Overview Methods at ZMB

Methods at ZMBOur well-established methods cover subcellular morphology in 2D and 3D, cryo techniques and correlative workflows for detecting proteins in 2D and 3D.Click on the of each method for a short description.For further details contact us:

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Method for imaging biological samples in 3D with isotropic resolution. Often 5x5x5 nm Principle: Sample is fixed chemically or by HPF method, followed by contrasting with heavy metals, dehydration and embedding in resins.The FIB (Focused Ion Beam) is used to ablate sample material, and consecutively the newly exposed surface (typically every 5nm) is imaged using the SEM (Scanning Electron Microscope).

FIB/SEM

Reference:

Knott G et al. J Neurosci. 2008

Method to study the surface topography of the sample. Whole samples can be visualized with simple sample preparation procedures. Principle:Sample is chemically fixed, followed by dehydration, special drying procedure, mounting on SEM stubs and heavy metal coating before imaging in a SEM.

Standard SEM

Method to study the subcellular protein localization. Ideal for cell pellets, small organism as well as tissue samples.Principle: Sample is chemically fixed, followed by cryoprotection in sucrose, freezing and ultracryosectioning.Ultrathin sections are immunostained and imaged with light microscopes before correlating the protein expression with the subcellular structures at the SEM.

Cryosectioning

Reference:Example:

Mateos et al., J. Vis. Exp. (2017):

Gomez-Gonzalez et al. Sci Adv. 2024

Method for imaging biological frozen hydrated samples in their native state. Principle: Sample is vitrified directly on an EM grid and transfer to the cryo-TEM. For single-particle analysis, high-resolution images obtained from different orientations of these particles are computationally processed to reconstruct the 3D structure of the molecule.

Cryo-TEM

Example:

Alvadia et al. Elife 2019

Method for imaging biological frozen hydrated samples in their native state. Principle: Sample is vitrified directly on an EM grid and transferred to the cryo-FIB-SEM for preparing a thin lamella, which is then imaged in cryoTEM.

Electron cryotomography

Review:

Berger et al. Nat Methods 2023

Method for imaging biological samples by light and electron microscopy with emphasis in 3D information Principle: Sample is chemically fixed and imaged by light microscopy.Contrasted with heavy metals, dehydrated and embedded in resins. The FIB (Focused Ion Beam) is used to ablate sample material, and consecutively the newly exposed surface (typically every 5nm) is imaged using the SEM (Scanning Electron Microscope).Correlation of the LM and EM brings the location of proteins and the subcellular morphology of the sample together.

CLEM FIB/SEM

Reference:

Maclachlan et al. Front. Neuroanat. 2018

Method for imaging biological tissue and cells with emphasis in 3D information about subcellular organelles.Principle: Sample is chemically fixed, followed by contrasting with heavy metals, dehydration and embedding in resins. After serial sectioning (50-100 nm) the ribbons are mounted on a silicon wafer and imaged in a SEM.

Array Tomography

Reference:Example:

White & Burden, Methods Cell Biol. 2023

Maheshwari et al. Brain Pathol. 2023

Method for imaging biological samples by light and electron microscopy with emphasis in 3D informationPrinciple: Sample is chemically fixed and imaged by light microscopy. Contrasted with heavy metals, dehydrated and embedded in resins. 3D EM imaging based on serial block-face imaging using an ultramicrotome integrated in the SEM (SBF-SEM).Correlation of the LM and EM brings the location of proteins and the subcellular morphology of the sample together.

CLEM Volumescope

Reference:

Maclachlan et al. Front. Neuroanat. 2018

Method to study the subcellular morphology of the sample. Ideal for monolayer cell cultures, cell pellets, small organism as well as tissue samples.Principle: Sample is chemically fixed, followed by contrasting with heavy metals, dehydration and embedding in resins. After ultrathin sectioning (50-100 nm) the sample, mounted on an EM grid, is imaged in a TEM.

Standard TEM

Example:

Zareba et al. Nat Comm. 2024

3D method for imaging biological tissue and cells based on serial block-face imaging using an ultramicrotome integrated in the SEM (SBF-SEM). Principle: Sample is chemically fixed, followed by contrasting with heavy metals, dehydration and embedding in resins.This slice-and-view method is based on removing the top layer of the sample with a diamond-knife ultramicrotome inside the SEM, followed by consecutive imaging of the block-face with an SEM.

Volumescope

Reference:

Lippens et al., Methods Cell Biol. 2019

Quick and simple method for assessing size and shape of small samples such as proteins, vesicles, isolated organelles, viruses, as well as bacterial flagella and pili.Principle: Sample is contrasted with a heavy metal salt solution, dried and observed in a TEM.

Negative staining

Example:

Gomez-Gonzalez et al., Sci Adv 2024

Method for imaging biological samples by light and electron microscopy with emphasis in 3D information Principle: Sample is chemically fixed and imaged by light microscopy.Contrasted with heavy metals, dehydrated and embedded in resins. Serial sectioned ribbons (50-100 nm) are collected on silicon wafers and imaged in a SEM.Correlation of the LM and EM brings the location of proteins and the subcellular morphology of the sample together.

CLEM Array Tomography

Reference:Reference

White & Burden, Methods Cell Biol. 2023

Maclachlan et al. Front. Neuroanat. 2018