Equipment
The Morphology and Image Analysis Core is located in lab area 3378 of the Biological Science Research Building (BSRB, see map). The laboratory contains bench space for specimen preparation as well as desk top computers and printers, an adjoining room for dedicated confocal, morphometric (MetaMorph), and Fluorescence Resonance Energy Transfer (FRET) microscopy. In addition, the Core has access to shared space including autoclave and dish washing facilities. Tissue culture incubators are present for short and long term storage of cells under a controlled environment.
The Core's basic aims are to provide service, access to specialized state-of-the-art instrumentation, education and training in morphological techniques.
Major Systems:
- Olympus FluoView 500 Laser Scanning Confocal Microscope (Reserve Time)
- Multi-wavelength Widefield Imaging System (Reserve Time)
- Live Cell Perfusion Chamber System
- High Resolution Image Processing Workstation
- MetaMorph Offline Workstation (Reserve Time)
Support Systems:
- CSMZ-U Zoom 1:10 Light Microscope
- Kodak Digital Science 8650 PS and Xerox Phaser 8200DPN Printers
- Ancillary Equipment
Additional Offsite Equipment:
The Olympus FluoView 500 Laser Scanning Confocal Microscope LSCM
The FV500 is controlled by a 2.4 Ghz personal computer under Windows XP and is capable of imaging 5 separate channels simultaneously (4 fluorescence + 1 transmitted light photomultiplier detectors) offering highly efficient, maximum emission sensitivity and the ability to record scanned images in 12 bits or 4096 gray levels, thus allowing quantitative linear measurement of fluorescence within regions of low contrast as well as very high contrast. Users are able to image a wide variety of fluorophores with laser excitation that includes Blue Violet (405nm) Multi-Line Argon Blue (458,488,515nm), Helium Neon Green (543nm) and Helium Neon Red (633nm) for standard Blue, Green, Red and Far-Red fluorochromes. The FV500’s acoustical optical tuning filter (AOTF) and adjustable scan speeds provides for minimal specimen fading, sequential scanning for reduced fluorescence cross talk, multiple regions of excitation, high resolution imaging (up to 2048 x 2048 pixels) of fixed or static samples, and rapid recording of kinetic events.
Optical sections in the z plane can be collected using a stop motor attached to the fine focus control of the microscope and driven by Fluoview software. The system is also equipped with Differential Interference Contrast (DIC) objectives and condensers and has the ability to capture transmitted light images with a highly sensitive photomultiplier (PMT) transmission detector.
Images can be saved to a peripheral hard drive for later analysis. Integral software allows for analysis of saved images in 2 dimensions ( e.g. brightness vs time ); confocal images obtained in a " z " series can be volume rendered and analyzed in 3 dimensions. Data can be archived on CD-R, CD-RW, DVD-R, DVD-RW or transferred to an alternate image analysis platform with greater storage capacity.
Click here to see confocal images taken in the MIAC
Multiwavelength Widefield Imaging System
The MIAC has a multiwavelength widefield imaging system based on an inverted Nikon Diaphot 200 fluorescent microscope. Standard blue, green and red fluorescent signals are visualized with Chroma filter cubes. Users have the option to use either Compix SimplePCI or MetaMorph (version 6.1r3, Molecular Devices) software to capture digital images with a Hamamatsu ORCA extended range digital CCD camera (C4742-95-12ER). The system is controlled by a Dell 2.4 GHz Pentium IV personal computer with 768 MB of RAM, Matrox Meteor II digital PCI frame grabber, and an 80 GB hard drive running Windows 2000 operating system. Images and data can be exported in convenient formats including tiffs and Microsoft Excel spread sheets and archived on an internal CD-RW drive.
This microscope is used as our state-of-the-art fluorescence resonance energy transfer (FRET) system. This type of microscopy enables members of the MDRTC to study a wide variety of biological events that influence the interaction between molecules. FRET technology involves the non-radiative transfer of energy from a fluorophore in an excited state to a nearby acceptor fluorophore. FRET has grown in popularity due to the emergence of GFP mutants with blue or yellow-shifted spectral properties. We have developed our system to take advantage of the fluorescent characteristics of enhanced cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). FRET microscopy relies on the ability to capture weak and transient fluorescent signals efficiently and rapidly from the interactions of labeled molecules in live samples. This necessitates the use of a digital camera that can perform under these stringent conditions. Our FRET system is equipped with a Hamamatsu ORCA extended range digital CCD camera (C4742-95-12ER) that can rapidly capture images at rates ranging from 8.3 – 45 frames per second with very high quantum efficiency resulting in shorter exposures of sensitive samples to fluorescent light. Thus, the ORCA digital camera allows us to get the maximum performance and utility from FRET microscopy. The acquisition and analysis of FRET data is semi-automated with the use of specialized Metamorph macros/journals developed by Dr. Joel Swanson, our FRET consultant. The customized macros also allow users the ability to perform stoichiometric FRET analysis.
This microscope is equipped with additional specialized excitation and emission filters to allow users to capture fluorescence based images of transfected green fluorescent protein chimeric molecules. There are also filters in the automated Sutter filter wheels to measure intracellular ion concentration in living cells. This replaces the Zeiss-Attofluor Fluorescence Digital Imaging System used previously for measurements of intracellular calcium.
Click here to see FRET images taken in the MIAC
Live Cell Perfusion Chamber System
The Core has a versatile live cell perfusion system from Warner Instruments Company (Series 20 PH-2 heated platform) with the option of having either a closed (RC-21BR) or open (RC-21BDW) perfusion chamber. Temperature is controlled automatically with a dual channel controller (TC-344B) that directly connects to the platform to monitor and regulate bath and solution temperature. Another available option is for users to grow cells in 35 mm dishes and connect to the temperature controller via a quick exchange platform (QE-1). Our component based system can be configured to work with either the Core’s Olympus confocal or Nikon wide-field fluorescent microscopes. They are interested in using a chamber system that will accommodate 35 mm dishes as well as specialized dishes.
High Resolution Image Processing Workstation
The Core has a high-end imaging workstation based on Windows XP Professional 64-bit operating system. This computer features two 64-bit 3.6 GHz Xeon processors, 8 GBs of RAM, and a GeForce 7800 GTX PCIe 512MB video card. This computer supports the use of deconvolution and 3D volumetric analysis software. The Core has AutoQuant software that specializes in image restoration and includes a deconvolution module for producing high resolution images by using cutting-edge algorithms to perform image restoration by removing out-of-focus haze, blur, noise and other problems from both 3D and 2D images. AutoQuant also includes alignment software that will correct for any drifts or shifts in images that are acquired in time or z-series data sets. Core users also have access to Volocity (Improvision, Lexington, MA) which is state-of-the-art multidimensional software to process and analyze z-series of high resolution images. The Iterative Restoration module eliminates noise and blur from the original confocal images for improvement in X, Y and Z resolution. The Visualization module provides high quality rendering in order to examine and rotate the 3D reconstructed images in real time with the detail and clarity necessary for working with volumetric data. Volocity Classification module is vital for identifying, classifying and measuring the morphological changes.
CSMZ-U Zoom 1:10 Light Microscope
The SMZ-U Zoom 1:10 is a parallel beam path stereo microscope with high resolution and wide zoom range, that offers high quality images by maintaining the natural shape and color of the specimen at any magnification. This instrument is equipped with a Sony AVC-D7 CCD Video Camera and PVM-135 Black and White Video Monitor.
Kodak Digital Science 8650 PS and Xerox Phaser 8200DPN Printers
The Core is equipped with Kodak Digital Science 8650 PS and Xerox Phaser 8200DPN Printers. The Kodak Digital Science PS Printer allows the user to switch between raster and POSTSCRIPT Language modes. It uses dye sublimation technology that allows for the printing of photographic-quality images, rich overhead transparencies, and page-size RGB proofs.
The Xerox Phaser 8200DPN color printer that allows color prints for documentation and presentation. Both printers are networked and accessible from all computer workstations.
Images can be analyzed and prepared for presentation using either PC-Windows or Macintosh workstations. Software includes Adobe Creative Suites and Microsoft Office. All workstations are networked and have CD-RW or DVD-RW drives.
Click here to see images analyzed with MetaMorph software in the MIAC
Other equipment includes an aging Reichert-Jung (2800 Frigocut) cryostat, a RMC MT-7 Ultramicrotome with a CR-21 Cryosectioning Attachment, and a Reichert-Jung KNIFEMAKER II. A Nikon Labphot2A microscope is available for viewing toluidine blue stained sections. The Core is also equipped with shakers, water baths, refrigerator, pH meter, tissue culture hood and incubators, centrifuges and other equipment.
Phillips CM-100 Transmission Electron Microscopy (TEM)
The Phillips CM-100 Transmission Electron Microscopy (TEM) is maintained and operated in the Microscopy and Image-analysis Laboratory (MIL, located in BSRB), part of the Department of Cell and Developmental Biology. The Phillips CM-100 is equipped with a motorized stage and a Kodak 1.6 megaplus digital camera capable of capturing electron images directly from the viewing screen. This microscope is used for the collection of digital EM images and for the direct transfer of data from the microscope to the image analysis software reducing the need for photographic film, chemicals, and paper. Use of this equipment is available to University of Michigan investigators on a recharge basis and the MIAC will cover half of this cost for approved projects. Routine film based imaging is also available for publication purposes for TEM film.
Click here to see Transmission Electron Microscopy (TEM) images taken in the MIAC
