HR Excellence in Science

Possible imaging techniques

 

Preparation and imaging of the biological specimens in TEM

Standard TEM sample preparation

Samples are prepared by HPF/FS method (high-pressure freezing, freeze substitution, infiltration, and epoxy resin embedding; methodology: s41598-020-74033-9) or by chemical method (glutaraldehyde fixation, OsO4 postfixation, dehydration, and epoxy resin embedding). Prior to the imaging, the specimens are ultrathin sectioned on an ultramicrotome and contrasted.

Replication of TBEV in the endoplasmic reticulum

 

Sample preparation for immunolocalization

Immunogold localization of proteins are done on the ultrathin sections - Tokuyasu method (methodology: DOI: 10.1016/j.cub.2018.09.008; or DOI: 10.1038/s41598-019-43284-6) or on the acrylic resins sections (LR White and Lowicryl K4M) after UV polymerization at low temperatures in an AFS unit. Immunogold localization of adhered specimens is possible (negative staining). Multiple localization of proteins is possible as well (DOI:10.1073/pnas.1909755117 a DOI: 10.1016/j.nano.2015.09.008)

 

 

Immunolocalization of ATPases in mitochondria of Trypanosoma

 

 

Electron tomography (ET)

ET is a method of 3D reconstruction done on contrasted sections with a maximal thickness of 200 nm with resolution up to ~ 1 nm. Reconstructed area is related to the magnification of TEM, going from 10x10 µm @ px=2,8 nm, up to 0,5×0,5 µm @ px=0,14 nm (DOI:10.1038/srep10745, DOI:10.1007/s00436-018-6001-9) . Reconstructed volume can be increased by stitching/connecting the tomograms in all axes (DOI:10.1016/j.cub.2018.09.008).

Left: Replication of TBEV in the endoplasmic reticulum, right: IRIDOVIRUS

 

SPA - single particle analysis

This method processes a big number of differently oriented particles of the studied macromolecule imaged by the TEM. These data are then used to reconstruct the structure of the studied macromolecule in 2D (RT-EM, negative staining) and in 3D (cryo-EM, plunge freezing) Cryo-EM, combined with the direct detector may reach in the reconstructions up to atomic resolution.

 

 

P4   

 

Cryo-ET

Cryo-ET is a method that allows the 3D reconstruction of native specimens immobilized in the amorphous ice with resolution ~ 4 nm  (depends on the radiative sensitivity of the sample). The simplest way of sample preparation is by plunge freezing method in
liquid ethane of the TEM grid covered with a thin layer of sample
suspension. Another method of preparation is by utilizing sectioning of
a bulk frozen sample at -140 °C (DOI:10.1016/j.ijpara.2020.09.010).

 

Extracellular vesicle excreted by the tapeworm Hymenolepis diminuta

 

 

Sub-tomogram averaging (STA)

STA is a computing method that allows an increase of resolution of ET and cryo-ET to decimals of nanometers. It can be applied to shape-similar objects, such as ribosomes, nuclear pores, and viruses.

 

 

 

STA analysis of chromosome lamellae formed from photosynthetic pigments

 

 

Preparation and imaging of biological specimens in SEM

Standard SEM sample preparation

The standard method of preparation includes glutaraldehyde fixation, OsO4 postfixation, dehydration, critical point drying, and sputter coating with metal (such as Au or Pt) (DOI:10.1016/j.protis.2010.02.004).

 

 

Nit on the hair

 

Cryo-SEM

This method serves for the imaging of surfaces and fractures of specimens vitrified with the HPF, plunge freezer in liquid ethane, or a nitrogen slush. Freeze fracturing, sublimation of ice,  and sputter coating are done in the chamber of CryoALTO 2500 (DOI: 10.1007/s10493-019-00442-9)

 

Trichome on the leaf

 

                        

SBF-SEM - Serial Block Face-Scanning Electron Microscopy is a method of volume SEM utilizing ultramicrotome mounted inside of the EM chamber that cuts off an ultrathin section from the sample (a pyramid approximately 200x200 µm) surface. The surface is scanned with an electron beam, detected by the backscatter electron detector. The resolution of the method is  6-15 nm in the X/Y-plane and 40-100 nm in the Z-axis. The set of images obtained is then processed via various software (MAPS, Amira, ImageJ, MIB, etc...) and can be segmented (Amira, MIB) for the precise measurements in nm/nm3 scale and a 3D model of the desired structure can be created.

Trypanosoma brucei, 3D model consisting of distinct organelles and cell parts

 

Array tomography

A method of the volume SEM utilizing scanning of serial sections deposited on glass or EM wafer. Obtained data are processed with the Amira software and a 3D model can be prepared from them (DOI:10.1038/s41598-019-56811-2).

Array tomography video.

 

Data processing

Our laboratory offers possibility of pre-processing, alignments of tomograms, subtomogram averaging, segmentation and 3D visualizations using AMIRA, MIB, IMOD, ImageJ of here acquired data.

 

CONTACT

Biology Centre CAS
Branišovská 1160/31
370 05 České Budějovice
Data box: r84nds8

 

+420 387 775 111 (switchboard)
+420 387 775 051 (secretariat)
+420 778 468 552 (for media)

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