Lifetime super-resolution optical fluctuation imaging.

In this paper, we propose a promising super-resolution imaging scheme in fluorescence lifetime domain (lifetime super-resolution optical fluctuation imaging, ltSOFI). ltSOFI has the potential to obtain super-resolution images by taking advantage of fluorescence lifetime blinking under wide-field lifetime detection. The proof-of-concept for ltSOFI was demonstrated through numerical simulation of high-order cumulant analysis on fluorescence lifetime blinking emitters. As a tentative experimental demonstration, we obtained super-resolution lifetime imaging from time-lapse FLIM recording of HeLa cells expressing a cAMP sensor using ltSOFI method. ltSOFI is expected to initiate a new dimension in the lifetime domain for blinking-based super-resolution microscopy. LAY DESCRIPTION: We report on a promising super-resolution imaging scheme in fluorescence lifetime domain (lifetime super-resolution optical fluctuation imaging, ltSOFI). ltSOFI has the potential to obtain super-resolution images by taking advantage of fluorescence lifetime blinking under wide-field lifetime detection. Past advances in super-resolution fluorescence microscopy primarily rely on the spatiotemporal modulation of the fluorescence intensity. Although the applications of the Q-dot blinking have been discussed in the literature, most of the discussions have focused on the blinking of fluorescence intensity. Few studies have shown the possibility of super-resolution imaging through fluorescence lifetime fluctuations. In this paper, we proposed the ltSOFI scheme that explored the possibility of super-resolution reconstruction from the blinking of fluorescence lifetime. The proof-of-concept for ltSOFI was demonstrated through numerical simulation of high-order cumulant analysis on fluorescence lifetime blinking emitters. As a tentative experimental demonstration, we obtained super-resolution lifetime imaging from time-lapse FLIM recording of HeLa cells expressing a cAMP sensor using ltSOFI method. The ltSOFI method is expected to initiate a new dimension in the lifetime domain for blinking-based super-resolution microscopy. Moreover, the existing fluorescence lifetime imaging microscopy and super-resolution nanoscopy can benefit from the implementation of ltSOFI to significantly improve the imaging spatial resolution of fluorescence lifetime images. In addition, the proof-of-concept demonstration achieved by the numerical simulation and tentative experiment will provide a new perspective for obtaining fluorescence lifetime images with much finer details.

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.

Multicolor Photo-Crosslinkable AIEgens toward Compact Nanodots for Subcellular Imaging and STED Nanoscopy.

Aggregation induced emission (AIE) has attracted considerable interest for the development of fluorescence probes. However, controlling the bioconjugation and cellular labeling of AIE dots is a challenging problem. Here, this study reports a general approach for preparing small and bioconjugated AIE dots for specific labeling of cellular targets. The strategy is based on the synthesis of oxetane-substituted AIEgens to generate compact and ultrastable AIE dots via photo-crosslinking. A small amount of polymer enriched with oxetane groups is cocondensed with most of the AIEgens to functionalize the nanodot surface for subsequent streptavidin bioconjugation. Due to their small sizes, good stability, and surface functionalization, the cell-surface markers and subcellular structures are specifically labeled by the AIE dot bioconjugates. Remarkably, stimulated emission depletion imaging with AIE dots is achieved for the first time, and the spatial resolution is significantly enhanced to ≈95 nm. This study provides a general approach for small functional molecules for preparing small sized and ultrastable nanodots.

Expansion enhanced nanoscopy.

The advance of optical super-resolution fluorescence microscopy has revolutionized our vision of the subcellular world. Further improvement in the spatial resolution is of great significance for structural and functional investigations. The recently developed expansion microscopy (ExM), which achieves sub-diffraction imaging via physical expansion of the sample, provides a great opportunity for further resolution enhancement of existing optical super-resolution techniques. However, although such combination seems apparent, several technical obstacles, especially the dramatic loss of fluorescence signal during ExM sample preparation, have hampered this goal. In this work, aiming at this challenge, we have developed new strategies to retain and increase the fluorescence of the expanded sample. With the new labeling methods, we have successfully made the labeling density of expanded samples sufficing the Nyquist sampling criteria for optical super-resolution imaging, such as stimulated emission depletion microscopy (STED) and super-resolution optical fluctuation imaging (SOFI). The newly developed expansion nanoscopic imaging (ExN) approaches, i.e. ExSTED and ExSOFI, demonstrated up to 4-fold resolution enhancement compared to standard STED and SOFI, providing a simple and effective way to realize high resolution imaging both at the cellular and tissue level.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells.

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.

Multicolor Super-resolution Fluorescence Microscopy with Blue and Carmine Small Photoblinking Polymer Dots.

Advances in the development of small photoblinking semiconducting polymer dots (Pdots) have attracted great interest for use in super-resolution microscopy. However, multicolor super-resolution imaging using conventional small photoblinking Pdots remains a challenge due to their limited color choice, broad emission spectrum, and heavy spectrum crosstalk. Here, we introduce two types of small photoblinking Pdots with different colors and relatively narrow emission spectra: blue PFO Pdots and carmine PFTBT5 Pdots for blinking-based statistical nanoscopy. Both of these probes feature ultrahigh single-particle brightness, very strong photostability, superior biocompatibility, and robust fluorescence fluctuation. In addition, these small photoblinking Pdots serve as excellent labels for dual-color super-resolution optical fluctuation imaging (SOFI) of specific subcellular structures, indicating their promise for long-term multicolor SOFI nanoscopy with high spatiotemporal resolution.

Small Photoblinking Semiconductor Polymer Dots for Fluorescence Nanoscopy.

Two types of small photoblinking Pdots with high brightness, strong photostability, and favorable biocompatibility, are designed. Super-resolution optical fluctuation imaging is achieved using these Pdots. Imaging of subcellular structures demonstrates that these small photoblinking Pdots are outstanding probes for fast, long-term super-resolution fluorescence imaging.

Superior performance with sCMOS over EMCCD in super-resolution optical fluctuation imaging.

Super-resolution optical fluctuation imaging (SOFI) is a fast and low-cost live-cell optical nanoscopy for extracting subdiffraction information from the statistics of fluorescence intensity fluctuation. As SOFI is based on the fluctuation statistics, rather than the detection of single molecules, it poses unique requirements for imaging detectors, which still lack a systematic evaluation. Here, we analyze the influences of pixel sizes, frame rates, noise levels, and different gains in SOFI with simulations and experimental tests. Our analysis shows that the smaller pixel size and faster readout speed of scientific-grade complementary metal oxide semiconductor (sCMOS) enables SOFI to achieve high spatiotemporal resolution with a large field-of-view, which is especially beneficial for live-cell super-resolution imaging. Overall, as the performance of SOFI is relatively insensitive to the signal-to-noise ratio (SNR), the gain in pixel size and readout speed exceeds the loss in SNR, indicating sCMOS is superior to electron multiplying charge coupled device in context to SOFI in many cases. Super-resolution imaging of cellular microtubule structures with high-order SOFI is experimentally demonstrated at large field-of-view, taking advantage of the large pixel number and fast frame rate of sCMOS cameras.

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.

Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy.

Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

Two-photon light-sheet nanoscopy by fluorescence fluctuation correlation analysis.

Advances in light-sheet microscopy have enabled the fast three-dimensional (3D) imaging of live cells and bulk specimens with low photodamage and phototoxicity. Combining light-sheet illumination with super-resolution imaging is expected to resolve subcellular structures. Actually, such kind of super-resolution light-sheet microscopy was recently demonstrated using a single-molecule localization algorithm. However, the imaging depth and temporal resolution of this method are limited owing to the requirements of precise single molecule localization and reconstruction. In this work, we present two-photon super-resolution light-sheet imaging via stochastic optical fluctuation imaging (2PLS-SOFI), which acquires high spatiotemporal resolution and excellent optical sectioning ability. 2PLS-SOFI is based on non-linear excitation of fluctuation/blinking probes using our recently developed fast two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM), which enables both deep penetration and thin sheet of light. Overall, 2PLS-SOFI demonstrates up to 3-fold spatial resolution enhancement compared with conventional two-photon light-sheet (2PLS) microscopy and about 40-fold temporal resolution enhancement compared with individual molecule localization-selective plane illumination microscopy (IML-SPIM). Therefore, 2PLS-SOFI is promising for 3D long-term, deep-tissue imaging with high spatiotemporal resolution.

Mirror-enhanced super-resolution microscopy.

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.

Hundred-thousand light holes push nanoscopy to go parallel.

With the application of the elements of all major super-resolution techniques including stimulated emission depletion, structure illumination microscopy, and photo-activated localization microscopy, the incoherent crossed standing-wave microscopy achieves parallel super-resolution imaging.

Fast super-resolution imaging with ultra-high labeling density achieved by joint tagging super-resolution optical fluctuation imaging.

Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, thereby bring it closer to single-molecule state, we can faithfully reconstruct the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods.

Development of a reversibly switchable fluorescent protein for super-resolution optical fluctuation imaging (SOFI).

Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins. The RSFPs that are currently used for SOFI are low in averaged fluorescence intensity in the fluctuation state, photostability, and on/off contrast ratio, thereby limiting the range of application of SOFI in biological super-resolution imaging. In this study, we developed a novel monomeric green RSFP termed Skylan-S, which features very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state. Taking advantage of the excellent optical properties of Skylan-S, a 4-fold improvement in the fluctuation range of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. Furthermore, super-resolution imaging of the actin or tubulin structures and clathrin-coated pits (CCPs) in living U2OS cells labeled with Skylan-S was demonstrated using the SOFI technique. Overall, Skylan-S developed with outstanding photochemical properties is promising for long-time SOFI imaging with high spatial-temporal resolution.