The discriminative bilateral filter: an enhanced denoising filter for electron microscopy data

Advances in three-dimensional (3D) electron microscopy (EM) and image processing are providing considerable improvements in the resolution of subcellular volumes, macromolecular assemblies and individual proteins. However, the recovery of high-frequency information from biological samples is hindered by specimen sensitivity to beam damage. Low dose electron cryo-microscopy conditions afford reduced beam damage but typically yield images with reduced contrast and low signal-to-noise ratios (SNRs). Here, we describe the properties of a new discriminative bilateral (DBL) filter that is based upon the bilateral filter implementation of Jiang et al. (Jiang, W., Baker, M.L., Wu, Q., Bajaj, C., Chiu, W., 2003. Applications of a bilateral denoising filter in biological electron microscopy. J. Struc. Biol. 128, 82-97.). In contrast to the latter, the DBL filter can distinguish between object edges and high-frequency noise pixels through the use of an additional photometric exclusion function. As a result, high frequency noise pixels are smoothed, yet object edge detail is preserved. In the present study, we show that the DBL filter effectively reduces noise in low SNR single particle data as well as cellular tomograms of stained plastic sections. The properties of the DBL filter are discussed in terms of its usefulness for single particle analysis and for pre-processing cellular tomograms ahead of image segmentation.     

Developing a denoising filter for electron microscopy and tomography data in the cloud

The low radiation conditions and the predominantly phase-object image formation of cryo-electron microscopy (cryo-EM) result in extremely high noise levels and low contrast in the recorded micrographs. The process of single particle or tomographic 3D reconstruction does not completely eliminate this noise and is even capable of introducing new sources of noise during alignment or when correcting for instrument parameters. The recently developed Digital Paths Supervised Variance (DPSV) denoising filter uses local variance information to control regional noise in a robust and adaptive manner. The performance of the DPSV filter was evaluated in this review qualitatively and quantitatively using simulated and experimental data from cryo-EM and tomography in two and three dimensions. We also assessed the benefit of filtering experimental reconstructions for visualization purposes and for enhancing the accuracy of feature detection. The DPSV filter eliminates high-frequency noise artifacts (density gaps), which would normally preclude the accurate segmentation of tomography reconstructions or the detection of alpha-helices in single-particle reconstructions. This collaborative software development project was carried out entirely by virtual interactions among the authors using publicly available development and file sharing tools.     

Evaluation of denoising algorithms for biological electron tomography

Tomograms of biological specimens derived using transmission electron microscopy can be intrinsically noisy due to the use of low electron doses, the presence of a "missing wedge" in most data collection schemes, and inaccuracies arising during 3D volume reconstruction. Before tomograms can be interpreted reliably, for example, by 3D segmentation, it is essential that the data be suitably denoised using procedures that can be individually optimized for specific data sets. Here, we implement a systematic procedure to compare various nonlinear denoising techniques on tomograms recorded at room temperature and at cryogenic temperatures, and establish quantitative criteria to select a denoising approach that is most relevant for a given tomogram. We demonstrate that using an appropriate denoising algorithm facilitates robust segmentation of tomograms of HIV-infected macrophages and Bdellovibrio bacteria obtained from specimens at room and cryogenic temperatures, respectively. We validate this strategy of automated segmentation of optimally denoised tomograms by comparing its performance with manual extraction of key features from the same tomograms.     

Scanning transmission electron microscopy of biological structures

The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen. Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies. Later the real applications have not followed the initial hopes. The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze-dried isolated macromolecules to unstained cryosections. Emphasis will be put on the mass-mapping, multi-signal and elemental mapping modes which are unique features of the STEM instruments.     

Applications of direct detection device in transmission electron microscopy

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120–400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721–739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 μm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.     

Immune electron microscopy in the study of biological matter

A brief review of literature data and our investigations on the antibodies used for specific labeling in electron microscopy is presented. Considered are the problems connected with structure and function of separate components of bacterial viruses revealed by means of specific antibodies. The results of fine differentiation of antigenic components in the case of phages of the colidysentery group allowed to elucidate the functional role of the adsorption apparatus in the course of phage interaction with the bacterial cell. The topology of structural proteins (gene-products 35, 36, 37) of the tail's long strands for phages T4, DDVI+h and DDVIh is determined. Antigenic properties of proteins that are found in the composition of two forms of Bacillus mycoides are demonstrated immune-electronmicroscopically. On the basis of this finding, a conclusion was made that one of these phages acted as precursor, the other--as satellite during the simultaneous development of these phages in the bacterial cell. It was also established that temperate and virulent phages are related antigenically, which proves that lysogenic bacteria can be one of the phage sources on the environmental conditions. Visualization of non-ribosomal genes of procaryots that code for structural proteins of a defective phage proves the efficiency of the immune-electronmicroscopic method for investigating of biological objects.     

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.     

Applications of high voltage electron microscopy to botanical ultrastructure

Although high voltage electron microscopes have been in general use over the past decade microscopists have tended to ignore the contribution their use could make to the study of plant ultrastructure. The majority of biological high voltage research has been restricted to the fields of zoology and bio-medicine.The high voltage electron microscope (HVEM) has several advantages over the conventional transmission electron microscope (CTEM) when applied to biological specimens. These include increased penetrating power of the electron beam, reduced chromatic abberation in thick specimens, and both reduced beam heating and ionization damage. All these factors permit the observation of thick sections, whole cells and hydrated specimens. Most botanical HVEM research has been restricted to the study of thick sectioned material. Various staining techniques have been applied to overcome the decrease in image contrast at high accelerating voltages, but the commonest have been modifications of lead and uranium stains previously developed for thin sections. Selective staining can simplify the mass of information in a thick specimen thus specific structures may be studied against an unstained background. Acidified phosphotungstic acid can be used to stain the plasma membrane and osmium impregnation will selectively stain many of the cytoplasmic membranes in a variety of specimens. Other techniques for the selective localization of cell components, such as enzyme cytochemistry and autoradiography have yet to be fully exploited by high voltage electron microscopists.Interpretation of the great quantity of information in a thick specimen can be facilitated by tilting the specimen and producing stereo pairs. Quantitative depth information can be extracted from stereo pairs by the use of measuring mirror stereoscopes or by direct measurement from each member of a stereo pair. Serial thick sectioning has been employed as an alternative to prolonged serial thin sectioning to aid in the reconstruction of large specimens.Stereo images can be viewed in a variety of ways with lenticular pocket stereoscopes, reflecting mirror stereoscopes, prismatic spectacles, polarized spectacles when projected onto a non depolarizing screen or presented on TV monitors.     

Viruses and the development of quantitative biological electron microscopy

The electron microscope has become an important tool for determining the structure of biological materials of all kinds. Many technical advances in specimen preparation and in sophisticated methods of image analysis, initially based on optical systems but latterly on computer processing, have contributed to the development of the subject. Viruses of various kinds have often provided a convenient and appropriate test specimen. This paper describes the major technical advances and shows how viruses have had an important role in most of the developments.     

Resolution as a function of accelerating voltage in electron microscopy of semithick biological specimens

In the past, biological sections ranging in thickness from 0.10- to 0.50-micron have usually been examined with high-voltage (greater than 500 kV) electron microscopes (HVEM). Now investigators are increasingly using intermediate voltage (200-500 kV) electron microscopes (IVEM), which are more readily available and demand less maintenance. In a study of "typical" plastic-embedded, stained sections of mouse liver ranging from 0.10 to 1.0 micron thick, we determined the resolution obtainable at 100, 200, and 1000 kV. At all three accelerating voltages the resolution (2.7 nm) for 0.10-micron sections was limited only by the sections stain granularity. For 0.25-micron thickness the resolutions were 5.8, 3.1, and 3.1 nm at 100, 200, and 1000 kV, respectively. The maximum usable thickness at 200 kV with resolution sufficient to resolve membranes clearly was between 0.75 and 1.0 micron, depending on the magnification. Resolution at 100 kV was adequate for screening sections up to 1.0-micron thick for preparation defects prior to examination with an IVEM or HVEM.     

Devices facilitating the preparation of biological specimens for electron microscopy

The construction of a matrix is described which facilitates the process of placing biological objects into polymer media in order to prepare ultrathin sections without the employment of gelatin, starch and polyethylene capsules that can be used only once. The construction of a reactor for cytochemical assays is presented. The apparatus can be used to locate enzymes within the cell, and to identify microorganisms. A modification of the dialysis technique for microbiological objects is proposed which accelerates and simplifies the process.