Description of the Imaging Core
The goal of the Imaging Core is to serve as a bridge to higher end imaging methodologies by allowing researchers access to microscopes and imaging software that enable them to obtain preliminary results and determine if more advanced imaging analysis is needed or warranted. Our user group images a wide variety of specimens ranging from yeast and platelets, cultured neurons and muscle and sections of whole organs. It should be noted that Eclipse E600 and the Axiovert 200M microscopes have been optimized for analysis of small cells such as yeast and platelets where high end microscopy (i.e., confocal) offers only limited benefit. The core also offers researchers an easy-access facility where simple analyses such as evaluation of transfection efficiencies can be monitored and documented. Finally access to a TIRF microscope offers a new horizon of technical capabilities. The recent addition of the Nikon A1R spinning disc confocal has significantly extended our capabilities.
The core currently maintains 4 standard epifluorescence microscopes, one TIRF scope and a spinning disc confocal. Three of the microscopes are housed in BBSRB B116C, designed to be a multi-user imaging room with four workstations and an experimental preparation bench. this facility we have a Nikon Eclipse 600 epifluorescence microscope equipped with 10X, to 100X objectives that is capable of DIC and three-color analysis (Dapi, Texas Red, FITC). A Spot RT SE6 digital camera is attached to the microscope and images are captured with the Spot software and analyzed with MetaMorph imaging software run on a dedicated Dell computer. This microscope is designed for analysis of fixed specimens and small cells, such as platelets and yeast.
At an adjacent workstation is a Zeiss Axiovert 100 inverted epifluoresence microscope equipped with 5x, l0x, 32x, and 40x objectives that is capable of two color analysis (Texas Red, FITC) and phase optics. An Insight 11.2 Color Mosaic digital camera is attached to the microscope and images are captured using Spot imaging software run on a designated Apple computer. This microscope is suitable for live cell analysis, histology sections and other fixed specimens.
In the same room there is also a Zeiss Axiovert 200M epifluorescence microscope with a motorized XYZ Stage (LudI) and a filterwheel housing filters for Dapi, FITC, TRITC and Cy5 as well as a DIC analyzer and has 10x to100x objectives. The system is fully programmable for the XYZ coordinates, filterwheel positions and exposure time with a given set of filters. The microscope is equipped with an Orca ER camera (Hamamtsu, Inc.) which is capable of detection over a broad range of fluorochromes (far red to UV). The microscope system is controlled by a Macintosh computer using Open Lab software. The system is designed for both live cells and fixed specimen imaging. Both the Bioptechs open and closed chamber heated stage as well as an objective heater are also available. Additional capabilities include time lapse and real time imaging of cellular events and deconvolution analysis of images.
The core also maintains an Olympus CKX31 housed in a nearby room, BBSRB B106B. This epifluorescent microscope is designed for everyday tissue culture needs, such as monitoring transfection efficiencies. The scope is capable of two color observation (GFP and RFP) and has 10x and 40x objectives.
The Total Internal Reflection Fluorescence (TIRF) microscope, housed in room B074B, is built on an Olympus 1X71 inverted platform. An Andor iXON multichannel detector is used for image capture and image acquisition and processing software is run on a dedicated Dell computer.
The Nikon A1 hybrid scanning confocal is housed in BBSRB, B074J. This system has 20x, 40x and 60x objectives and four lasers for excitation of DAPI, FITC, TRITC, and Cy5. Transmitted light acquisition is also available with DIC optics. A dedicated Dell computer is used for image acquisition using NIS Elements software. The system has a resonant scanner of high speed imaging of live cellular events and a nonresonant scanner for high resolution imaging. This system is designed for both live and fixed specimens and is capable of real time imaging.
Each computer associate with tehg imaging core has full Internet access as well as access to laser printers and color laser printers. All of the computers have DVD drives and USB ports to back up imaging data. Additional filter cubes are available for the Axioverts including CyFP, YFP and dsRed. We have developed and on-line microscope reservation systems and users are required to check the key to microscope rooms from departmental staff.
Instruction on the use of the microscopes and interpretation of data are offered by Dr. Moncman, the core director, on an individual basis as requested. Some remedial instruction is also available. Those individuals using the core receive instruction in microscope care and maintenance procedures. The core coordinates a series of instructional talks given by vendor representatives to better train users on the capabilities of the imaging software packages.