The role of backscattering in SHG tissue imaging

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.     

High-resolution confocal imaging and three-dimensional rendering

Intrinsic opacity and inhomeogeniety of most biological tissues have prevented the efficient light penetration and signal detection for high-resolution confocal imaging of thick tissues. Here, we summarize recent technical advances in high-resolution confocal imaging for visualization of cellular structures and gene expression within intact whole-mount thick tissues. First, we introduce features of the FocusClear technology that render biological tissue transparent and thus improve the light penetration and signal detection. Next, a universal fluorescence staining method that labels all nuclei and membranes is described. We then demonstrate the postrecording image processing techniques for 3D visualization. From these images, regions of interest in the whole-mount brain can be segmented and volume rendered. Together, these technical advances in confocal microscopy allow visualization of structures within whole-mount tissues up to 1mm thick at a resolution similar to that of the observation of single cells in culture. Practical uses and limitations of these techniques are discussed.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning

Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning

Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane.     

Progress in applying the high-voltage electron microscope to biomedical research

This review attempts a physical definition of the technical problems and achievements in applying the high-voltage electron microscope (HVEM) to biological and medical research. It is hoped that the review will summarize for biologists, funding agencies, and institutions the achievements of the HVEM, its future prospects, and the main problem areas that still need to be explored. At present it is not known whether future HVEMs will favor the fixed beam or the scanning transmission electron microscopy (STEM) mode. The STEM mode offers reduced radiation damage as a result of more efficient electron detection and ease of manipulation of the collected signals by separating the elastic and inelastic signals. Energy filtration to remove the inelastic signal provides a means to enhance the contrast and improve the resolution for thick specimens. Several prototype STEM-mode HVEMs are now under development and it is expected that, in a few years, comparisons of fixed beam and STEM modes will be possible. The review discusses several HVEM instrument features that remain poorly developed. In the area of image recording a photographic emulsion has been designed to give optimized performance at an acceleration voltage of 1 MV. However, this remains unavailable commercially. Conversion of the HVEM electron image to a usable light image by phosphors etc., involves some difficulties, making it difficult to obtain good performance from TV systems. Since the HVEM is particularly useful for three-dimensional imaging, the further development of improved goniometers for stereo viewing and image reconstruction is important. The large volume available in the objective specimen volume and the increased penetration at high acceleration voltages make the HVEM particularly suitable for the application of environmental chambers in the microscopy and electron diffraction of thick wet specimens. An improved signal-to-noise ratio improves the prospects for elemental analysis at high acceleration voltages. When carefully carried out, improved resolution can be obtained in dark-field over that obtainable at 100 kV. Dark-field provides the easiest way to obtain high contrast on weakly stained or unstained objects. Its further improvement requires the use of specially thick and shaped beam stops and apertures that are not penetrated by the 1 MV beam. Recent HVEM studies of whole cells and microorganisms are reviewed. These studies already show that the former thin-section approach led to some incorrect ideas about the shape of some organelles and their three-dimensional relationships. This new information is proving important in helping to establish the function of fibrillar and membranous components of the cell. The most important limitation in examining thick sections is the large depth of field that causes excessive overlap of in-focus structures in stereo views of thick sections. In a few cases special specific heavy metal stains have been developed to overcome this problem, but an optical solution would be more generally applicable. Attempts are now being made to unscramble overlapped detail by applying the image reconstruction techniques of tomography and holography. It is concluded that even with existing techniques, the HVEM examination of thick sections provides a very useful improvement in sampling statistics and in three-dimensional imaging of cell structures over that obtainable by examining thin sections at a lower acceleration voltage (100 kV). Randomized author sequence.     

Concepts in imaging and microscopy. Exploring biological structure and function with confocal microscopy

Confocal microscopy is providing new and exciting opportunities for imaging cell structure and physiology in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focusing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantitative analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell morphology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function.     

Recent advances in X-ray imaging of breast tissue: From two- to three-dimensional imaging

Breast cancer is a globally widespread disease whose detection has already been significantly improved by the introduction of screening programs. Nevertheless, mammography suffers from low soft tissue contrast and the superposition of diagnostically relevant anatomical structures as well as from low values for sensitivity and specificity especially for dense breast tissue. In recent years, two techniques for X-ray breast imaging have been developed that bring advances for the early detection of breast cancer. Grating-based phase-contrast mammography is a new imaging technique that is able to provide three image modalities simultaneously (absorption-contrast, phase-contrast and dark-field signal). Thus, an enhanced detection and delineation of cancerous structures in the phase-contrast image and an improved visualization and characterization of microcalcifications in the dark-field image is possible. Furthermore, latest studies about this approach show that dose-compatible imaging with polychromatic X-ray sources is feasible. In order to additionally overcome the limitations of projection-based imaging, efforts were also made towards the development of breast computed tomography (BCT), which recently led to the first clinical installation of an absorption-based BCT system. Further research combining the benefits of both imaging technologies is currently in progress. This review article summarizes the latest advances in phase-contrast imaging for the female breast (projection-based and three-dimensional view) with special focus on possible clinical implementations in the future.     

Lung tumors on multimodal radiographs derived from grating-based X-ray imaging – A feasibility study

PurposeThe purpose of this study was to assess whether grating-based X-ray imaging may have a role in imaging of pulmonary nodules on radiographs.Materials and methodsA mouse lung containing multiple lung tumors was imaged using a small-animal scanner with a conventional X-ray source and a grating interferometer for phase-contrast imaging. We qualitatively compared the signal characteristics of lung nodules on transmission, dark-field and phase-contrast images. Furthermore, we quantitatively compared signal characteristics of lung tumors and the adjacent lung tissue and calculated the corresponding contrast-to-noise ratios.ResultsOf the 5 tumors visualized on the transmission image, 3/5 tumors were clearly visualized and 1 tumor was faintly visualized in the dark-field image as areas of decreased small angle scattering. In the phase-contrast images, 3/5 tumors were clearly visualized, while the remaining 2 tumors were faintly visualized by the phase-shift occurring at their edges. No additional tumors were visualized in either the dark-field or phase-contrast images. Compared to the adjacent lung tissue, lung tumors were characterized by a significant decrease in transmission signal (median 0.86 vs. 0.91, p = 0.04) and increase in dark-field signal (median 0.71 vs. 0.65, p = 0.04). Median contrast-to-noise ratios for the visualization of lung nodules were 4.4 for transmission images and 1.7 for dark-field images (p = 0.04).ConclusionLung nodules can be visualized on all three radiograph modalities derived from grating-based X-ray imaging. However, our initial data suggest that grating-based multimodal X-ray imaging does not increase the sensitivity of chest radiographs for the detection of lung nodules.     

Epi-illumination dark-field microscopy enables direct visualization of unlabeled small organisms with high spatial and temporal resolution

Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an “open top” for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans.     

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

Background

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.

Methods

Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.

Results

We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.

Conclusion

Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.

General significance

This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.     

Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane.     

Biological applications of synchrotron radiation infrared spectromicroscopy

Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (< 10 μm) with a high signal to-noise ratio. IR spectroscopy is a powerful technique to investigate biological systems and offers many new imaging opportunities. The field of infrared biological imaging covers a wide range of fundamental issues and applied researches such as cell imaging or tissue imaging. Molecular maps with a spatial resolution down to the diffraction limit may be now obtained with a synchrotron radiation IR source also on thick samples. Moreover, changes of the protein structure are detectable in an IR spectrum and cellular molecular markers can be identified and used to recognize a pathological status of a tissue. Molecular structure and functions are strongly correlated and this aspect is particularly relevant for imaging. We will show that the brilliance of synchrotron radiation IR sources may enhance the sensitivity of a molecular signal obtained from small biosamples, e.g., a single cell, containing extremely small amounts of organic matter. We will also show that SR IR sources allow to study chemical composition and to identify the distribution of organic molecules in cells at submicron resolution is possible with a high signal-to-noise ratio. Moreover, the recent availability of two-dimensional IR detectors promises to push forward imaging capabilities in the time domain. Indeed, with a high current synchrotron radiation facility and a Focal Plane Array the chemical imaging of individual cells can be obtained in a few minutes. Within this framework important results are expected in the next years using synchrotron radiation and Free Electron Laser (FEL) sources for spectro-microscopy and spectral-imaging, alone or in combination with Scanning Near-field Optical Microscopy methods to study the molecular composition and dynamic changes in samples of biomedical interest at micrometric and submicrometric scales, respectively.     

Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues

The deep tissue penetration and submicron spatial resolution of multiphoton microscopy and the high detection efficiency and nanometer spectral resolution of a spectrograph were utilized to record spectral images of the intrinsic emission of mouse skin tissues. Autofluorescence from both cellular and extracellular structures, second-harmonic signal from collagen, and a narrowband emission related to Raman scattering of collagen were detected. Visualization of the spectral images by wavelength-to-RGB color image conversion allowed us to identify and discriminate tissue structures such as epidermal keratinocytes, lipid-rich corneocytes, intercellular structures, hair follicles, collagen, elastin, and dermal cells. Our results also showed morphological and spectral differences between excised tissue section, thick excised tissue, and in vivo tissue samples of mouse skin. Results on collagen excitation at different wavelengths suggested that the origin of the narrowband emission was collagen Raman peaks. Moreover, the oscillating spectral dependency of the collagen second-harmonic intensity was experimentally studied. Overall, spectral imaging provided a wealth of information not easily obtainable with present conventional multiphoton imaging systems.     

isoSTED microscopy with water-immersion lenses and background reduction

Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few 10 nm at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO₂ supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm³. Because fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method that allows us to identify this unspecific background signal and to remove it from the image.     

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.     

Bi-Directional X-Ray Phase-Contrast Mammography

Phase-contrast x-ray imaging is a promising improvement of conventional absorption-based mammography for early tumor detection. This potential has been demonstrated recently, utilizing structured gratings to obtain differential phase and dark-field scattering images. However, the inherently anisotropic imaging sensitivity of the proposed mono-directional approach yields only insufficient diagnostic information, and has low diagnostic sensitivity to highly oriented structures. To overcome these limitations, we present a two-directional x-ray phase-contrast mammography approach and demonstrate its advantages by applying it to a freshly dissected, cancerous mastectomy breast specimen. We illustrate that the two-directional scanning procedure overcomes the insufficient diagnostic value of a single scan, and reliably detects tumor structures, independently from their orientation within the breast. Our results indicate the indispensable diagnostic necessity and benefit of a multi-directional approach for x-ray phase-contrast mammography.     

偏振光成像技术用于肿瘤病变检测的研究进展

偏振光成像是一种非标记、无损伤检测技术,它与现有非偏振光学方法硬件兼容,但能提供更丰富的样品结构和光学信息,并且对亚波长微观结构变化十分敏感.最近,偏振光成像方法在生物医学,特别是肿瘤癌症检测领域显示出很好的应用前景.本文介绍了常用的偏振光散射成像方法,包括偏振差、偏振度、旋转线偏振成像、偏振显微、穆勒矩阵成像等,并展示这些偏振方法在生物医学领域,特别是癌症检测方面的最新研究进展.目前偏振差、偏振度等成像方法已被初步用于皮肤癌的诊断,而穆勒矩阵包含更为丰富的组织微观结构信息,因而具有更好的诊断应用前景.通过对穆勒矩阵进行分解、变换等处理,可获得具有明确物理意义的成像参数,并发展为针对不同应用的特异性方法.目前,随着新型光源、偏振器件和探测器的出现,特别是数据计算处理能力的急剧提升,偏振成像在数据解释和测量方法方面的研究快速发展,已经在生物医学领域显示出很好的应用前景.