Stochastic Optical Fluctuation Imaging Research Articles

SOGO-SOFI, light-modulated super-resolution optical fluctuation imaging using only 20 raw frames for high-fidelity reconstruction

Taking advantage of the stochastic photoswitching of genetically encodable reversibly photoswitchable fluorescent proteins (RSFPs), super-resolution optical fluctuation imaging (SOFI) and its variant photochromic stochastic optical fluctuation imaging (pcSOFI) are valuable tools for wide field super-resolution (SR) imaging. Live-cell (pc)SOFI, which requires a small number of original frames to reconstruct an SR image, is prone to structural discontinuity artifacts and low spatial resolution. Herein, we developed a repeated synchronized on- and gradually off-switching SOFI (SOGO-SOFI) that maximized the photoswitching frequency of RSFPs by light modulation and required only 20 frames for high-quality reconstruction. Live-cell SOGO-SOFI imaging of the endoplasmic reticulum (ER) exhibited 10 times higher temporal resolution (100 fps) and fewer artifacts than pcSOFI. Moreover, a combination of SOGO-SOFI with Airyscan further increased the image contrast and the resolution of Airyscan by a factor of 1.5 from 140 nm to 91 nm. The capabilities of SOGO-SOFI were further demonstrated by dual-color imaging of nucleolar proteins in mammalian cells and deep imaging of ER structures in thick brain slices (20.6 µm).

Hysteresis and Stochastic Fluorescence by Aggregated Ensembles of Graphene Quantum Dots.

“Blinking” behavior of fluorophores, being harmful for the majority of super-resolved techniques, turns into a key property for stochastic optical fluctuation imaging and its modifications, allowing one to look at the fluorophores already used in conventional microscopy, such as graphene quantum dots, from a completely new perspective. Here we discuss fluorescence of aggregated ensembles of graphene quantum dots structured at submicron scale. We study temperature dependence and stochastic character of emission. We show that considered quantum dots ensembles demonstrate rather complicated temperature-dependent intermittent emission, that is, “blinking” with a tendency to shorten “blinking” times with the increase of temperature. We verify “blinking” mechanism demonstrating hysteresis of the optical response under pulsed excitation timed to expected rates of dots transition to “dark” nonemitting states. Experimental results are well fitted by a simple qualitative model of transitions to the “dark” states. The obtained results suggest that this type of standardized quantum dots and even their submicron-size agglomerations can be useful as controlled fluorophores for super-resolution microscopy and, particularly, for SOFI-like microscopy.

Wide-Field Super-Resolution Optical Fluctuation Imaging through Dynamic Near-Field Speckle Illumination.

Stochastic optical fluctuation imaging (SOFI) generates super-resolution fluorescence images by emphasizing the positions of fluorescent emitters via statistical analysis of their on-and-off blinking dynamics. In SOFI with speckle illumination (S-SOFI), the diffraction-limited grain size of the far-field speckles prevents independent blinking of closely located emitters, becoming a hurdle to realize the full super-resolution granted by SOFI processing. Here, we present a surface-sensitive super-resolution technique exploiting dynamic near-field speckle illumination to bring forth the full super-resolving power of SOFI without blinking fluorophores. With our near-field S-SOFI technique, up to 2.8- and 2.3-fold enhancements in lateral spatial resolution are demonstrated with computational and experimental fluorescent test targets labeled with conventional fluorophores, respectively. Fluorescent beads separated by 175 nm are also super-resolved by near-field speckles of 150 nm grain size, promising sub-100 nm resolution with speckle patterns of much smaller grain size.

Super-resolution optical fluctuation imaging—fundamental estimation theory perspective

We provide a quantitative analysis of super-resolution imaging techniques which exploit temporal fluctuations of luminosity of the sources in order to beat the Rayleigh limit. We define an operationally justified resolution gain figure of merit, that allows us to connect the estimation theory concepts with the ones typically used in the imaging community, and derive fundamental resolution limits that scale at most as the fourth-root of the mean luminosity of the sources. We fine-tune and benchmark the performance of state-of-the-art methods, focusing on the cumulant-based image processing techniques (known under the common acronym stochastic optical fluctuation imaging), taking into account the impact of limited photon number and sampling time.

Fluctuation‐based Fluorescence Labeling and Biosensing for Multi‐color Super‐resolution Imaging

Multi-color super-resolution microscopy has become a desirable method for exploring the dynamic interaction and compartmentalization of biomolecules in living cells. Among numerous super-resolution microscopy concepts, photochromic stochastic optical fluctuation imaging (pcSOFI) has been widely recognized and applied in the life sciences due to advantages such as use of off-the-shelf equipment and high temporal resolution. Furthermore, with reversibly switchable fluorescent proteins (RSFP), pcSOFI could be used to localize biomolecules in high resolution in living systems. Previously, we discovered a phenomenon called fluorescence fluctuation increase by contact (FLINC), in which the RSFP Dronpa can specifically enhance the ability of fluorescence fluctuations of non-RSFP TagRFP-T when they are spatially close to each other. We developed FLINC-based A kinase-activity reporter (AKAR) and achieved super-resolution PKA activity imaging by pcSOFI in living cells. However, FLINC-based sensors contain two FPs, presenting challenges in multi-color super-resolution imaging. To overcome this issue, we identified mutations that disrupt fluorescence of Dronpa but maintain FLINC. We then introduced a Dronpa-removed FLINC (DrFLINC) as an enhanced red label, in combination with pcSOFI, to localize biomolecules in super-resolution in different compartments of living cells. Applying this new red fluorescent label in dual-color pcSOFI, we revealed that type II PKA regulatory subunit-α (RIIα) mainly located on microtubule in MIN6 beta cells. Furthermore, we developed DrFLINC-AKAR and demonstrated the multiplexed super-resolution imaging to reveal the regulatory context of PKA activity microdomains in living cells.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

ABSTRACTFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

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.

Live‐cell super‐resolution imaging of PKA activity reveals microdomain organization and regulation

Compartmentalized biochemical activities are crucial to many signaling processes in the cell. However, it has been difficult to visualize dynamic protein activities in living cells at the super‐resolution required to see their sub‐diffraction‐limit spatial regulation. Recently, we introduced a novel class of fluorescent biosensors that can detect biochemical activities in living cells at a resolution up to three‐fold better than the diffraction limit. Harnessing a specific binding‐induced change in protein fluorescence dynamics called Fluorescent fLuctuation INcrease by Contact (FLINC), these biosensors translate kinase activities or protein‐protein interactions into changes in fluorescence fluctuations. These fluctuations can then be quantified through Stochastic Optical Fluctuation Imaging (SOFI), permitting us to follow the protein activity dynamics, over tens of minutes, at super‐resolution. Utilizing one such biosensor for Protein Kinase A (PKA), we resolved minute activity microdomains on the plasma membrane of living cells, and discovered the role of clustered anchoring proteins in organizing these active PKA microdomains. We further demonstrate a new version of FLINC – REmoved Dronpa FLINC (red‐FLINC) – and use it to contextualize the PKA activity microdomains with the location of an additional protein. With red‐FLINC, live cell super‐resolution activity imaging requires only a single channel of the visible spectrum, freeing the others for imaging other proteins, or perturbing their activities with optogenetics. Using this technology. we determined the spatial relationship between membrane PKA activity microdomains and important participants in the PKA pathway such as AKAP79 and L‐type calcium channel. Our studies suggest that biochemical activities of the cell are spatially organized into an activity architecture.Support or Funding InformationNIH DP1 CA174423, R35 CA197622 and R01 DK073368This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Observing the Assembly of Protein Complexes in Living Eukaryotic Cells in Super-Resolution Using refSOFI.

Few approaches are currently available that allow the detection of protein-protein interactions (PPIs) in super-resolution, and the observation of the assembly of protein complexes in living cells has been particularly challenging. We developed reconstituted fluorescence-based stochastic optical fluctuation imaging (refSOFI), which is based on bimolecular fluorescence complementation (BiFC) and SOFI, allowing us to detect protein complex assembly 30 min after the induction of complex formation. Here we describe how to use refSOFI to map the assembly of two proteins of interest into a complex within living cells at super-resolution.

Biosensor maps localized cell activity

A new type of fluorescent biosensor makes it possible to visualize enzymatic and cell-signaling activities occurring at highly specific locations in live cells. Such activities often occur at 100-nm-sized sites and observing them is currently difficult or impossible. For example, the diffraction limit of visible light prevents light microscopy from capturing dynamic events at sites smaller than 200 to 250 nm. Superresolution techniques such as SOFI (stochastic optical fluctuation imaging) break the diffraction limit of light microscopes. But they can image only static structures in cells, not dynamic bioactivities. Now, Jin Zhang of the University of California, San Diego, and coworkers have developed biosensors that light up cellular processes in a new way and are SOFI-detectable, down to a resolution of about 100 nm (Nat. Methods 2017, DOI: 10.1038/nmeth.4221). They discovered a new biosensing phenomenon called FLINC—Fluorescence fLuctuation INcrease by Contact—in which fluorescence fluctuations speed u...

Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.

Model-free uncertainty estimation in stochastical optical fluctuation imaging (SOFI) leads to a doubled temporal resolution.

Stochastic optical fluctuation imaging (SOFI) is a super-resolution fluorescence imaging technique that makes use of stochastic fluctuations in the emission of the fluorophores. During a SOFI measurement multiple fluorescence images are acquired from the sample, followed by the calculation of the spatiotemporal cumulants of the intensities observed at each position. Compared to other techniques, SOFI works well under conditions of low signal-to-noise, high background, or high emitter densities. However, it can be difficult to unambiguously determine the reliability of images produced by any superresolution imaging technique. In this work we present a strategy that enables the estimation of the variance or uncertainty associated with each pixel in the SOFI image. In addition to estimating the image quality or reliability, we show that this can be used to optimize the signal-to-noise ratio (SNR) of SOFI images by including multiple pixel combinations in the cumulant calculation. We present an algorithm to perform this optimization, which automatically takes all relevant instrumental, sample, and probe parameters into account. Depending on the optical magnification of the system, this strategy can be used to improve the SNR of a SOFI image by 40% to 90%. This gain in information is entirely free, in the sense that it does not require additional efforts or complications. Alternatively our approach can be applied to reduce the number of fluorescence images to meet a particular quality level by about 30% to 50%, strongly improving the temporal resolution of SOFI imaging.

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.

RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells.

It has become increasingly clear that protein-protein interactions (PPIs) are compartmentalized in nanoscale domains that define the biochemical architecture of the cell. Despite tremendous advances in super-resolution imaging, strategies to observe PPIs at sufficient resolution to discern their organization are just emerging. Here we describe a strategy in which PPIs induce reconstitution of fluorescent proteins (FPs) that are capable of exhibiting single-molecule fluctuations suitable for stochastic optical fluctuation imaging (SOFI). Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells. Using this strategy, termed reconstituted fluorescence-based SOFI (refSOFI), we investigated the interaction between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the pore-forming channel subunit ORAI1, a crucial process in store-operated Ca(2+) entry (SOCE). Stimulating SOCE does not appear to change the size of existing STIM1/ORAI1 interaction puncta at the ER-plasma membrane junctions, but results in an apparent increase in the number of interaction puncta.

Fourier interpolation stochastic optical fluctuation imaging

Stochastic Optical Fluctuation Imaging (SOFI) is a super-resolution fluorescence microscopy technique which allows to enhance the spatial resolution of an image by evaluating the temporal fluctuations of blinking fluorescent emitters. SOFI is not based on the identification and localization of single molecules such as in the widely used Photoactivation Localization Microsopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM), but computes a superresolved image via temporal cumulants from a recorded movie. A technical challenge hereby is that, when directly applying the SOFI algorithm to a movie of raw images, the pixel size of the final SOFI image is the same as that of the original images, which becomes problematic when the final SOFI resolution is much smaller than this value. In the past, sophisticated cross-correlation schemes have been used for tackling this problem. Here, we present an alternative, exact, straightforward, and simple solution using an interpolation scheme based on Fourier transforms. We exemplify the method on simulated and experimental data.

Diffraction-unlimited fluorescence microscopy of living biological samples using pcSOFI.

The complex microscopic nature of many live biological processes is often obscured by the diffraction limit of light, requiring diffraction-unlimited fluorescence microscopy to resolve them. Because of the vast range of different processes that can be studied, sub-diffraction imaging should work efficiently under many different conditions. Photochromic stochastic optical fluctuation imaging (pcSOFI) is a recent addition to the field of diffraction-unlimited fluorescence microscopy. This robust and versatile method employs a statistical analysis of random fluctuations in the emission of single labels, in this case reversibly switchable fluorescent proteins (RSFPs), to retrieve super-resolution information. Added to the resolution enhancement, pcSOFI also offers contrast enhancement and background reduction in a practical and convenient way. Here, we describe the necessary steps to obtain diffraction-unlimited images, including multicolor and three-dimensional imaging, and highlight the advantages of pcSOFI together with the circumstances under which pcSOFI can be favorably applied.

Refsofi for Imaging Protein-Protein Interactions in Living Cells in Super-Resolution

Virtually all cellular processes depend on networks of highly dynamic protein-protein interactions (PPIs). Hence, deciphering cellular functions at the molecular level requires investigating the spatiotemporal characteristics of PPIs precisely within the context of these signaling networks. However, there is accumulating evidence that protein complexes are often organized into microdomains or nanodomains whose size is below the diffraction limit and thus cannot be sufficiently characterized using standard imaging techniques. Despite the tremendous progress in establishing and refining super-resolution imaging techniques for fluorescently labeled proteins in live cells, observation of protein-protein interactions (PPIs) in super-resolution remains challenging. To address this problem, we developed a strategy in which PPIs induce reconstitution of different fluorescent proteins, whereupon these interactions are resolved in super-resolution using Stochastic Optical Fluctuation Imaging (SOFI) within 30 minutes after complex formation. We employed this reconstituted fluorescence based SOFI (refSOFI) to investigate the interaction between the endoplasmic reticulum Ca2+ sensor STIM1 and the pore forming channel subunit ORAI1, a process crucial for store-operated Ca2+ entry. We found that STIM1-ORAI1 interaction puncta exhibit a smaller size than that suggested by current fluorescence microscopy-based assessments. Moreover, we discovered that the density of these punctate structures is not necessarily dependent on their size. In fact, we observed that STIM1-ORAI1 activation predominantly leads to the formation of new, smaller puncta instead of larger complexes. However, we identified that other perturbing factors can significantly change the size of puncta. These insights critically depend on the obtained super-resolution information. In conclusion, refSOFI represents a versatile tool to examine PPIs in living cells in super-resolution.

High-resolution single-molecule fluorescence imaging of zeolite aggregates within real-life fluid catalytic cracking particles.

Fluid catalytic cracking (FCC) is a major process in oil refineries to produce gasoline and base chemicals from crude oil fractions. The spatial distribution and acidity of zeolite aggregates embedded within the 50–150 μm-sized FCC spheres heavily influence their catalytic performance. Single-molecule fluorescence-based imaging methods, namely nanometer accuracy by stochastic chemical reactions (NASCA) and super-resolution optical fluctuation imaging (SOFI) were used to study the catalytic activity of sub-micrometer zeolite ZSM-5 domains within real-life FCC catalyst particles. The formation of fluorescent product molecules taking place at Brønsted acid sites was monitored with single turnover sensitivity and high spatiotemporal resolution, providing detailed insight in dispersion and catalytic activity of zeolite ZSM-5 aggregates. The results point towards substantial differences in turnover frequencies between the zeolite aggregates, revealing significant intraparticle heterogeneities in Brønsted reactivity.

Widely accessible method for superresolution fluorescence imaging of living systems

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.