Super-resolution Fluorescence Microscopy Techniques Research Articles

Clinical application of the HM-1000 image processing for HER2 fluorescence in situ hybridization signal quantification in breast cancer

BackgroundAccurate quantification of human epidermal growth factor receptor 2 (HER2) gene amplification is important for predicting treatment response and prognosis in patients with breast cancer. Fluorescence in situ hybridization (FISH) is the gold standard for the diagnosis of HER2 status, particularly in cases with equivocal status on immunohistochemistry (IHC) staining, but has some limitations of non-classical amplifications and such cases are diagnosed basing on additional IHC and FISH. This study investigated the clinical utility of a novel super-resolution fluorescence microscopy technique for the better FISH signal visualization and HER2 FISH classification.MethodsFourteen breast cancer tissue samples were retrospectively collected between September 2018 and February 2022, and FISH HER2 signal quantification was evaluated by determining the HER2/chromosome 17 centromere (CEP17) ratio and the number of HER2 signals per nucleus in super- versus conventional-resolution images.ResultsSuper-resolution images maintained the same overall HER2 diagnosis from routine, but HER2 FISH amplification changed negative to monosomy in two cases. Two Letrozole non-response relapses coincided to monosomy samples. The median number of HER2 signals per nucleus was 7.5 in super-resolution images and 4.0 in conventional-resolution images in HER2-positive samples and 2.8 and 2.1 signals per nucleus, respectively, in HER2-negative samples.ConclusionsSuper-resolution images improved signal visualization, including a significant difference in the number of countable HER2 and CEP17 signals in a single nucleus compared with conventional-resolution images. Increased accuracy of signal quantification by super-resolution microscopy may provide clinicians with more detailed information regarding HER2 FISH status that allows to better FISH classification such as HER2-low samples.

3D Super-Resolution Fluorescence Imaging of Microgels.

Super-resolution fluorescence microscopy techniques are powerful tools to investigate polymer systems. In this review, we address how these techniques have been applied to hydrogel nano- and microparticles, so-called nano- or microgels. We outline which research questions on microgels could be addressed and what new insights could be achieved. Studies of the morphology, shape, and deformation of microgels; their internal compartmentalization; the cross-linker distribution and polarity inside them; and their dynamics and diffusion are summarized. In particular, the abilities to super-resolve structures in three dimensions have boosted the research field and have also allowed researchers to obtain impressive 3D images of deformed microgels. Accessing information beyond 3D localization, such as spectral and lifetime properties and correlative imaging or the combination of data with other methods, shines new light onto polymer systems and helps us understand their complexity in detail. Such future trends and developments are also addressed. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 74 is April 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Super-Resolution Microscopy to Study Interorganelle Contact Sites

Interorganelle membrane contact sites (MCS) are areas of close vicinity between the membranes of two organelles that are maintained by protein tethers. Recently, a significant research effort has been made to study MCS, as they are implicated in a wide range of biological functions, such as organelle biogenesis and division, apoptosis, autophagy, and ion and phospholipid homeostasis. Their composition, characteristics, and dynamics can be studied by different techniques, but in recent years super-resolution fluorescence microscopy (SRFM) has emerged as a powerful tool for studying MCS. In this review, we first explore the main characteristics and biological functions of MCS and summarize the different approaches for studying them. Then, we center on SRFM techniques that have been used to study MCS. For each of the approaches, we summarize their working principle, discuss their advantages and limitations, and explore the main discoveries they have uncovered in the field of MCS.

Imaging live cells simplified with super-resolution fluorescence microscopy technique

Algorithm significantly improves structured illumination microscopy by reducing raw frames and speeding up processing times.

A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every super-resolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging super-resolution methods. Photoactivatable dyes offer substantial improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group-free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogues, yielding a family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell labelling for conventional microscopy, as well as the coordinate-stochastic and deterministic nanoscopies STED, PALM and MINFLUX.

Application of super-resolution fluorescence microscopy in hematologic malignancies

Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells. The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type, treatment and prognosis of hematologic malignancies. Super-resolution fluorescence microscopy (SRM) is a special kind of optical microscopy technology, which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014. With the development of SRM, many related technologies have been applied to the diagnosis and treatment of clinical diseases. It was reported that a major type of SRM technique, single molecule localization microscopy (SMLM), is more sensitive than flow cytometry (FC) in detecting cell membrane antigens’ expression, thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools. Furthermore, SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases. In this paper, we mainly discuss the application of SRM in clone hematological malignancies.

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy.

The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.

Fast image scanning microscopy with efficient image reconstruction

Image Scanning Microscopy is an advanced linear super-resolution fluorescence microscopy technique. Unlike traditional methods that introduce complex optical system designs, ISM is capble of taking the advantage of computational method to achieve super-resolution. As ISM involves large-scale localizations to all scanned-positions in most experiments, robust and accurate algorithms are required to avoid artifacts. Meanwhile, the existing ISM image reconstruction method is mainly based on post-processing technique due to high computing complexity. Therefore, large-scale images processing in ISM image reconstruction becomes too computationally intensive, which impedes the development of fast ISM. In this article, a robust ISM scanning-position localization technique and an efficient accurate image reconstruction technique for large scale ISM reconstruction are proposed. In particular, the localization of the ISM scanning position is realized by efficient image correlation and accurate robust Gaussian fitting. For simplicity and accuracy, the image reconstruction step introduces an efficient sub-pixel reassignment technique, by which image up-sampling is completely avoided. The simulation results show high performance in the proposed localization technique. The experimental result shows that the proposed image reconstruction technique can reach 20 times faster than existing method, which makes it very suitable for large-scale image processing in ISM image reconstruction, especially in a real-time ISM system.

Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10–20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Precision of fiducial marker alignment for correlative super\u2010resolution fluorescence and transmission electron microscopy

Recent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

Nanoelectromechanical photothermal polarization microscopy with 3 Å localization precision

Single-molecule microscopy has become an indispensable tool for biochemical analysis. The capability of characterizing distinct properties of individual molecules without averaging has provided us with a different perspective for the existing scientific issues and phenomena. Recently, super-resolution fluorescence microscopy techniques have overcome the optical diffraction limit by the localization of molecule positions. However, the labeling process can potentially modify the intermolecular dynamics. Based on the highly sensitive nanomechanical photothermal microscopy reported previously, we propose optimizations on this label-free microscopy technique toward localization microscopy. A localization precision of 3 Å is achieved with gold nanoparticles, and the detection of polarization-dependent absorption is demonstrated, which opens the door for further improvement with polarization modulation imaging.

Live-SIMBA: an ImageJ plug-in for the universal and accelerated single molecule-guided Bayesian localization super resolution microscopy (SIMBA) method.

Live-cell super-resolution fluorescence microscopy techniques allow biologists to observe subcellular structures, interactions and dynamics at the nanoscale level. Among of them, single molecule-guided Bayesian localization super resolution microscopy (SIMBA) and its derivatives produce an appropriate 50 nm spatial resolution and a 0.1-2s temporal resolution in living cells with simple off-the-shelf total internal reflection fluorescence (TIRF) equipment. However, SIMBA and its derivatives are limited by the requirement for dual-channel dataset or single-channel dataset with special design, the time-consuming calculation for extended field of view and the lack of real-time visualization tool. Here, we propose a universal and accelerated SIMBA ImageJ plug-in, Live-SIMBA, for time-series analysis in living cells. Live-SIMBA circumvents the requirement of dual-channel dataset using intensity-based sampling algorithm and improves the computing speed using multi-core parallel computing technique. Live-SIMBA also better resolves the weak signals inside the specimens with adjustable background estimation and distance-threshold filter. With improved fidelity on reconstructed structures, greatly accelerated computation, and real-time visualization, Live-SIMBA demonstrates its extended capabilities in live-cell super-resolution imaging.

TIRF Microscopy Studies of Axial Protein Organization using Photoswitchable Fluorescent Proteins

The spatial organization of proteins at the plasma membrane remains an intense focus of investigation and has prompted the development of numerous super-resolution Total Internal Reflection Fluorescence (TIRF) microscopy techniques. For example, some of the new technologies allow very high resolution in both axial and lateral directions, albeit often at the expense of performing live cell imaging. Live cell TIRF has been improved by multi-angle TIRF imaging providing more information in the axial direction and TIRF-Structured Illumination Microscopy methods improving lateral resolutions but these approaches are often incompatible. Here, we propose to utilize the photoswitching kinetics of photoswitchable fluorescent proteins located within the TIRF evanescent wave to determine their relative axial positions. Since the intensity of the evanescent wave formed at the interface between the glass coverslip and the sample decays exponentially in depth (the evanescent field is typically <200 nm in depth), we rely on the linear excitation intensity dependence of photoswitching to provide a readout of axial depth differences for proteins of interest. In testing its accuracy and utility, we performed photoswitching TIRF (psTIRF) imaging of several proteins located in focal adhesions, which we can compare with previously published interferometric Photoactivated Localization Microscopy (iPALM) studies of the same proteins.

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.

Total internal reflection fluorescence pattern-illuminated Fourier ptychographic microscopy

We report a widefield super-resolution (SR) fluorescence microscopy technique termed total internal reflection fluorescence pattern-illuminated Fourier ptychographic microscopy (TIRF-piFPM). It employs pattern-illuminated Fourier ptychography (piFP) under the total internal reflection fluorescence (TIRF) mode, providing a lateral resolution of ∼100 nm, reducing the background level, and correcting unknown optical aberrations. Like the total internal reflection fluorescence structure illuminated microscopy (TIRF-SIM), the illumination field of TIRF-piFPM is modulated by sinusoidal patterns generated by evanescent wave interference. It differs from TIRF-SIM in that TIRF-piFPM reconstructs the SR image by FP iteration. To demonstrate the performance of TIRF-piFPM, we compare it with TIRF-SIM by conducting simulations and experiments and prove that TIRF-piFPM can provide a better result than TIRF-SIM in terms of its robustness to noise and aberration correction ability. In addition, dynamic changes of mitochondria in U2OS cells are captured with a temporal resolution of 2 s, demonstrating its live-cell imaging capability. The addvantages may enable TIRF-piFPM to serve as an alternative to SR fluorescence microscopes in the TIRF family, with promising applications in biological and biomedical imaging.

Höchstmögliche räumlich-zeitliche Auflösung in der Lichtmikroskopie

Super resolution (SR) fluorescence microscopy techniques STED, SIM and PALM/STORM have revolutionized light microscopy and provide resolution of imaging way below the general limit of resolution as defined by the Abbe law. Single molecule tracking (SMT) allows the pursuit of movement of single proteins or lipids in real time, revealing in vivo dwell times and binding kinetics, a further milestone in live cell imaging. A combination of SR and SMT is a logical next evolution step in the goal to study dynamic processes in live cells at a maximal spatial and temporal resolution.

Spectrally Resolved Photodynamics of Individual Emitters in Large-Area Monolayers of Hexagonal Boron Nitride.

Hexagonal boron nitride (h-BN) is a 2D, wide band gap semiconductor that has recently been shown to display bright room-temperature emission in the visible region, sparking immense interest in the material for use in quantum applications. In this work, we study highly crystalline, single atomic layers of chemical vapor deposition grown h-BN and find predominantly one type of emissive state. Using a multidimensional super-resolution fluorescence microscopy technique we simultaneously measure spatial position, intensity, and spectral properties of the emitters, as they are exposed to continuous wave illumination over minutes. As well as low emitter heterogeneity, we observe inhomogeneous broadening of emitter line-widths and power law dependency in fluorescence intermittency; this is strikingly similar to previous work on quantum dots. These results show that high control over h-BN growth and treatment can produce a narrow distribution of emitter type and that surface interactions heavily influence the photodynamics. Furthermore, we highlight the utility of spectrally resolved wide-field microscopy in the study of optically active excitations in atomically thin two-dimensional materials.

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery—also named injectisome—assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.

Probing the Growth Kinetics for the Formation of Uniform 1D Block Copolymer Nanoparticles by Living Crystallization-Driven Self-Assembly.

Living crystallization-driven self-assembly (CDSA) is a seeded growth method for crystallizable block copolymers (BCPs) and related amphiphiles in solution and has recently emerged as a highly promising and versatile route to uniform core-shell nanoparticles (micelles) with control of dimensions and architecture. However, the factors that influence the rate of nanoparticle growth have not been systematically studied. Using transmission electron microscopy, small- and wide-angle X-ray scattering, and super-resolution fluorescence microscopy techniques, we have investigated the kinetics of the seeded growth of poly(ferrocenyldimethylsilane)- b-(polydimethylsiloxane) (PFS- b-PDMS), as a model living CDSA system for those employing, for example, crystallizable emissive and biocompatible polymers. By altering various self-assembly parameters including concentration, temperature, solvent, and BCP composition our results have established that the time taken to prepare fiber-like micelles via the living CDSA method can be reduced by decreasing temperature, by employing solvents that are poorer for the crystallizable PFS core-forming block, and by increasing the length of the PFS core-forming block. These results are of general importance for the future optimization of a wide variety of living CDSA systems. Our studies also demonstrate that the growth kinetics for living CDSA do not exhibit the first-order dependence of growth rate on unimer concentration anticipated by analogy with living covalent polymerizations of molecular monomers. This difference may be caused by the combined influence of chain conformational effects of the BCP on addition to the seed termini and chain length dispersity.

Mapping Transient Protein Interactions at the Nanoscale in Living Mammalian Cells

Protein-protein interactions (PPIs) form the basis of cellular processes, regulating cell behavior and fate. PPIs can be extremely transient in nature, which hinders their detection. In addition, traditional biochemical methods provided limited information on the spatial distribution and temporal dynamics of PPIs that is crucial for their regulation in the crowded cellular environment. Given the pivotal role of membrane micro- and nanodomains in the regulation of PPIs at the plasma membrane, the development of methods to visualize PPIs with a high spatial resolution is imperative. Here, we present a super-resolution fluorescence microscopy technique that can detect and map short-lived transient protein-protein interactions on a nanometer scale in the cellular environment. This imaging method is based on single-molecule fluorescence microscopy and exploits the effect of the difference in the mobility between cytosolic and membrane-bound proteins in the recorded fluorescence signals. After the development of the proof of concept using a model system based on membrane-bound modular protein domains and fluorescently labeled peptides, we applied this imaging approach to investigate the interactions of cytosolic proteins involved in the epidermal growth factor signaling pathway (namely, Grb2, c-Raf, and PLCγ1). The detected clusters of Grb2 and c-Raf were correlated with the distribution of the receptor at the plasma membrane. Additionally, the interactions of wild type PLCγ1 were compared with those detected with truncated mutants, which provided important information regarding the role played by specific domains in the interaction with the membrane. The results presented here demonstrate the potential of this technique to unravel the role of membrane heterogeneity in the spatiotemporal regulation of cell signaling.