Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

双光子显微镜在生物医学中的应用及其进展

光学显微镜的发展历史是一段不断提高显微镜的分辨率和对比度的历史。双光子显微镜是近30年来非线性显微镜的研究发展的代表。它在分辨率上与共聚焦显微镜相当,但在成像的层析穿透深度上有显著提高,并且大大减少了光毒性与光漂白。由于生物细胞组织中富有各种自家荧光源,因此双光子显微镜被广泛应用于皮肤组织甚至癌组织以及细胞的成像。基于共聚焦扫描显微镜的双光子显微镜可以很容易的与二次谐波显微镜组合,对皮肤组织中的重要成分胶原纤维进行成像。双光子显微镜还可以结合其他非线性光学现象对组织以及细胞进行成像,显示其强大的生命力。将来随着携带方便且廉价的双光子显微镜的出现,双光子显微镜有望在临床医学上发挥其有效的作用。     

Microscopy basics and the study of actin–actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin–ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.     

High data output and automated 3D correlative light-electron microscopy method

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.     

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

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.     

Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology

A. Evered and N. Dudding Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology Objective: To evaluate virtual microscopy in terms of diagnostic performance and acceptability among practising cytologists. Methods: Twenty‐four experienced cytologists were recruited to examine 20 SurePath® cervical cytology slides by virtual microscopy. Diagnostic accuracy was compared with glass slide microscopy using an unbiased crossover experimental design. Responses were allocated a score of one for a correct identification of normal or abnormal (borderline/atypical changes in squamous or glandular cells or worse) and a score of zero for an incorrect response (a normal slide reported as abnormal or vice versa). Perceptions of virtual microscopy were assessed by questionnaire analysis. Results: Participants yielded a total of 285 responses for the virtual slide set and 300 for the glass slide set. The approximate time to screen a virtual slide was 18 minutes, compared with 8 minutes or less for a glass slide. Overall there was no significant difference between virtual microscopy and glass slide microscopy in terms of diagnostic accuracy (P = 0.22). Virtual microscopy under‐performed when images were captured over a narrow focal range (P = 0.01). Diagnostic accuracy of virtual microscopy equalled that of glass slide microscopy when participants were able to focus through the full thickness of the slide images (P = 0.07). The most common difficulties experienced by participants with virtual microscopy were freezing of the computer screen during image download, slow response of the computer during slide movement and, in some instances, ‘fuzzy’ images. Cytologists have a strong preference for glass slides over virtual microscopy despite the overall equal diagnostic performance of the two viewing modalities. Conclusions: Diagnostic accuracy of virtual microscopy can equal that of glass slide microscopy. However, without good computer network connections, wide focal range and software that permits effortless navigation across virtual slides, cytologists are unlikely to be convinced of the utility of this technology for cytology screening and diagnosis.     

In situ Imaging of Pea Starch in Seeds

A method has been developed for the in situ imaging of starch in dry seeds by exploiting the tight packing of the starch and protein storage reserves within the cells of the embryo. The method can be adapted to prepare seed samples which are suitable for light microscopy (birefringence and iodine staining), scanning electron microscopy and atomic force microscopy. Its potential for imaging the internal structure of starch granules without any prior isolation process is demonstrated for round smooth peas. Using a standard ultramicrotome, thin sections were cut directly from selected regions of dry pea seeds and examined by light microscopy before and after hydration. The sectioning procedure left a planed surface with the internal structure of the starch granules exposed. This material was examined by scanning electron microscopy and atomic force microscopy directly or after controlled hydration. In the hydrated pea samples, the growth ring structure and blocklet sub-structure of individual starch granules within the seed were visualised directly by atomic force microscopy. Furthermore, the effects of hydration and staining were monitored and have been used to introduce contrast into the images. The observations have revealed new information on the blocklet distribution within pea starch granules and the physical origins of the growth ring structure of the granules: the blocklet distribution suggests that the granules contain alternating bands with different levels of crystallinity, rather than alternating amorphous and crystalline growth rings.     

三维反卷积显微成像技术浅谈

三维宽场反卷积显微成像技术是应用光学切片方法获取三维标本的二维图像序列,然后通过反卷积图像处理方法进行图像恢复,进而进行三纺重建的一种以光学技术和图像处理技术为核心的业微成橡方法。本讲述了光学切片的基本原理,给出了反卷积处理中点扩展函数的理论模型和实验测试方法,然后对现存的反卷积算法做了对比。对这一领域的发展趋势做了预测。     

三维反卷积显微成像技术浅谈

三维宽场反应卷积显微成像技术是应用光学切片方法获取三维标本的二维图像序列,然后通过反卷积图像处理方法进行图像恢复,进而进行三维重建的一种以光学技术和图像处理技术为核心的显微成像方法。本文讲述了光学切片的基本原理,给出了反卷积处理中点扩展函数的理论模型和实验测试方法,然后对现存的反卷积算法做了对比。最后,文章对这一领域的发展趋势作了预测。     

Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy

We have imaged microtubules, essential structural elements of the cytoskeleton in eukaryotic cells, in physiological conditions by scanning force microscopy. We have achieved molecular resolution without the use of cross-linking and chemical fixation methods. With tip forces below 0.3 nN, protofilaments with ~6 nm separation could be clearly distinguished. Lattice defects in the microtubule wall were directly visible, including point defects and protofilament separations. Higher tip forces destroyed the top half of the microtubules, revealing the inner surface of the substrate-attached protofilaments. Monomers could be resolved on these inner surfaces.Abbreviations APTS (3-aminopropyl)triethoxysilane - DETA N¹-[3-(trimethoxysilyl)propyl]diethylenetriamine - EM electron microscopy - MT microtubule - SFM scanning force microscopy     

Ex vivo confocal laser scanning microscopy: An innovative method for direct immunofluorescence of cutaneous vasculitis

Ex vivo confocal laser scanning microscopy (ex vivo CLSM) offers an innovative diagnostic approach through vertical scanning of skin samples with a resolution close to conventional histology. In addition, it enables fluorescence detection in tissues. We aimed to assess the applicability of ex vivo CLSM in the detection of vascular immune complexes in cutaneous vasculitis and to compare its diagnostic accuracy with direct immunofluorescence (DIF) microscopy. Eighty‐two sections of 49 vasculitis patients with relevant DIF microscopy findings were examined using ex vivo CLSM following staining with fluorescent‐labeled IgG, IgM, IgA, C3 and fibrinogen antibodies. DIF microscopy showed immunoreactivity of vessels with IgG, IgM, IgA, C3 and Fibrinogen in 2.0%, 49.9%, 12.2%, 59.2% and 44.9% of the patients, respectively. Ex vivo CLSM detected positive vessels with the same antibodies in 2.0%, 38.8%, 8.2%, 42.9% and 36.7% of the patients, respectively. The detection rate of positive superficial dermal vessels was significantly higher in DIF microscopy as compared to ex vivo CLSM (P < .05). Whereas, ex vivo CLSM identified positive deep dermal vessels more frequently compared to DIF microscopy. In conclusion, ex vivo CLSM could identify specific binding of the antibodies in vessels and showed a comparable performance to conventional DIF microscopy in diagnosing vasculitis.     

Raising the speed limits for 4D fluorescence microscopy

Three-dimensional time-lapse (4D) fluorescence microscopy is becoming a routine experimental tool. This article summarizes current technologies, and describes a new method for speeding image acquisition during 4D confocal microscopy.     

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)     

A Review of Scanning Probe Microscopy Investigations of Liposome-DNA Complexes

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.     

多房棘球绦虫光镜及扫描电镜的观察

本文对甘肃漳县草滩乡家犬小肠所获检的棘球绦虫,做了光镜及扫描电镜(SEM)观察。根据其形态特征及流行病学资料,初步认定为多房棘球绦虫(Echinococcus multicularis)。对扫描电镜所记述的多房棘球绦虫成虫不同部位的表面微毛的形态结构做了较为详细的观察和比较;此外还观察了成熟节片侧缘生殖孔及孕节片内虫卵表面的超微结构。     

Fluorescence Tracing of Intracellular Proteins

Developments in fluorescence microscopy and the availability of fluorescently labeled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci allowing deep insight into cell and organelle biology. Furthermore, confocal microscopy permits analysis of the three dimensional architecture of cells that could not be accomplished by conventional light microscopy. The goal of fluorescence protein tracing by microscopy is to visualize cellular constituents and general cytoarchitecture as close to native organization as possible. To achieve this, and to preserve cellular structure in the best possible manner, the specimen is usually fixed chemically. Here I review several standard fixation, permeabilization and labeling schemes followed by examples of several standard imaging techniques.     

角质层的离析及显微观察方法

植物角质层的离析方法很多,获取化石植物的角质层可以同时用几种化学处理方法。舒氏液浸解是最常用的方法,也是几种方法共用时最关键的步骤。浸解液浓度过大或处理时间过长有可能破坏角质层的结构。对于已经获取到的角质层,利用相差光学显微技术可以增加反差。利用微分干涉光学显微技术不仅反差增加,而且有立体感,背景颜色也可以调节。荧光显微分析技术在不破坏标本的前提下,可以获取到表皮的特征。扫描电子显微镜具有观察不透明物体,放大范围广以及高的分辨力等优越性,可作为光学显微镜的必要补充。     

Cryo-electron microscopy reveals macromolecular organization within biological liquid cyrstals seen in the polarizing microscope

A method is described for examining water dispersible biopolymers in the frozen, hydrated state by electron microscopy using the filamentous bacterial viruses Pf1 and fd as examples. The technique reveals liquid-crystalline textures that correlate well with polarizing microscopy of magnetically oriented specimens. At higher magnification the packing of the virus particle is revealed to a spatial resolution of better than 30 Å, thus linking directly with data from X-ray diffraction and optical microscopy. Electron diffraction confirms that the structure is preserved to high resolution (4 Å). The technique permits a detailed understanding of the processes involved in the orientation of these samples in a strong magnetic field and clarifies the long-range bi-axial properties of some fibres as seen by X-ray diffraction and optical microscopy.