Vermont Cancer Center
Microscopy Imaging Center
Douglas J. Taatjes, Ph.D.
Michele von Turkovich
Douglas J. Taatjes, Ph.D.
Microscopy Imaging Center
University of Vermont
Health Science Research Facility
89 Beaumont Avenue, Room 203
Burlington, VT 05405
203 Health Science Research Facility
The Microscopy Imaging Center (MIC) is a College of Medicine Core Facility designed as a multi-user resource for the collection and analysis of biological images for clinical and research applications. The MIC consists of ten microscopy-based imaging systems. At the light and electron microscopic levels, it provides state of the art, quality assured, morphologically oriented services. The MIC is operated on a fee for service basis and provides professional consultation and assistance with equipment use, experimental design and interpretation.
The Microscopy Imaging Center provides the following services and equipment to projects directed toward elucidation of determinants of carcinogenesis. The facility is staffed by experienced laboratory technologists and research professionals who can provide guidance with experimental design, tissue sectioning, sample preparation, data analysis and interpretation, and guidance as to optimal or appropriate use of the instrumentation available in the facility.
- Morphologic services and consultation at the light and electron microscopy level
- Light and electron microscopic immunocytochemistry
- Confocal scanning laser microscopy
- Laser scanning cytometry
- Atomic force microscopy
- Scanning and transmission electron microscopy
- Laser capture microdissection
- Super resolution microscopy
- Preparation of paraffin and frozen sections
- Whole animal imaging
- Electric Cell Substrate Impedance Sensing
- Image analysis and processing
- Training for use of the above equipment
- Special histological staining
- Testing of new antibodies and developing new staining techniques
The following equipment is available in the Microscopy Imaging Center:
JEOL 1400 Transmission Electron Microscope - The JEOL 1400 Transmission Electron Microscope is a high-contrast, high-resolution instrument utilizing a LaB6 filament, and capable of operating at accelerating voltages up to 120 kV. It incorporates a newly designed electron optics system which is optimized for integrated use with a digital ccd camera imaging device. Image acquisition provided by an AMT XR611 high resolution 11 megapixel mid-mount ccd camera. This camera is positioned near the normal film plane of the TEM affording a wide field of view for image capture.
JEOL 6060 Scanning Electron Microscope - The JEOL 6060 scanning electron microscope for investigations of bulk specimens. The JSM-6060 is a high-performance, compact, scanning electron microscope with excellent SE and BSE resolution. Standard automated features include auto focus, auto gun (Bias), auto stig, auto saturation, and automatic contrast and brightness. Images are captured in digital format, ready for computer-assisted processing and analysis. The JEOL 6060 SEM also includes an Energy Dispersive X-ray Microanalysis System from Oxford Instruments (High Wycombe, UK). Oxford’s INCA Energy 350 software package (Windows XP TM platform) allows users to perform element analysis of points or line scans, as well as larger area element and phase mapping with image drift correction. Both qualitative and quantitative data can be attained.
The Olympus BX50 Light Microscope is an upright microscope with 4X, 10X, 20X, 40X (with collar correction for coverslip thickness), and optional 60X (oil) and 100X (oil) objectives. It is fitted with Mercury epi-fluorescence and Halogen direct illumination systems and binocular infinity optics eye pieces. The microscope is equipped with filter blocks to visualize 488 (excites blue, emits green, such as FITC), 568 (excites green, emits red, such as Rhodamine), with a multi filter cube which provides 488, 568 and UV light (used for DAPI). The blank setting is used for UV and bright field. Visit Chroma.com for further information.
MFP-3D-BIOTM Atomic Force Microscope By Asylum Research - The MFP-3D-BIOTM is mounted on an Olympus IX71 inverted microscope that resides on a Herzan AVI 350-S Active Vibration Isolation Table which dynamically dampens the most extreme vibrational disturbances; and the system is totally enclosed in an air tight state-of-the-art BCH-45 Large Acoustic Isolation Hood with air temperature control capabilities. The closed looped system allows investigation of dry or hydrated biological/materials surface features at very high resolution to include dynamic 3D visual analysis, force curves, optical (fluorescence and phase) image overlays precisely registered over AFM topographical information; plus advanced modes such as nanomanipulation(s), nanoindentation(s), Dual AC and more. The system software modules are written in Igor Pro, an integrated program by Wavemetrics which provides very advanced features for visualizing, analyzing, transforming and presenting experimental data.
CompuCyte Laser Scanning Cytometer. This system is a hybrid instrument between a flow cytometer and a microscope-based imaging system. It consists of an Olympus BX50 upright light microscope attached to a laser scanning system. The system is equipped with two lasers: an argon ion (approximately 488 nm) and a helium/neon (approximately 633 nm), and three filter block/photomultiplier tube combinations allowing for simultaneous imaging of up to three fluorophores, or two fluorophores and forward scatter. Software for analysis and data processing are part of the system, as well as a color ccd camera for image capture.
Arcturus XT-Ti Laser Capture Microdissector - Arcturus (Life Technologies) XT-Ti Laser Capture Microdissector system. This system is used for specifically removing desired cells from a tissue section for subsequent molecular analysis. Both gentle infrared cell capture and rapid ultraviolet laser cutting are included on this one instrument. Cells are captured automatically using an interactive pen-display monitor and trackball-actuated stage. The microdissector is integrated onto a Nikon Eclipse-Ti inverted microscope, equipped with 2X, 10X, and 40X objective lenses. Fluorescence illumination is accomplished with a Lumen Dynamics X-Cite lamp module with liquid guide. Fluorescence cubes for uv excitation (325-375nm Ex/420 Em), blue excitation (450-490nm Ex/515nm Em), green excitation (510-560 nm Ex/590nm Em), and red excitation (590-650nm Ex/663-738nm Em) provide the ability to microdissect and capture fluorescently labeled cells within tissues. A second ccd camera has been added to a side port on the microscope for high resolution acquisition of brightfield and fluorescence images. Tissue sections can be microdissected from plain glass slides, PEN membrane coated slides, and Framed, coated slides.
Zeiss LSM 510 META Confocal Laser Scanning Imaging System - Zeiss LSM 510 META confocal scanning laser microscope. This is a state-of-the-art multi-spectral imaging system, consisting of a confocal scanning system attached to an Axiovert 200M inverted light microscope. A combination of diode and argon lasers provide excitation lines of 405 nm, 458 nm, 477 nm, 488 nm, 514 nm, 543 nm and 633 nm. The system incorporates two conventional detectors with the META spectral detector, allowing for the visualization of up to eight fluorophores per specimen. An environmental chamber attached to the microscope stage provides for the control of temperature, gas and humidity for long duration live-cell experiments. Software packages for physiology experiments and 3-D reconstruction of image sets are also part of the system.
Electric Cell-substrate Impedance Sensing - An ECIS system (from Applied BioPhysics, Inc. Troy, NY) measures impedance, resistance, and capacitance in cultured cells grown on specially-fabricated chamber slides fitted with gold film-coated microelectrode. The cell electrical parameters are measured at various frequencies from 64-64,000 Hz, and indicate barrier function (adherence of cells to each other through junctional complexes), and adherence of cells to the substrate (capacitance). This system provides a continuous temporal assay of the physiologic state of cultured cells. The effect of the addition of chemical compounds, pharmaceuticals, or other reagents on cell physiology and morphology can then be monitored. The ECIS system in the MIC is housed adjacent to an Olympus IX70 inverted microscope, for convenient visual assessment of the cells.
IVIS Whole Animal Imager - Caliper LifeSciences IVIS Lumina II Whole Animal In Vivo Imaging System is used to image quantitative fluorescent and bioluminescent probes in live mice. The system consists of a light-tight animal imaging chamber with integrated gas anesthesia capabilities, a high-resolution back-thinned CCD camera, and computer controlled automation and analysis capabilities. The field of view can be adjusted to image up to three mice simultaneously, or zoomed in for higher resolution partial body imaging. The system is housed in the Animal Care Facility in the basement of the Health Science Research Facility (two floors directly below the MIC).
Nikon STORM Super-Resolution Microscope - Nikon N-STORM super-resolution microscope system consisting of a Nikon Ti-E TIRF inverted microscope base with laser modules delivering excitation at 405 nm, 488 nm, 561 nm, and 647 nm, and a high sensitivity Andor iXON3 DU897 EMCCD camera. This instrument provides resolution in the fluorescence mode of 20 nm lateral and 50 nm axial. Both fixed and live cell preparations can be imaged, as well as imaging in three dimensions.
Universal Imaging MetaMorph Workstation - The MetaMorph software is on a dedicated computer using a 20 inch flat screen monitor with associated color printer. MetaMorph Offline software (Universal Imaging Corporation, a subsidiary of Molecular Devices) is a high end software package used extensively for image analysis, including colocalization, measurements of areas and distances and object counting and cell tracking with interactive user created regions of interest or automated analysis using an intensity threshold. All measurements are logged onto an Excel spreadsheet for subsequent analysis of data.
PerkinElmer Volocity 3D Rendering Software - Volocity (PerkinElmer Waltham, MA) is a high-performance 3D imaging software package providing visualization, exploration and analysis capabilities of multi-channel 3D volumes over time. The software can be used for 2D, 3D, and 4D imaging, and will accept image sequences from both confocal and wide-field microscopes. The Volocity Visualization module provides an extensive array of visualization and publication features. The Volocity 3D view enables the user to interactively explore a 3D rendered object. The viewing point can be placed either inside or outside of the 3D object, and changes of structures over time monitored. Animated movie volumes can be created with the Movie Sequencer module. The Volocity Classification module will identify, measure and track biological structures in 2D, 3D, and 4D. Co-localization analysis can also be performed for two fluorophores in 3D.
MBF StereoInvestigator Software - StereoInvestigator (MBF Bioscience Williston, VT) image analysis software is designed to count cells, quantify lengths, area, and volumes in biological samples while employing stereological principles. With 13 stereological probes, it will efficiently allow you to obtain precise and unbiased estimates of cell number (Optical Fractionator probe), volume fractions (Cavalieri Estimator probe), or spatial distribution (Nearest Neighbor probe) of tissue components. Results can be exported directly into Microsoft Excel.
Policies & Scheduling
Guidelines of Use
The Microscopy Imaging Center is designed to facilitate investigators in their research endeavors. We will offer training and expertise for using the microscopes and workstations in the facility. Initial training is free. As a multiuser facility, we ask that all users follow these guidelines. If you have equipment questions, please ask the facility staff for assistance.
Safety Policies. Please see the bulletin board in the hallway outside of the imaging rooms for lab safety procedures. Note: For health and safety reasons, as well as to protect the many computer assisted microscopy systems, we do not allow food or drink in the facility.
Working Alone/After Hours Policy. Due to increasing safety standards the Microscopy Imaging Center is no longer allowing tissue processing, incubations or work with chemicals to be done between the hours of 5:00 pm and 7:00 am weekdays or on weekends. The main lab and the cutting room (ultramicrotomes, paraffin microtome and cryostats) will be closed during these hours. The imaging rooms are the only ones available after hours and on weekends. We strongly encourage users doing imaging after hours to take safety precautions. Safety precautions for anyone working alone should be:
1. always notify someone if you are working alone
2. keep a laboratory phone (911 for UVM police services) - or cell phone on you at all times with the UVM police services number - 802-656-3473
3. contact MIC staff (numbers located in lab)
Log Book. All pieces of facility equipment have a log book. All users need to log all computer workstation time, laser scanning time, prints made, film used, etc. for each session. Please continue to use the paper log in for each instrument as well as the MIC Depot post-time notification system until further notice.
Hard Drives. The hard drives must remain free for optimal use. Therefore, we ask that all images be saved to a zip disk, CDR or USB memory stick during collection or transferred at the end of each session and deleted from the hard drive. If you have questions about how to do this, contact a staff person to help you. We have both zip disks and CDRs available for you at cost. The hard drive (C drive) is checked weekly and any images left on this drive are deleted without notice.
The facility's imaging equipment is accessible 24 hours a day, seven days a week. After normal working hours, the facility is accessible through a card swipe system. All equipment has a 15 minute rule which is as follows: If you are signed up to use a piece of equipment and do not show up for the first 15 minutes of your time, another user may take your time slot. If you know you cannot make your appointment, please delete your time from the online schedule or call 656-0813 to cancel and we will free up your time for another user. For those people who would like assistance using the confocal, please contact the facility personnel prior to signing up so that we can coordinate scheduling and have someone available to help you.
Last modified February 13 2013 01:37 PM