Computer-aided microtomography with true 3-D display in electron microscopy

A novel research system has been designed to permit three-dimensional (3-D) viewing of high resolution image data from transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The system consists of front-end primary data acquisition devices, such as TEM and SEM machines, which are equipped with computer-controlled specimen tilt stages. The output from these machines is in analogue form, where a video camera attached to the TEM provides the sequential analogue image output while the SEM direct video output is utilized. A 10 MHz digitizer transforms the video image to a digital array of 512 X 512 pixel units of 8 bits deep-stored in a frame buffer. Digital images from multiple projections are reconstructed into 3-D image boxes in a dedicated computer. Attached to the computer is a powerful true 3-D display device which has hardware for graphic manipulations including tilt and rotate on any axis and for probing the image with a 3-D cursor. Data editing and automatic contouring functions are used to enhance areas of interest, and specialized software is available for measurement of numbers, distances, areas, and volumes. With proper archiving of reconstructed image sequences, a dynamic 3-D presentation is possible. The microtomography system is highly versatile and can process image data on-line or from remote sites from which data records would typically be transported on computer tape, video tape, or floppy disk.     

Utility of 2-D and 3-D virtual microscopy in cervical cytology education and testing

OBJECTIVE: To evaluate the effectiveness of 3-D vs. 2-D virtual microscopy as adjuncts to education and assessment in cervical cytology. STUDY DESIGN: Five cervical cytology slides were acquired in 2-D; then the identical area of the slide was acquired in 3-D, resulting in 2 sets of virtual slides for comparison with the original glass slide. Seventy-nine paid volunteer cytologists and cytotechnology students participated. Approximately half were sent the 2-D set of slides via the Web, and the others a 3-D set of slides on a DVD. Evaluators examined the virtual slides and committed to an interpretation. After receipt of the original glass slides, a second interpretation was made, if different from the virtual slide interpretation. RESULTS: Diagnostic accuracy using virtual cytology slides was similar to that for glass slides (94% vs. 96%). There was no difference in diagnostic accuracy between 2-D and 3-D slides (p = 0.28); however, the ability to focus 3-D slides in the z-axis was strongly endorsed by the participants because of the uncertainty and frustration of having some cells out of focus on 2-D virtual slides. CONCLUSION: There was consensus that virtual cervical cytology slides would be a useful augmentation to education and testing.     

A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy

Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a “toolbox” of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.     

3-D reconstruction of bluetongue virus tubules using cryoelectron microscopy.

Bluetongue virus (BTV) forms tubules in infected mammalian cells. These tubules are virally encoded entities which can be formed with only one protein, NS1. The NS1 protein does not form a part of virus particles, and its function in viral infection is uncertain. Expression of the NS1 gene in insect cells by recombinant baculovirus yields high amounts of NS1 tubules (ca. 50% of cellular proteins) which are morphologically and immunologically similar to authentic BTV NS1 and can be isolated to about 90% purity. The structure of these synthetic NS1 tubules was investigated by cryoelectron microscopy. NS1 tubules are on average 52.3 nm in diameter and up to 100 nm long. The structure of their helical surface lattice has been determined using computer image processing to a resolution of 40 A. The NS1 protein is about 5.3 nm in diameter and forms a dimer-like structure, so that the tubules are composed of helically coiled ribbons of NS1 "dimers," with 21 or 22 dimers per turn. The surface lattice displays P2 symmetry and forms a one-start helix with a pitch of 9.1 nm. The NS1 tubules exist in two slightly different pH-dependent conformational states.     

小动物体内可见光三维成像技术研究进展

活体动物体内可见光成像是采用生物发光和荧光为标记物,利用灵敏的仪器来监控活体动物体内的细胞活动、蛋白表达情况和基因行为。近年来,可见光成像在生物医学的各个方面得到了广泛的应用。随着成像技术和检测仪器的不断发展,现已从平面二维成像逐渐发展为立体三维成像。三维成像技术在靶点的空间定位、与器官的关系,及绝对定量方面都有了很大的进展。本文就三维成像技术的原理、应用和发展前景进行了简要的综述。     

Fluorescent stains for quantification of DNA by confocal laser scanning microscopy in 3-D

Confocal microscopy requires the use of fluorophores to visualize structures of interest within a specimen. To perform reliable measurements of the intensity of fluorescence, the stain should be specific, penetrate well into tissue sections, and bind stoichiometrically. Furthermore, emission must be linear with respect to DNA content and brightness, and fluorescence should be stable. Confocal microscopy is used to determine DNA ploidy and to analyze texture of nuclei, which is accomplished in three dimensions, because nuclei can be measured within the original tissue context. For this purpose the sample must be stained with a DNA binding fluorophore with the properties described above. Stains with different properties have been developed for different applications. We review here the advantages and disadvantages of these different stains for analyzing DNA ploidy and nuclear texture using three-dimensional microscopy. We conclude that SYBR green I and TO-PRO-3 are the most suitable stains for this purpose at present.     

Comprehensive volumetric optical microscopy in vivo

Comprehensive volumetric microscopy of epithelial, mucosal and endothelial tissues in living human patients would have a profound impact in medicine by enabling diagnostic imaging at the cellular level over large surface areas. Considering the vast area of these tissues with respect to the desired sampling interval, achieving this goal requires rapid sampling. Although noninvasive diagnostic technologies are preferred, many applications could be served by minimally invasive instruments capable of accessing remote locations within the body. We have developed a fiber-optic imaging technique termed optical frequency-domain imaging (OFDI) that satisfies these requirements by rapidly acquiring high-resolution, cross-sectional images through flexible, narrow-diameter catheters. Using a prototype system, we show comprehensive microscopy of esophageal mucosa and of coronary arteries in vivo. Our pilot study results suggest that this technology may be a useful clinical tool for comprehensive diagnostic imaging for epithelial disease and for evaluating coronary pathology and iatrogenic effects.     

An ab initio algorithm for low-resolution 3-D reconstructions from cryoelectron microscopy images

A statistical method for determining low-resolution 3-D reconstructions of virus particles from cryoelectron microscope images by an ab initio algorithm is described. The method begins with a novel linear reconstruction method that generates a spherically symmetric reconstruction, which is followed by a nonlinear reconstruction method implementing an expectation-maximization procedure using the spherically symmetric reconstruction as an initial condition and resulting in a reconstruction with icosahedral symmetry. An important characteristic of the complete method is that very little need be known about the particle before the reconstruction is computed, in particular, only the type of symmetry and inner and outer radii. The method is demonstrated on synthetic cowpea mosaic virus data, and its robustness to 5% errors in the contrast transfer function, 5% errors in the location of the center of the particles in the images, and 5% distortion in the 3-D structure from which the images are derived is demonstrated numerically.     

Quantifying anisotropic solute transport in protein crystals using 3-D laser scanning confocal microscopy visualization

The diffusion of a solute, fluorescein into lysozyme protein crystals has been studied by confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy makes it possible to non-invasively obtain high-resolution three-dimensional (3-D) images of spatial distribution of fluorescein in lysozyme crystals at various time steps. Confocal laser scanning microscopy gives the fluorescence intensity profiles across horizontal planes at several depths of the crystal representing the concentration profiles during diffusion into the crystal. These intensity profiles were fitted with an anisotropic model to determine the diffusivity tensor. Effective diffusion coefficients obtained range from 6.2 x 10(-15) to 120 x 10(-15) m2/s depending on the lysozyme crystal morphology. The diffusion process is found to be anisotropic, and the level of anisotropy depends on the crystal morphology. The packing of the protein molecules in the crystal seems to be the major factor that determines the anisotropy.     

Wearable optical resolution photoacoustic microscopy

Optical resolution photoacoustic microscopy (ORPAM) is an emerging imaging technique, which has been extensively used to study various brain activities and disorders of the anesthetized/restricted rodents with a special focus on the morphological and functional visualization of cerebral cortex. However, it is challenging to develop a wearable photoacoustic microscope, which enables the investigation of brain activities/disorders on freely moving rodents. Here, we report a wearable and robust optical resolution photoacoustic microscope (W‐ORPAM), which utilizes a small, light, stable and fast optical scanner. This wearable imaging probe features high spatiotemporal resolution, large field of view (FOV) and easy assembly as well as adjustable optical focus during the in vivo experiment, which makes it accessible to image cerebral cortex activities of freely moving rodents. To demonstrate the advantages of this technique, we used W‐ORPAM to monitor both morphological and functional variations of vasculature in cerebral cortex during the induction of ischemia and reperfusion of a freely moving rat.     

Near-field scanning optical microscopy in cell biology

Near-field optics has produced the highest optical resolution that has ever been achieved. The methods involved lie at the interface of far-field optical microscopy and scanned probe microscopy. This article describes the principles behind near-field scanning optical microscopy (NSOM) and highlights its potential in cell biology.     

Deep learning-based image processing in optical microscopy

Optical microscopy has emerged as a key driver of fundamental research since it provides the ability to probe into imperceptible structures in the biomedical world. For the detailed investigation of samples, a high-resolution image with enhanced contrast and minimal damage is preferred. To achieve this, an automated image analysis method is preferable over manual analysis in terms of both speed of acquisition and reduced error accumulation. In this regard, deep learning (DL)-based image processing can be highly beneficial. The review summarises and critiques the use of DL in image processing for the data collected using various optical microscopic techniques. In tandem with optical microscopy, DL has already found applications in various problems related to image classification and segmentation. It has also performed well in enhancing image resolution in smartphone-based microscopy, which in turn enablse crucial medical assistance in remote places.Graphical abstract     

Nonlinear optical microscopy in decoding arterial diseases

Pathological understanding of arterial diseases is mainly attributable to histological observations based on conventional tissue staining protocols. The emerging development of nonlinear optical microscopy (NLOM), particularly in second-harmonic generation, two-photon excited fluorescence and coherent Raman scattering, provides a new venue to visualize pathological changes in the extracellular matrix caused by atherosclerosis progression. These techniques in general require minimal tissue preparation and offer rapid three-dimensional imaging. The capability of label-free microscopic imaging enables disease impact to be studied directly on the bulk artery tissue, thus minimally perturbing the sample. In this review, we look at recent progress in applications related to arterial disease imaging using various forms of NLOM.     

Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase.