Applications for Cryo FIB-SEM
Courtesy of R. Niessner, N. Ivleva, M. Seidel, A. Kunze, Institute of Hydrochemistry, Chair for Analytical Chemistry, TU Muenchen, Germany.
Overview

Applications for Cryo FIB-SEM

Explore inspiring examples

Using FIB-SEM, samples are imaged with the electron beam of the SEM before a focused ion beam mills away as little as 3-10 nm. The sample is then imaged again and the process is repeated to build up a high resolution view of the complete 3D volume. Cryogenic FIB-SEM generates these volumes from vitrified samples and enables the capture of 3D ultrastructure in the near native state.

Understanding the Role of Mitochondrial Fission Events in Cancer

Cancer cells exhibit a strong phenotype towards mitochondrial fission, which can potentially explain their resistance to drugs. Understanding how and why this phenotype develops in cancerous cells and how therapeutic methods could combat such a phenotype could assist in the quest for methods to mitigate progression of the disease.

In this application example, Cryo FIB-SEM is used as part of a complete workflow that connects fluorescence imaging with high resolution cryo electron microscopy. The approach ensures comprehensive and precise assessment of mitochondrial fission events.

Plunge-frozen Adenocarcinoma Cells

Plunge-frozen Adenocarcinoma Cells

Plunge-frozen Adenocarcinoma Cells

Plunge-frozen adenocarcinoma cells

Plunge-frozen adenocarcinoma cells

Using the combined information that can be gained from fluorescence imaging and ultrastructural imaging it is possible to visualize the mitochondrial network and confirm that in this adenocarcinoma cell line there is an increased incidence of mitochondrial fission.

This correlative dataset includes both fluorescent and structural information from the same cells, displayed in context using ZEN Connect. The cells were grown on sapphire discs and then plunge-frozen. The fluorescence data was captured using ZEISS LSM 900 with the cryo stage (the red is actin-mCherry and the green is Mitotracker (mitochondria)) and the SEM data was captured with ZEISS Crossbeam.

By preparing specimens using cryo fixation, artefacts that can be caused by chemical fixation, such as accumulation of mitochondria, are avoided and the sample is preserved in the near-to-native state.

Plunge-frozen Adenocarcinoma Cells

Plunge-frozen Adenocarcinoma Cells

Plunge-frozen Adenocarcinoma Cells

Plunge-frozen adenocarcinoma cells

Plunge-frozen adenocarcinoma cells

When planning experiments involving multiple imaging platforms such as Cryo FIB-SEM and confocal microscopy, the ability to move easily from one technology to the next and find exactly the same location for image acquisition significantly increases the efficiency of the workflow. In this example, a deeper understanding of the adenocariconoma cells could be reached because of the seamless combination of fluorescence insights (mitotracker) from ZEISS LSM 900 and mitochondrial structural information from the cryo Crossbeam.

The sample was cryogenically preserved and imaged at cryo temperatures, both on ZEISS LSM 900 and in ZEISS Crossbeam. The ease of the workflow makes this kind of approach attainable even for a less experienced user of the different technologies.

Understanding Cellular Structure at the Ultrastructural Level

The process and extent of axon myelination is a great importance to the efficient functioning of the nervous system. Monitoring changes to myelin thickness in disease or under different conditions can assist in understanding the processes of myelination and demyelination. This in turn can lead to the development of therapeutic approaches for treating conditions where myelination has been disrupted.

High resolution visualization of myelin and axon structure is vital for quantitative assessment of myelin thickness. Using cryogenic fixation, the native state of the cellular structure is maintained and with high contrast acquisition using cryo FIB-SEM it is possible to precisely measure myelin thickness in 3D without needing to add any heavy metal contrast agents.

High Pressure Frozen Mouse Optic Nerve at 14 Days

High Pressure Frozen Mouse Optic Nerve at 14 Days

High Pressure Frozen Mouse Optic Nerve at 14 Days

High pressure frozen mouse optic nerve at 14 days (−150°C). Image captured with ZEISS Crossbeam.

High pressure frozen mouse optic nerve at 14 days (−150°C). Image captured with ZEISS Crossbeam.

FIB-SEM can generate these high-resolution, high contrast volumetric insights of cryo preserved specimens without needing the addition of any heavy metal contrast agents.

The 3D dataset from which this single 2D image was taken was captured at −150°C so the sample could be imaged in its near to native state. Using the ZEISS Crossbeam, imaging and milling of the sample was performed simultaneously and this significantly increased the efficiency of the complete acquisition.

Numerous myelinated axons (ax) and the cell body with nucleus (nu) and organelles like mitochondria and Golgi complex are visible. The golgi complex (g) within the process of another cell is also discernible as well as the collagen of the extracellular matrix (em) forming the pia of the nerve.

Exploring the Biomineralization Process of Calcite Crystals in a Coccolithophorid Alga

The ultrastructural environment of soluble, amorphous calcium phases of coccolithophores is difficult to image with the classical, water-based preparation protocols. However, using FIB-SEM operated under cryo conditions, vitrified marine algae can be imaged in the near to native state to elucidate their ultrastructure in 3D.

The ZEISS FIB-SEM enables high-contrast volumetric imaging without heavy metal staining. Using the energy selective backscattered (EsB) electron detector of the ZEISS Crossbeam, 3D images can be acquired that enable the differentiation of mature coccoliths and coccoliths in statu nascendi. At the same time, the in-lens secondary electron images are also able to reveal membrane-bound compartments.

The video shows a 3D visualization of the coccolithophore Emiliania huxleyi. E. huxleyi cells were collected by centrifugation, high-pressure frozen and stored in liquid N2. Throughout the preparation and data acquisition process, the temperature was never higher than −150 °C. The 3D reconstruction shows the mature coccoliths (in yellowish), a coccolith in statu nascendi (blue) and lipid bodies (red).

The sample surface is removed with the focused ion beam, followed by imaging the freshly exposed sample surface with the electron beam. This milling and imaging process is repeated again and again (image resolution: 2048 x 1536 nm).

Understanding the Structure and Function of Bacteria Such as E. Coli

Escherichia coli (E. coli) normally lives in the intestines of healthy people and animals. Usually this bacterium is harmless, however, certain strains can cause medical symptoms such as diarrhea, stomach pain and low-grade fever. Understanding the structure of E.-coli and how that structure responds and changes in the presence of different medical agents can assist in the development of new treatments and preventative approaches.

Electron microscopy of bacteria enables high resolution analysis that helps in understanding their structure and function. This is often difficult to achieve using optical microscopy. Expanding structural analysis to 3D ensures that a complete picture of the bacterium can be generated so no details in the volume are missed. Different approaches are available but many of these require the addition of contrast enhancing agents. This is where cryo FIB-SEM is a vital technology since it provides the capability to image the bacterium in the near to native state without the addition of contrast agents.

Escherichia coli

Escherichia coli

Escherichia coli

Escherichia coli. Sample: courtesy of R. Niessner, N. Ivleva, M. Seidel, A. Kunze, Institute of Hydrochemistry, Chair for Analytical Chemistry, TU Muenchen, Germany.

Escherichia coli. Sample: courtesy of R. Niessner, N. Ivleva, M. Seidel, A. Kunze, Institute of Hydrochemistry, Chair for Analytical Chemistry, TU Muenchen, Germany.

The native morphology of this E.Coli bacterial specimen was of interest here. The 3D structure of the E. coli can be seen in the image as well as some silver nanoparticles which coat the outside of the bacteria.

The specimen was imaged using cryo FIB-SEM using the Inlens detector.

Escherichia coli

Escherichia coli

Escherichia coli

Escherichia coli. Sample: courtesy of R. Niessner, N. Ivleva, M. Seidel, A. Kunze, Institute of Hydrochemistry, Chair for Analytical Chemistry, TU Muenchen, Germany.

Escherichia coli. Sample: courtesy of R. Niessner, N. Ivleva, M. Seidel, A. Kunze, Institute of Hydrochemistry, Chair for Analytical Chemistry, TU Muenchen, Germany.

The silver nanoparticles are particularly visible in this dataset and when this information is combined with the images taken with the InLens detector, this provides a great deal of insight into the 3D structure and surface of the bacteria. This image was taken with the EsB detector.

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