Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.     

Interactive visual exploration of overlapping similar structures for three-dimensional microscope images

Background

Recent advances in microscopy enable the acquisition of large numbers of tomographic images from living tissues. Three-dimensional microscope images are often displayed with volume rendering by adjusting the transfer functions. However, because the emissions from fluorescent materials and the optical properties based on point spread functions affect the imaging results, the intensity value can differ locally, even in the same structure. Further, images obtained from brain tissues contain a variety of neural structures such as dendrites and axons with complex crossings and overlapping linear structures. In these cases, the transfer functions previously used fail to optimize image generation, making it difficult to explore the connectivity of these tissues.

Results

This paper proposes an interactive visual exploration method by which the transfer functions are modified locally and interactively based on multidimensional features in the images. A direct editing interface is also provided to specify both the target region and structures with characteristic features, where all manual operations can be performed on the rendered image. This method is demonstrated using two-photon microscope images acquired from living mice, and is shown to be an effective method for interactive visual exploration of overlapping similar structures.

Conclusions

An interactive visualization method was introduced for local improvement of visualization by volume rendering in two-photon microscope images containing regions in which linear nerve structures crisscross in a complex manner. The proposed method is characterized by the localized multidimensional transfer function and interface where the parameters can be determined by the user to suit their particular visualization requirements.     

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei

Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed.     

A boundary delimitation algorithm to approximate cell soma volumes of bipolar cells from topographical data obtained by scanning probe microscopy

Background  

Cell volume determination plays a pivotal role in the investigation of the biophysical mechanisms underlying various cellular processes. Whereas light microscopy in principle enables one to obtain three dimensional data, the reconstruction of cell volume from z-stacks is a time consuming procedure. Thus, three dimensional topographic representations of cells are easier to obtain by scanning probe microscopical measurements.     

From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

Background

In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM).

Methodology

To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression.

Conclusion

Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.     

NetMHCIIpan-2.0 - Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure

Background

Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively.

Results

We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest.

Conclusions

The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.     

Method: automatic segmentation of mitochondria utilizing patch classification,contour pair classification,and automatically seeded level sets

Background  

While progress has been made to develop automatic segmentation techniques for mitochondria, there remains a need for more accurate and robust techniques to delineate mitochondria in serial blockface scanning electron microscopic data. Previously developed texture based methods are limited for solving this problem because texture alone is often not sufficient to identify mitochondria. This paper presents a new three-step method, the Cytoseg process, for automated segmentation of mitochondria contained in 3D electron microscopic volumes generated through serial block face scanning electron microscopic imaging. The method consists of three steps. The first is a random forest patch classification step operating directly on 2D image patches. The second step consists of contour-pair classification. At the final step, we introduce a method to automatically seed a level set operation with output from previous steps.     

TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

Background  

Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.     

Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

Background  

Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.     

Statistical learning methods as a preprocessing step for survival analysis: evaluation of concept using lung cancer data

Background

Despite its superb lateral resolution, flat-panel-detector (FPD) based tomosynthesis suffers from low contrast and inter-plane artifacts caused by incomplete cancellation of the projection components stemming from outside the focal plane. The incomplete cancellation of the projection components, mostly due to the limited scan angle in the conventional tomosynthesis scan geometry, often makes the image contrast too low to differentiate the malignant tissues from the background tissues with confidence.

Methods

In this paper, we propose a new method to suppress the inter-plane artifacts in FPD-based tomosynthesis. If 3D whole volume CT images are available before the tomosynthesis scan, the CT image data can be incorporated into the tomosynthesis image reconstruction to suppress the inter-plane artifacts, hence, improving the image contrast. In the proposed technique, the projection components stemming from outside the region-of-interest (ROI) are subtracted from the measured tomosynthesis projection data to suppress the inter-plane artifacts. The projection components stemming from outside the ROI are calculated from the 3D whole volume CT images which usually have lower lateral resolution than the tomosynthesis images. The tomosynthesis images are reconstructed from the subtracted projection data which account for the x-ray attenuation through the ROI. After verifying the proposed method by simulation, we have performed both CT scan and tomosynthesis scan on a phantom and a sacrificed rat using a FPD-based micro-CT.

Results

We have measured contrast-to-noise ratio (CNR) from the tomosynthesis images which is an indicator of the residual inter-plane artifacts on the focal-plane image. In both cases of the simulation and experimental imaging studies of the contrast evaluating phantom, CNRs have been significantly improved by the proposed method. In the rat imaging also, we have observed better visual contrast from the tomosynthesis images reconstructed by the proposed method.

Conclusions

The proposed tomosynthesis technique can improve image contrast with aids of 3D whole volume CT images. Even though local tomosynthesis needs extra 3D CT scanning, it may find clinical applications in special situations in which extra 3D CT scan is already available or allowed.     

Impact of image segmentation on high-content screening data quality for SK-BR-3 cells

Background  

High content screening (HCS) is a powerful method for the exploration of cellular signalling and morphology that is rapidly being adopted in cancer research. HCS uses automated microscopy to collect images of cultured cells. The images are subjected to segmentation algorithms to identify cellular structures and quantitate their morphology, for hundreds to millions of individual cells. However, image analysis may be imperfect, especially for "HCS-unfriendly" cell lines whose morphology is not well handled by current image segmentation algorithms. We asked if segmentation errors were common for a clinically relevant cell line, if such errors had measurable effects on the data, and if HCS data could be improved by automated identification of well-segmented cells.     

Correlation of 4Pi and Electron Microscopy to Study Transport Through Single Golgi Stacks in Living Cells with Super Resolution

Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited resolution and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern high-resolution light microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electron microscopy in cell biology applications. In this study, we provide the first comparative correlative analysis of three-dimensional structures obtained by 4Pi microscopy and segmented by a zero-crossing procedure with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein procollagen 3 was mapped by both 4Pi microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for the structures, indicating the particular accuracy of the 4Pi microscopy. Furthermore, for the first time, transport of a cargo molecule (vesicular stomatitis virus G protein-pEGFP) through individual Golgi stacks (labeled by galactosyl transferase-venusYFP) was visualized by 4Pi microscopy. Following the procedures validated by the correlative analysis, our transport experiments show that (i) VSVG-pEGFP rapidly enter/exit individual Golgi stacks, (ii) VSVG-pEGFP never fills the GalT-venusYFP compartments completely and (iii) the GalT-venusYFP compartment volume increases upon VSVG-pEGFP arrival. This morphological evidence supports some previous TEM-based observations of intra-Golgi transport of VSVG-pEGFP and provides new insights toward a better understanding of protein progression across Golgi stacks. Our study thus demonstrates the general applicability of super-resolution fluorescence microscopy, coupled with the zero-crossing segmentation procedure, for structural studies of suborganelle protein distributions under living cell conditions.     

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists.This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method.Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

Characterizaton of the Vessel Geometry,Flow Mechanics and Wall Shear Stress in the Great Arteries of Wildtype Prenatal Mouse

Introduction

Abnormal fluid mechanical environment in the pre-natal cardiovascular system is hypothesized to play a significant role in causing structural heart malformations. It is thus important to improve our understanding of the prenatal cardiovascular fluid mechanical environment at multiple developmental time-points and vascular morphologies. We present such a study on fetal great arteries on the wildtype mouse from embryonic day 14.5 (E14.5) to near-term (E18.5).

Methods

Ultrasound bio-microscopy (UBM) was used to measure blood velocity of the great arteries. Subsequently, specimens were cryo-embedded and sectioned using episcopic fluorescent image capture (EFIC) to obtain high-resolution 2D serial image stacks, which were used for 3D reconstructions and quantitative measurement of great artery and aortic arch dimensions. EFIC and UBM data were input into subject-specific computational fluid dynamics (CFD) for modeling hemodynamics.

Results

In normal mouse fetuses between E14.5–18.5, ultrasound imaging showed gradual but statistically significant increase in blood velocity in the aorta, pulmonary trunk (with the ductus arteriosus), and descending aorta. Measurement by EFIC imaging displayed a similar increase in cross sectional area of these vessels. However, CFD modeling showed great artery average wall shear stress and wall shear rate remain relatively constant with age and with vessel size, indicating that hemodynamic shear had a relative constancy over gestational period considered here.

Conclusion

Our EFIC-UBM-CFD method allowed reasonably detailed characterization of fetal mouse vascular geometry and fluid mechanics. Our results suggest that a homeostatic mechanism for restoring vascular wall shear magnitudes may exist during normal embryonic development. We speculate that this mechanism regulates the growth of the great vessels.     

CellProfiler: Novel Automated Image Segmentation Procedure for Super-Resolution Microscopy

Background

Super resolution (SR) microscopy enabled cell biologists to visualize subcellular details up to 20 nm in resolution. This breakthrough in spatial resolution made image analysis a challenging procedure. Direct and automated segmentation of SR images remains largely unsolved, especially when it comes to providing meaningful biological interpretations.

Results

Here, we introduce a novel automated imaging analysis routine, based on Gaussian, followed by a segmentation procedure using CellProfiler software (www.cellprofiler.org). We tested this method and succeeded to segment individual nuclear pore complexes stained with gp210 and pan-FG proteins and captured by two-color STED microscopy. Test results confirmed accuracy and robustness of the method even in noisy STED images of gp210.

Conclusions

Our pipeline and novel segmentation procedure may benefit end-users of SR microscopy to analyze their images and extract biologically significant quantitative data about them in user-friendly and fully-automated settings.

     

A novel thin section preparation and staining protocol to increase contrast and resolution of cell details for light microscopy

ABSTRACT

We developed a novel sectioning and staining method to make high contrast, high resolution sections of plant tissue for light microscopy. Specimens of teosinte (Zea mays L., ssp. mexicana) root tips were fixed and embedded in Technovit 7100? plastic resin. Thin sections, 1?2.5 μm, were cut and mounted on glass slides. The sections were either treated with RNase or not, then stained with 0.1% toluidine blue O and observed through ∞/0 objective lenses. For light microscopy, the enzyme staining procedure increased resolution and contrast. High magnification ∞/0 objective lenses produced high quality images for digital photography without using a coverslip or immersion oil. Our slide preparation and microscopic analysis were less labor intensive and more rapid than previous methods and enabled rapid and precise alignment of serial transverse sections for both tracking cell lineages and tissue measurements.     

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.