Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.     

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.     

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA² (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes'' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.     

Live-cell imaging in the era of too many microscopes

At the time of this writing, searching Google Scholar for ‘light-sheet microscopy’ returns almost 8500 results; over three-quarters of which were published in the last 5 years alone. Searching for other advanced imaging methods in the last 5 years yields similar results: ‘super-resolution microscopy’ (>16 000), ‘single-molecule imaging’ (almost 10 000), SPIM (Single Plane Illumination Microscopy, 5000), and ‘lattice light-sheet’ (1300). The explosion of new imaging methods has also produced a dizzying menagerie of acronyms, with over 100 different species of ‘light-sheet’ alone, from SPIM to UM (Ultra microscopy) to SiMView (Simultaneous MultiView) to iSPIM (inclined SPIM, not to be confused with iSPIM, inverted SPIM). How then is the average biologist, without an advanced degree in physics, optics, or computer science supposed to make heads or tails of which method is best suited for their needs? Let us also not forget the plight of the optical physicist, who at best might need help with obtaining healthy samples and keeping them that way, or at worst may not realize the impact their newest technique could have for biologists. This review will not attempt to solve all these problems, but instead highlight some of the most recent, successful mergers between biology and advanced imaging technologies, as well as hopefully provide some guidance for anyone interested in journeying into the world of live-cell imaging.     

Single-Molecule Light-Sheet Imaging of Suspended T Cells

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.     

Light-Sheet Confined Super-Resolution Using Two-Photon Photoactivation

Light-sheet microscopy is a useful tool for performing biological investigations of thick samples and it has recently been demonstrated that it can also act as a suitable architecture for super-resolution imaging of thick biological samples by means of individual molecule localization. However, imaging in depth is still limited since it suffers from a reduction in image quality caused by scattering effects. This paper sets out to investigate the advantages of non-linear photoactivation implemented in a selective plane illumination configuration when imaging scattering samples. In particular, two-photon excitation is proven to improve imaging capabilities in terms of imaging depth and is expected to reduce light-sample interactions and sample photo-damage. Here, two-photon photoactivation is coupled to individual molecule localization methods based on light-sheet illumination (IML-SPIM), allowing super-resolution imaging of nuclear pH2AX in NB4 cells.     

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.     

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.     

膨胀显微成像技术的原理及应用

膨胀显微成像技术（expansion microscopy,ExM）是一种新型超分辨成像技术。该技术借助可膨胀水凝胶均匀地物理放大生物样本,在常规光学成像条件下实现超分辨成像。ExM适用于细胞、组织切片等多种类型生物样本。蛋白质、核酸、脂质等生物大分子均可借助ExM进行超分辨成像。ExM可与共聚焦显微镜、光片显微镜、超高分辨显微镜联合使用,进一步提高成像分辨率。近年来,多种从基础ExM拓展而来的衍生技术进一步促进了该技术的实际应用。本文综述了ExM及其衍生技术的基本原理、ExM与不同成像技术联用的研究进展及ExM在不同类型生物样本中的应用进展,并对ExM技术的发展前景做出展望。     

Light-sheet based fluorescence microscopy: the dark side of the sample finally revealed

Light-sheet based fluorescence microscopy (LSM) is an optical technique that becomes more and more popular for multi-view imaging of in vivo sample in its physiological environment. LSM combines the advantages of the direct optical sectioning to the ones of optical tomography by angular scanning. In fact, a thin light-sheet illuminates laterally a section of the sample, thus limiting the effects of photobleaching and phototoxicity only to the plane of interest. The spatial resolution can be improved by combining multiple views obtained along different angle into a single data, leading to a 3D isotropic rendering of the sample. Such an approach provides several advantages in comparison to conventional 3D microscopic techniques: confocal and multiphoton microscopies. It makes LSM an optical tool suited for imaging specimens with a subcellular resolution even inside an embryo and with temporal resolution adapted for real-time monitoring of biological processes.     

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.     

Faster STORM using compressed sensing

In super-resolution microscopy methods based on single-molecule switching, the rate of accumulating single-molecule activation events often limits the time resolution. Here we developed a sparse-signal recovery technique using compressed sensing to analyze images with highly overlapping fluorescent spots. This method allows an activated fluorophore density an order of magnitude higher than what conventional single-molecule fitting methods can handle. Using this method, we demonstrated imaging microtubule dynamics in living cells with a time resolution of 3 s.     

PALM and STORM: What hides beyond the Rayleigh limit?

Super-resolution imaging allows the imaging of fluorescently labeled probes at a resolution of just tens of nanometers, surpassing classic light microscopy by at least one order of magnitude. Recent advances such as the development of photo-switchable fluorophores, high-sensitivity microscopes and single particle localization algorithms make super-resolution imaging rapidly accessible to the wider life sciences research community. As we take our first steps in deciphering the roles and behaviors of individual molecules inside their living cellular environment, a new world of research opportunities beckons. Here we discuss some of the latest developments achieved with these techniques and emerging areas where super-resolution will give fundamental new “eye” sight to cell biology.     

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.     

Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.     

Coordinate-based colocalization analysis of single-molecule localization microscopy data

Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.     

Integrated optical molecular imaging system for four-dimensional real-time detection in living single cells

A novel, multifunctional optical imaging system was developed by integrating four-dimensional (4D) real-time confocal microscopy (RT-CM), multicolor total internal reflection microscopy (TIRFM), and Nomarski differential interference contrast (DIC) microscopy based on an epifluorescence microscope platform. A microcell incubator was combined with the imaging system for extended, real-time monitoring of living cells. The 4D images were generated by a combination of 3D images and multiple spatial or time images of a specimen, obtained at 10 nm intervals. Optical sectioning was accomplished with a z-motor, which obtained 4D information with sequential layered sections. The integrated imaging system showed excellent detection sensitivity at the single-molecule level and 3D-spatial resolution (20 nm x-y and 10 nm z-axis) without moving the cell sample. This could be a tool for obtaining crucial information needed to develop approaches for characterizing and understanding the dynamics of biomolecules and nanoparticles in individual living cells and molecular interactions at the single-molecule level.     

几种超分辨率荧光显微技术的原理和近期进展

在生命科学领域,人们常常需要在细胞内精确定位特定的蛋白质以研究其位置与功能的关系．多年来,宽场/共聚焦荧光显微镜的分辨率受限于光的阿贝/瑞利极限,不能分辨出200 nm以下的结构．近年来,随着新的荧光探针和成像理论的出现,研究者开发了多种实现超出普通共聚焦显微镜分辨率的三维超分辨率成像方法．主要介绍这些方法的原理、近期进展和发展趋势．介绍了光源的点扩散函数(point spread function, PSF)的概念和传统分辨率的定义,阐述了提高xy平面分辨率的方法．通过介绍单分子荧光成像技术,引入了单分子成像定位精度的概念,介绍了基于单分子成像的超分辨率显微成像方法,包括光激活定位显微技术(photoactivated localization microscopy, PALM)和随机光学重构显微技术(stochastic optical reconstruction microscopy, STORM)．介绍了两大类通过改造光源的点扩散函数来提高成像分辨率的方法,分别是受激发射损耗显微技术(stimulated emission depletion, STED)和饱和结构照明显微技术(saturated structure illumination microscopy, SSIM)．比较了不同的z轴提取信息的方法,并阐述了这些方法与xy平面上的超分辨率显微成像技术相结合所得到的各种三维超分辨率显微成像技术的优劣．探讨了目前超分辨率显微成像的发展极限和方向．     

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets¹, monitor dynamics in living cells^2-4, and visualize the three dimensional evolution of entire organisms^5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo⁷ and are currently being applied to disease imaging and diagnosis in clinical settings^8,9.Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole¹, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.Download video file.^{(90M, mov)}     

A simple, versatile method for GFP-based super-resolution microscopy via nanobodies

We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.