT nanoscopy is still hindered mainly because of the unsatisfactory properties of existing RSFPs. In this work, we report GMars-Q, a monomeric RSFP with low residual off-state fluorescence and strong fatigue resistance attributed to a biphasic photobleaching process. We further demonstrate that GMars-Q is particularly suitable for long-term parallelized RESOLFT nanoscopy as it supports an order of magnitude longer imaging durations than existing RSFPs. The excellent photophysical properties of GMars-Q also suggest that it would be of general interest for other RESOLFT nanoscopic methods.

CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

Two‐Color RESOLFT Nanoscopy with Green and Red Fluorescent Photochromic Proteins

Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy of living cells have relied on the use of reversibly switchable fluorescent proteins (RSFP) emitting in the green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red-emitting RSFP enabling a spatial resolution up to four times higher than the diffraction barrier. By co-expressing green and red RSFPs in living cells we demonstrate two-color RESOLFT imaging both for single ("donut") beam scanning and for parallelized versions of RESOLFT nanoscopy where an array of >23,000 "donut-like" minima are scanned simultaneously.

Nanoscopy with more than 100,000 'doughnuts'.

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm-sized fields of view in <1 s using 116,000 such doughnuts.

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.