- Presentations by specialists reviewing CLEM technologies and applications
- Interactive tour of EM core facility and introduction of a new Focal Charge Compensation module from ZEISS in a serial block face scanning electron microscope system.
- Partnership Signing Ceremony
- Reception and networking with specialists
Advances in 3D Correlative and Contextual Imaging for Connected and High-Content Microscopy
Kirk J. Czymmek (Vice President, Global ZEISS Microscopy Customer Centers)
All biological specimens are three-dimensional, from organelles to bacteria, somatic cell layers to tissue and model organisms. A greater understanding of the structure-function relationship in cells and tissues is now well within reach through high resolution correlation of chemical markers and structural components in three dimensions. In particular, rapid advances have been made in 3D spatial localization of fluorescent probes with various optical sectioning and super-resolution techniques and provide the powerful ability to specifically label proteins, structures and/or organelles. Likewise, three-dimensional EM reconstructions have traditionally been very labor intensive, comprising mostly of manual collection of the thin sections and manual montaging of large area sequential images. Now, remarkable productivity and automation of EM instrumentation to generate create high resolution 3D tomograms (3D images) are producing considerable amounts of information, previously too difficult or time consuming to collect. This presentation will review various correlative approaches and highlight new to paradigms to “connect” light, electron and X-ray microscopy platforms in a targeted correlative multi-scale approach with special emphasis on ambient and cryo-light to electron microscopy workflows.
Mapping structure-function at synaptic resolution
Benjamin Scholl (Research Fellow, Fitzpatrick Lab) and Connon Thomas (EM Assistant, EM Core Facility)
Understanding how single neurons integrate synaptic inputs from numerous sources to generate somatic responses remains a challenge. Current methods to map the synaptic inputs arriving onto an individual neuron rely either on examining ultrastructural anatomy or recording how individual synapses are activated by specific stimuli. While both approaches are useful in unraveling connectivity underlying the properties of neural circuits, these approaches have not yet been combined and it is unknown how the activity of individual synapses relates to their anatomical connectivity. Here we combined in vivo two-photon calcium imaging and serial block-face scanning electron microscopy (SBFSEM) to correlate ultrastructural and functional properties of the same subcellular features of individual cells. As compromises made during sample preparation to accommodate our correlative methods can affect tissue conductivity during SBFSEM imaging, we implemented Focal Charge Compensation module (FCC) which significantly improves image quality and reduces beam damage. This presentation will highlight our correlative workflow, the challenges we faced, and techniques we implemented to overcome them.
Nanoscale 3-D imaging of cells and tissue by FIB-SEM
Kedar Narayan (Group Leader, Center for Molecular Microscopy NCI/NIH & Frederick National Laboratory for Cancer Research)
Focused ion beam (FIB) technology, already a powerful tool in materials sciences, is now carving a niche in nanoscale-resolution 3-D imaging of biological specimens. Combined with a scanning electron microscope (SEM) and a host of powerful hardware and software advances, a FIB-SEM instrument can now be used to generate near artifact-free and near TEM-resolution 3-D ultrastructural images of stained, resin-embedded biological samples too thick to be easily imaged by conventional EM methods. The FIB can also be used as a sample preparation tool under room temperature or cryogenic conditions to render biological specimens accessible to chemical imaging and cryoTEM approaches. Together, these advances make FIB technology an increasingly significant addition to the imaging toolkit in biology.
At the Center for Molecular Microscopy (CMM) at the National Cancer Institute (NCI) and Frederick National Laboratory for Cancer Research, we visualize cells and tissue in 3-D primarily by FIB-SEM imaging in conjunction with TEM and fluorescence techniques. In partnership with Zeiss Inc and Fibics Inc, we have developed and applied technical advances to image a variety of biological samples, leading to insights into cell biology and pathology. These data have helped confirm hypotheses and also reveal unexpected and new biology; in parallel, we continue to make advances in increasing both the scope and scale of FIB-SEM imaging as well as its integration with other imaging modalities.
Seminar in the Auditorium
|1:00pm||Opening Remarks||Matthias Haury (MPFI)|
|1:05pm||Advances in 3D Correlative and Contextual Imaging for Connected and High-Content Microscopy||Kirk J. Czymmek (Carl Zeiss)|
|1:45pm||EM Facility Technology Review||Naomi Kamasawa (MPFI)|
|2:00pm||Mapping structure-function at synaptic resolution||Ben Scholl & Connon Thomas (MPFI)|
|2:30pm||Nanoscale 3-D imaging of cells and tissue by FIB-SEM||Kedar Narayan (Invited speaker from NCI/NIH)|
Ceremony in Atrium
|3:10pm||Announcement by Scientific Director and CEO of MPFI|
|3:20pm||Announcement by Vice President of Research Microscopy Solutions of Carl Zeiss Microscopy LLC|
|3:40pm||Signing of the partnership|
|4:45pm||Optional Demo for the Focal Charge Compensation Module (FCC) in Electron Microscopy Facility|
Open to faculty, staff, students, and post-doctoral researchers who are interested in utilizing the latest advances in electron microscopy.