Would you like to learn more about correlative light- and electron- microscopy (CLEM)?
The Electron Microscopy Core from Max Planck Florida Institute for Neuroscience will host this exciting 2-day workshop, providing you with educational lectures by experts in the field and hands-on training that you can adapt to your own projects. Attendees will explore the basics of CLEM applications, and learn practical workflows for how to correlate functional fluorescence imaging to high-resolution EM imaging in the hands-on demonstration using our state-of-the-art equipment.
Workshop Highlights (topics include)
- Presentations by specialists reviewing CLEM technologies and applications
- Casual reception with specialists for Q & A
- Interactive tour of EM Core facilities and brief demo of the latest equipment
- Hands-On Session (2nd Day) for ATUMtome (RMC/Boeckeler), Atlas5 AT with Merlin VP Compact & Gemini SEM300 (Carl Zeiss), and others.
|REGISTRATION FEES||First day lectures only: Free
Lectures and hands-on demonstration (Limited to 12 seats): $30
|CONTACT||Naomi Kamasaw (Head, EM Core Facility) firstname.lastname@example.org|
From Correlative to 3D – Recent Advances in Bio Scanning Electron Microscopy
Kirk J. Czymmek (Director of North American Applications and Labs, Global Coordinator ZEISS Microscopy Labs, Carl Zeiss Microscopy, LLC)
Biological structures, such as organelles, bacteria, somatic cell layers, tissues and model organisms are inherently three-dimensional (3D) and technologies that aid visualization and analysis of 3D biological samples is growing rapidly. Specifically, recent developments in Bio Scanning Electron Microscopy allow high-resolution imaging of samples embedded in resin, then physically sliced and the resulting sections collected in a serial ribbon on tape, glass slide or slotted grid. Each approach has unique benefits and allows access to antibody and other affinity probes for interrogation of specific molecules. Alternatively, 3D tomograms of entire sliced volumes can be automated with serial block face imaging, where an ultramicrotome is either integrated directly into the electron microscope chamber or a focused ion beam (FIB) is used to mill away the specimen. Block face images have significantly lower distortions associated with material removal than the compression seen in thin sections, thus final 3D EM reconstructions from this approach align closely with the 3D LM volume image. Furthermore, a far greater understanding of the structure-function relationship in cells and tissues is now achievable via high resolution correlation of chemical markers and structural components in both 2D and 3D dimension. This presentation will focus on contemporary methods and probes, methods and workflow for array tomography, serial block face imaging, correlative light and electron microscopy (CLEM) and correlative array tomography (CAT) and demonstrate with practical examples how these tools can provide powerful and novel insights into biological problems.
CLEM Applications in Neuroscience
1. Challenges of CLEM applications
Different EM techniques can be applied to correlate fluorescence images of target proteins, organelles or cells. I will introduce three approaches; 1) with the re-emergence of array tomography, (semi-) thin sections of resin embedded samples are used for immuno-fluorescence labeling followed by SEM imaging. 2) To visualize surface structure of cultured neurons that have fluorescence signals in SEM, the use of Ionic liquid allows preservation of fragile processes, protrusions and ruffling like structures without dehydration and drying artifacts. 3) Advancements in freeze-fracture replica Immunogold labeling allow for correlation of functional data obtained by electrophysiology with two-dimensional localization of membrane proteins such as channels and receptors. Our current challenges and trouble shootings will be discussed.
2. ATUM-Atlas workflow using preembedding immuno-EM
The Automated Tape-collecting Ultramicrotome (ATUM)-Atlas workflow combines fluorescence microscopy functional imaging (2-photon, epifluorescence, confocal) with electron microscopy level resolution and allows for advanced investigation of neuronal activity, morphology, and connectivity. After functional imaging is performed either in organotypic cultured slices or in vivo, these brain tissues go through preembedding immuno-EM sample preparation. Ultrathin sections are collected using the ATUMtome and placed on a silicon wafer, allowing larger section size and lower risk when collecting thousands of sections. The SEM is then used to find the target cell, and serial section imaging is semi-automatically performed using Atlas 5 AT software. Using multiple imaging sessions with different magnifications, three-dimensional analysis of the target cells can be performed in relatively larger area of interest of the samples compared to the conventional serial thin-section reconstructions.
Day One - Nov 2
|01:30 - 02:15 pm||From Correlative to 3D – Recent Advances in Bio Scanning Electron Microscopy
Kirk J. Czymmek
|02:20 - 02:40 pm||CLEM Applications in Neuroscience - Challenges of CLEM applications
|02:45 - 03:05 pm||CLEM Applications in Neuroscience - ATUM-Atlas workflow using preembedding immuno-EM
|03:15 - 04:25 pm||EM Core Facility Tour and Demo|
|4:30 pm||Reception for Q&A|
Day Two - Nov 3
|09:00 - 12:00 pm||Hands-On Session 1|
|12:00 - 01:00 pm||Lunch|
|01:00 - 04:00 pm||Hands-On Session 2|