Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.     

Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging

In fluctuation‐based optical nanoscopy, investigating high‐density labeled subcellular structures with high fidelity has been a significant challenge. In this study, based on super‐resolution radial fluctuation (SRRF) microscopy, the joint tagging (JT) strategy is employed to enable fast high‐density nanoscopic imaging and tracking. In fixed cell experiment, multiple types of quantum dots with distinguishable fluorescence spectra are jointly tagged to subcellular microtubules. In each spectral channel, the decrease in labeling density guarantees the high‐fidelity super‐resolution reconstruction using SRRF microscopy. Subsequently, the combination of all spectral channels achieves high‐density super‐resolution imaging of subcellular microtubules with a resolution of ~62 nm using JT assisted SRRF technique. In the live‐cell experiment, 3‐channel JT is utilized to track the dynamic motions of high‐density toxin‐induced lipid clusters for 1 minute, achieving the simultaneous tracking of many individual toxin‐induced lipid clusters spatially distributed significantly below the optical diffraction limit in living cells.      

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy

Super‐resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction‐unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared with that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P‐labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.      

Twinkle,twinkle little star: Photoswitchable fluorophores for super-resolution imaging

Photoswitchable fluorescent probes are key elements of newly developed super-resolution fluorescence microscopy techniques that enable far-field interrogation of biological systems with a resolution of 50 nm or better. In contrast to most conventional fluorescence imaging techniques, the performance achievable by most super-resolution techniques is critically impacted by the photoswitching properties of the fluorophores. Here we review photoswitchable fluorophores for super-resolution imaging with discussion of the fundamental principles involved, a focus on practical implementation with available tools, and an outlook on future directions.     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Structured illumination imaging with quasi‐periodic patterns

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.     

Development a flexible light‐sheet fluorescence microscope for high‐speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure

We report a flexible light‐sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination‐detection modes: the first uses angle‐dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video‐rate volumetric imaging; the second combines digitally‐scanned light‐sheet illumination with an axially‐swept light‐sheet waist and stage‐scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination‐detection mode achieves dual spectral‐channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time‐lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination‐detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t‐tubule network in a fixed cardiomyocyte cell.     

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

Novel organic dyes for multicolor localization‐based super‐resolution microscopy

Precise multicolor single molecule localization‐based microscopy (SMLM) requires bright probes with compatible photo‐chemical and spectral properties to resolve distinct molecular species at the nanoscale. The accuracy of multicolor SMLM is further challenged by color channel crosstalk and chromatic alignment errors. These constrains limit the applicability of known reversibly switchable organic dyes for optimized multicolor SMLM. Here, we tested 28 commercially available dyes for their suitability to super‐resolve a known cellular nanostructure. We identified eight novel dyes in different spectral regimes that enable high quality dSTORM imaging. Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing‐based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3‐color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.

A major limitation of multicolor single molecule localization based super‐resolution microscopy (SMLM) is the availability of suitable photo‐switchable fluorescent dyes. By screening 28 commercially available dyes, novel dyes in different spectral regimes were identified that are well suited for dual and triple color SMLM with low crosstalk. These novel dyes are employed to image the relative nanoscale distribution of sub‐cellular components.     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

X10 expansion microscopy enables 25‐nm resolution on conventional microscopes

Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.     

Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca²⁺‐sensitive protein kinase C α fusion protein are demonstrated.     

Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances

Photoswitchable fluorescent proteins (PSFPs) with controllable spectral shifts in emission in response to light have led to breakthroughs in cell biology. Conventional photoswitching, however, is not applicable to weakly fluorescent proteins. As an alternative, photothermal (PT) and photoacoustic (PA) spectroscopy have demonstrated a tremendous potential for studying absorbing nonfluorescent proteins and nanoparticles. However, little progress has been made in the development of switchable PT and PA probes with controllable spectral shifts in absorption. Here, we introduce the concept of photothermally switchable nanoparticles (PTSNs). To prove the concept, we demonstrated fast, reversible magnetic–PT switching of conventional and gold‐coated magnetic nanoparticle clusters in cancer cells in vitro and PT switching of nonlinear ultrasharp plasmonic resonances in gold nanorods molecularly targeted to circulating cells in vivo. We showed that genetically encoded PSFPs with relatively slow switching can serve as triple‐modal fluorescent, PT, and PA probes under static conditions, while PTSNs with ultrafast switching may provide higher PA sensitivity in the near‐infrared window of tissue transparency under dynamic flow conditions. Application of nonlinear phenomena for super‐resolution spectral PT and PA cytometry, microscopy, and spectral burning beyond the diffraction and spectral limits are also proposed. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Dual‐color STED super‐resolution microscope using a single laser source

STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies，due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples.     

Fluorescent proteins for single‐molecule fluorescence applications

We present single‐molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single‐molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single‐molecule applications, demonstrated by high photostability and rare fluorescence‐intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single‐molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy

Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.