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MDRC Morphology and Image Analysis Equipment

The Morphology and Image Analysis Core Core is located in Room 6245 on the 6th floor of the Brehm Center for Diabetes Research, a new clinical and research facility located to the west of the UM Kellogg Eye Center Building ( see map). The Core consists of a suite of interior rooms (6245, 6245A, 6245B and 6245C) and each microscope is housed in an individual room to maximize imaging of fluorescent signals.  The main area of the core consists of a laboratory with 2 benches for specimen preparation, a fume hood and space for two computer workstations and printers. 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

Confocal Microscopes


Epi-Fluorescence Microscope


Live Cell Perfusion Chamber System


High Resolution Image Processing Workstations and Supporting Software


Network Attached Storage (NAS) Server

 

Support Systems

 

Other Specialized Equipment on Medical Campus

 


Confocal Microscopes

Olympus FluoView 500 Laser Scanning Confocal Microscope LSCM


image2The 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 are saved to local secondary 250 GB hard drive and then transferred to a 1.5 TB mirrored RAID-1 network-attached storage (NAS) device that is accessible from core workstations or individual laboratory computers through the UM medical campus 4 Gbps network.  Integral Fluoview 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. Fluoview software saves images in a non-proprietary format to facilitate the use of the raw data files in 3rd party image analysis or visualization software such as ImageJ, MetaMorph, AutoQuant, Volocity and Imaris. 

Strengths of the Olympus FluoView 500 Laser Scanning Confocal Microscope

Click here to see confocal images taken in the MIAC

 


Nikon A1 Confocal Microscope


image4The Nikon A1 confocal combined with an inverted Ti-E microscope will accommodate both fixed and live samples.  It is capable of imaging 5 separate channels (4 fluorescence + 1 transmitted light photomultiplier detectors) to capture signals from blue, green, red and far-red fluorochromes.  The Nikon A1 is a filter based system to direct wavelengths to the PMT detectors and has the following laser lines to excite fluorescent signals, 405 nm laser diode,  457, 488, 514 nm argon laser, 543 nm HeNe, and 640 nm red diode.  The system is controlled through Nikon’s Elements Confocal Microscopy software and is built on a HP computer with an i7 Intel 2.8 GHz CPU, 16 GB RAM running Windows 7 64-bit OS.  All software and data are located on a single 1 TB hard drive and data can be transferred to the core’s 1.5 TB mirrored RAID-1 NAS. 

The modular platform of the A1 system offers the greatest degree of flexibility, allowing for future upgrades to enhance the system.  The Ti-E microscope was upgraded to support live cell imaging.  These upgrades consisted of Nikon’s Perfect Focus System (PFS) to maintain focus on samples by compensating for thermal drifts in the microscope, a Prior motorized stage for tiling large areas and multi-point imaging and a 60x (1.2 NA) water immersion objective.  A specialized stage adaptor was manufactured by the Vision Core’s machine shop to accommodate a Tokai Hit environmental chamber to regulate temperature and CO2 to support live cell imaging.

Strengths of the Nikon A1 Confocal Microscope

 

Leica SP5 Confocal TCS Microscope


The Leica TCS SP5 confocal system is shared instrument between the Diabetes and the Kellogg Eye Centers.  The Leica SP5 system is housed in the Leonard G. Miller Microscopy Suite Room 7054 of the Brehm Tower.  One of the strengths of the system is its AOBS Acousto Optical Beam Splitter (AOBS), a tunable crystal that allows for precise laser line excitation for up to 8 lines simultaneously, supporting excitation with the following laser lines, Laser Lines: 405 nm laser diode, 458, 476, 488, 496, 514 nm argon laser, 543 nm HeNe, 594 nm HeNe and 633 nm HeNe.  The system has four spectral PMT detectors for variable emission wavelength selection and lambda (wavelength) scanning for spectral unmixing. 

The DMI6000 inverted microscope has a scanning stage SuperZ for precise control over Z scans, tiling of large areas and multi-point mark and find.  The Leica Application Suite Advanced Fluorescence software (version 2.5.1.6757) is easy to use and has integrated wizards for programming specialized techniques like FRET, FRAP and photoactivation.  The system is controlled by a HP computer with a Xeon 3 GHz CPU and 2 GB RAM.  The Windows XP professional OS and software are run on a 150 GB hard drive and data are stored on a secondary 1 TB hard drive and then transferred to the core’s 1.5 TB mirrored RAID-1 NAS.  A Tokai Hit environmental chamber was purchased to regulate temperature and CO2 to support live cell imaging.

Strengths of the Leica SP5 Confocal TCS Microscope:

 

Epi-Fluorescence Microscope

Multiwavelength Widefield Imaging System

Description: Nikon Diaphot 200The 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 (version 6.5.2, Hamamatsu), MetaMorph (version 7.7, Molecular Devices) or MetaFluor (version 7.7, 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 MDRC 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.  The system was updated to incorporate specialized fura-2 filters into the automated Sutter filter wheels to give users the ability to measure changes in calcium in live cells.  MetaFluor Ratio Fluorescence Imaging Software (version 7.7, Molecular Devices) was added to the Core’s computer to simplify acquisition and analysis.  This was coordinated with the purchase of an ALA Scientific Instruments profusion system that fits our existing Warner Instruments chambers and can be used with both widefield and confocal microscopes. 

Strengths of the Nikon Diaphot200 Epi-Fluorescent Microscope

Click here to see FRET images taken in the MIAC

 

 

Live Cell Perfusion Chamber System

Description: Live Cell ProfusionThe 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 confocal or 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 Workstations and Supporting Software


Description: IMG_0494.JPGDescription: IMG_0496.JPGThe Core has two high-end imaging workstations based on Windows 64-bit operating systems.  One of these computers features an Intel i7-965 quad core CPU at 3.3 GHz, 12 GB RAM, an EVGA GTX 285 1024MB video card and two hard drives (a 300 GB drive at 10,000 RPM for the 64-bit OS and a 500 GB 7,200 RPM drive for the 32-bit OS). The workstation dual boots to either 64-bit or 32-bit Windows XP professional OS to allow this single computer to support software that does not currently have a 64-bit counterpart such as offline versions of Fluoview and Leica Application Suites Advanced Fluorescence software packages.  The second computer has two 3.6 GHz Xeon processors, 8 GBs of RAM, and a GeForce 7800 GTX PCIe 512MB video card and a 128 GB solid state hard drive running Windows 7 64-bit OS.  This computer has multiple secondary hard drives for data (two 450 GB and 1 750 GB hard drives). 

These computers support the use of image analysis software packages such as for deconvolution and 3D volumetric analysis.  The Core has AutoQuant (version X2.2, Media Cybernetics) 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 (version 5.02 64-bit, Perkin Elmer) 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.  The MIAC has also purchased Imaris software, Bitplane’s cutting-edge 3D and 4D imaging software.  Imaris allows visualization of original data objects in a real time interactive manner so investigators can quickly make visual assessments of their experiments in 3D and 4D to discover relationships that are otherwise hidden.  Its rendering quality, speed, precision and interactivity are unrivalled. With a large variety of segmentation options, Imaris provides investigators with the most effective tools to segment even the toughest datasets to identify, separate, and visualize individual objects and then retrieve a comprehensive array of measurements from the objects.  The software includes the ability to colocalize signals and tracking particles and link to MATLAB (v2011, Mathworks) for specialized or customable analyses.   The core continues to provide access to offline version of MetaMorph (version 7.6.3, Molecular Devices).  This software provides sophisticate morphometric analysis tools and features some very useful semi-automated processes such as cell counting.  These workstations also have additional analysis open source packages installed such as ImageJ (U. S. National Institutes of Health, Bethesda, Maryland), Fuji and Virtual Dub.

 

PC Workstation #1: Windows XP, 32-bit and 64-bit dual Operating System

Specs

Intel i7-965 quad core CPU at 3.3 GHz, 12 GB RAM, an EVGA GTX 285 1024MB video card and two hard drives (a 300 GB drive at 10,000 RPM for the 64-bit OS and a 500 GB 7,200 RPM drive for the 32-bit OS), Dell UltraSharp U2408 24” Monitor 1920x1200

Software

 

PC Workstation #2: Windows 7, 64-bit Operating System

Specs

Two 3.6 GHz Xeon processors, 8 GBs of RAM, a GeForce 7800 GTX PCIe 512MB video card and a 128 GB solid state hard drive running Windows 7 64-bit OS.  This computer has multiple secondary hard drives for data (two 450 GB and 1 750 GB hard drives), Dell UltraSharp U2410 24” Monitor 1920x1200

Software

 

Network Attached Storage (NAS) Server

Image data sets are becoming increasingly larger with the advancement of detectors, computers and storage devices associated with the core’s microscopes.  The MIAC owns a network attached storage (NAS) server to improve the storage and transfer of data collected in the core.  The QNAP TS-259 Pro Turbo NAS is a powerful 2-bay server that has two 1.5 TB hard drives that provide 1.5 TBs of mirrored (RAID1) storage space.  Investigators are able to safely store data acquired on the core’s equipment up to 1 month and then remotely access the server to quickly and efficiently transfer their data to their laboratory computers.  This unit is expandable to accommodate additional external hard drives connected to one of its four USB 2.0 or two eSATA ports.

Specs

Expansion Ports

 

Ancillary Equipment


Description: Printers The Core is equipped with Kodak Digital Science 8650 PS Printer that 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 CMYK proofs.  The Core also has a Xerox ColorQube 8570DN printer that allows color prints for documentation and presentation.  All printers are networked and accessible from all computer workstations. 

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 incubators, centrifuges and other equipment.

Other equipment includes 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.

The MIAC maintains partial interest in a Leica CM1950 Cryostat that is located in 3488 BSRB.  Please contact Dr. Stephen Ernst (e-mail: ernst@umich.edu or Telephone: 734-763-8109) for access and use of the Leica cryostat.

 

 

Other Specialized Equipment on Medical Campus

The core can advise and in some cases assist investigators who wish to use imaging instrumentation outside the core.  Investigators interested in these services should make special arrangements with the MIAC.  Please contact Dr. Stephen Lentz (e-mail: lentzs@umich.edu or Telephone: 734-647-8233) for more information.

 

Leica Multi-photon Confocal Microscopy System in Ophthalmology and Visual Sciences

The Leica Multi-photon Confocal Microscope is maintained and operated in the Department of Ophthalmology and Visual Sciences located in the adjoining Kellogg Eye Center research building next to the Brehm Center. 

Microscope

Objectives

Laser Lines

Detectors

Features

 

Electron Microscopy in the Microscopy and Image Analysis Laboratory

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.  Routine film based imaging is also available for publication purposes for TEM film.