Analytical Imaging Technologies
An open access facility, the Analytical Imaging Core Facility is located in the Diabetes Research Institute and contains 1,031 square feet of laboratory, office, and storage space. The core is equipped with state-of-the-art technologies and instrumentation including:
Leica MP-NDD4/SP5/FCS/FLIM multiphoton/confocal upright F-Techniques microscope (MP/SP5)
Includes Coherent Chameleon Ultra II Ti:Sapphire laser with 3.8 Watts peak average power, 4 external non-descanned detectors (NDD4), SP5 spectral confocal scanhead with high resolution and high speed resonant scanners, 405, 458, 476, 488, 496, 514, 561, 594, and 633 nm laser lines, ISS fluorescence correlation spectroscopy (FCS) with two avalanche photodiode detectors, Becker & Hickl time correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM), and three high performance internal photomulitplier tubes (PMTs). The resonant scanner enables 14 frames per second at 512×512 pixels for 2 channels, up to 210 frames per second for 512×16 pixels for 2 channels, or 8000 lines per second. The MP/SP5 is equipped with a full set of water saline immersion dipping lenses. Z-series, timelapse, and spectral series are available. This multiphoton microscope was one of the first in the world with Leica’s NDD4 non-descanned detector module for deep tissue imaging by multiphoton excitation fluorescence microscopy. The NDD4 module features simultaneous blue, cyan green and red fluorescence acquisition. The Coherent Chameleon Ultra II Ti: Sapphire multiphoton laser has 3.8 Watts peak average power, enabling deep tissue imaging.
Leica SP5 spectral confocal inverted microscope (SP5 inverted)
Includes motorized stage, standard and high resolution Z-focus, SP5 spectral confocal scanhead with high resolution and high speed resonant scanners, 405, 458, 476, 488, 496, 514, 561, 594, and 633 nm laser lines. The resonant scanner enables 14 frames per second at 512×512 pixels for 2 channels, up to 210 frames per second for 512×16 pixels for 2 channels, or 8000 lines per second. The SP5 is equipped with a full set of oil immersion lenses. Z-series, timelapse, and spectral series are available. The high speed Z-galvonometer enables high speed XZ scans (200 um range), standard Leica DMI6000 Z-drive for large distance Z-series (limited by the opacity of your specimen), automated high resolution scanning stage with stage tiling and mark & find.
BD Pathway Bioimager High Content Screening System
The Pathway is a high content screening system that is optimized for high throughput, high speed, live cell physiology with 96-well and 384-well plates. The Pathway includes 2 mercury arc lamps, each with its own excitation filter wheel, a high speed excitation wavelength switcher, an optional spinning disk confocal attachment for optical sectioning, and an emission filter wheel. The Pathway is able to spritz a reagent into a well during imaging. A typical experiment is to image Fura-2 using high speed 340/380 nm excitation ratioing, and to add a reagent while scanning at high speed. The Pathway uses a Hamamatsu ORCA-ER camera, which enables optimizing spatial and temporal resolution by adjusting the binning settings. The Pathway can also scan microscope slides.
Zeiss ApoTome Axiovert 200M
In the area of fluorescence microscopy (widefield), the Zeiss ApoTome is an innovative slider module that provides improved image quality and enhanced contrast without using lasers. The automated microscope and Hamamatsu ORCA-ER Firewire digital camera are operated by the Zeiss AxioVision software for image capturing, analysis, and deconvolution. This is compatible with several image formats acquired with other instruments available through the core. Advantages of the ApoTome include:
- Digital optical section images using the same filter sets as viewed by eye
- Images free of artifacts, out-of-focus information is removed
- Improved signal-to-noise ratio
- Improves resolution in axial (Z) direction
- 3D visualizations of specimens
- Deconvolution of image stacks
The microscope images can be acquired in standard widefield fluorescence or in ApoTome optical sectioning mode. The ORCA-ER camera also can be operated from MetaMorph for high speed image capture with simultaneous graphing of regions of interest.
Leica AS LMD laser microdissection
Laser capture microdissection using the Leica AS LMD allows investigators to obtain essentially pure samples of a desired cell population from tissue sections for phenotypic analysis of dissected cells in their native state. The dissected cells can then be used to extract RNA, DNA, or proteins for further studies. Thus, LCM offers unprecedented access to specific cells, including cancer cells, for defining their pattern of gene expression, in combination with powerful techniques such as gene array and real-time PCR. The microscope has a motorized stage and camera that enables “specimen overview” images of entire tissue sections.
Leica DMIRB Inverted Microscope
Standard widefield fluorescence and brightfield “pathology” microscopy can be performed using the Leica DMIRB inverted microscope equipped to perform triple fluorescence, phase contrast and histology/immunohistochemistry light microscopy.
We have upgraded our Leica DMIRB inverted microscope to a new Leica DFC495 color digital camera, Leica LAS image acquisition software, HP30w 2560×1600 pixel computer monitor and new Dell PC with fast RevoDrive solid state drive (SSD) PCI-Express card. The new DFC495 camera has much better color rendering than the old MicroPublisher RTV5 camera. The LAS software has an excellent stitching command.
MetaMorph Imaging System
The MetaMorph Imaging System (MIS) consists of hardware and software that enables the capture and analysis of microscopy or digital images obtained through the use of the instruments described above. MetaMorph offers more sophisticated capturing and analytical capabilities than the software provided with those instruments. The MIS can be used for live cell imaging, multi-label fluorescence, confocal microscopy, motion analysis, co-localization studies, FISH, FRET, FRAP, and live/dead cell assays. Most image analysis is done with MetaMorph, and the core can help users on projects involving Adobe Photoshop, NIH ImageJ, and additional software. Dr. McNamara is a MetaMorph expert, having used and supported it since 1992 when he was applications scientist for the manufacturer.
Pathscan4 microscope slide scanner
The Pathscan4 is the latest generation microscope slide scanner from Meyer Instruments. The Pathscan4 scans at 7,200 dpi (3.6 um pixel size) and can scan an entire brightfield histology or immunohistochemistry microscope slide in 1 minute. Users have the option of using the Quikscan software or LaserSoft SilverFast Ai.
Epson V750-M flatbed scanner
The Epson V750-M is the latest generation flatbed scanner. The V750-M features an integrated full lid transparency adapter. Users have the option of using the EpsonScan software, LaserSoft SilverFast Ai, or Hamrick VueScan Pro. The AICF has a complete set of optical density calibration standards for the V750-M.
CompuCyte iCys Imaging Laser Scanning Cytometer
The CompuCyte iCys Laser Scanning Cytometer (LSC) allows “flow cytometer-like” fluorescent histograms and scatterplots, gating and quantitation of tissue sections on a microscope slide. The LSC measures multicolor fluorescence and light scatter of cells on slides. Multiple biochemical, immunological, and morphological measurements are made on each cell. In addition to fluorescence to phenotype cells, LSC offers many possible applications, including immunofluorescence with listmode data output, fluorescence in situ hybridization (FISH), detection quantitation of apoptosis (TUNEL, annexin V), the study of cell adhesion, cell cycle, and DNA content, most of which apply to cell biology applied to many diseases, including cancer.
The Analytical Imaging Core Facility retired our Zeiss LSM510 confocal microscope scanhead in December 2011 after 8+ years of heavy use. We have a Hamamatsu ORCA-ER CCD camera for the Axiovert 200M fluorescence microscope and this instrument was converted into a widefield microscope in 2012.