 imaging of neuronal mitochondria using a serial block-face scanning electron microscope

 Imaging of Neuronal Mitochondria Using a Serial Block-Face Scanning Electron Microscope

Trim the samples to the area of interest and mount them onto an aluminum pin using gelling cyanoacrylate superglue. Then coat the sample with colloidal silver paste around the sides of the block to provide a conductive path to the aluminum pin. Examine the tissue specimens using a scanning electron microscope system equipped with an in-chamber ultramicrotome stage and a low kilovolt backscattered electron detector.Image the samples with these settings. Begin analysis by selecting image type, then 8-bit to convert the images to 8-bit TIFF format from the original proprietary 16-bit. If automatic contrast and/or brightness conversion during this step is not ideal for images, reopen the 16-bit images and press image. Adjust brightness contrast.Select a range that works for all images and press Apply. Then perform the conversion. If there was unacceptable image movement between slices, align the image stacks by selecting Plug-ins, Registration and linear stack alignment with SIFT and set the alignment for translation only mode rather than rigid body.Enlarge the Canvas size prior to registration by selecting image adjust Canvas size. Alternatively, reduce the image to an area of interest by selecting the desired region and then cropping it. If necessary, scale images to a smaller, more manageable size using image j, then selecting image and scale.

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Synaptic & Cellular Imaging

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Source:&#160;Mukherjee, K.,&#160;et. al. Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.&#160;J. Vis. Exp. (2016)This ...

Take a trimmed, resin-embedded mouse brain tissue section containing the hippocampus.&#160;The section is stained with heavy metals to enhance contrast under an electron microscope.Mount the section onto an aluminum pin using cyanoacrylate glue.&#160;Coat the block sides with colloidal silver paste to create a conductive path to the aluminum pin.&#160;Examine the tissue block using a serial block-face scanning electron microscope (SBFSEM).Within the SBFSEM, the ultramicrotome sequentially slices the block into ultrathin layers while a low-voltage electron beam scans the exposed surface.A backscattered electron detector captures high-resolution 2D images, revealing mitochondria and surrounding cellular structures.&#160;Using appropriate software, adjust the brightness and contrast to improve image clarity.Align the sequential images to reconstruct a 3D volume of the brain tissue. This 3D reconstruction allows precise mapping of mitochondrial morphology, spatial distribution, and their relationships within neuronal compartments.

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Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Mitocondrias y endoplasmática retícula imagen por microscopía electrónica de luz y volumen correlativa

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Investigación

JoVE Journal

Bioquímica

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

Estudios dirigidos mediante la cara de bloque serie y la microscopía electrónica de escaneo de haz de iones enfocado

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

Biología

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

Microscopía Electrónica De Barrido Bloque-Cara Serial (SBF-SEM) De Muestras De Tejido Biológico

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Imaging of Neuronal Mitochondria Using a Serial Block-Face Scanning Electron Microscope

Transcripción

Imaging of Neuronal Mitochondria Using a Serial Block-Face Scanning Electron Microscope