Department of Pathology: Confocal Facility
About:
Our facility currently houses a Radiance 2000 laser confocal microscope with an argon, green He/Ne, and Red diode laser. Three PMT's are present for capture of up to three distinct fluorescent emissions simultaneously. Currently we are capable of exciting at the following wavelengths: 457, 476, 488, 514, 543, and 637. Filters to capture specific fluorescent emissions include: 485 ± 30, 488 ± 10, 515 ±30, 530 ± 60, 545 ± 40, 590 ± 70, 600 ± 50, and 660 and up. In the next few months we expect to add a Blue Diode laser. With the installation of this final laser, we will be equipped to capture fluors with excitations ranging from 340 to 650 and emission from 400 to over 700. This would allow co-localization of 3 fluorescent markers in a single image, but more markers could be captured in sequential images to yield up to 6 distinct fluorescent signatures per image.
The microscope attached to the confocal is an inverted Nikon microscope equipped with 40X air, 60X oil, and 100X oil objectives and filters to view fluors with emission peaks ranging from 450 to over 700 including FITC, TRITC, and Cy5 for example. The scope is also outfitted with Differential Interference Contrast (DIC) optics for transmission images of the tissue or cells as well as 10X, 20X, and 40X phase objectives with an associated long working distance lens and phase rings to capture transmission images by phase microscopy. This combination of objectives, light imaging, and fluorescent filters can serve a variety of functions and meets the needs of all the investigators in the center grant. With the addition of the Blue diode laser, we will also add a fluorescent filter for UV range fluors (ie. DAPI or Hoechst 34442) further expanding the versatility of the system.
The software used to run the confocal microscope is Lasersharp 2000 which is distributed by BioRad as part of the Radiance package. This software automates the image collection process eliminating the need for users to change filters or add beam splitters depending on the application. The software allows programming for capture of up to 3 fluors at a time over a time course or 3 dimensional space and vertical sectioning. Analysis of the images using Lasersharp 2000 includes 3-D reconstruction and histogram analysis of fluorescent intensity. This software is available in an “off-line” version for installation on computers of any user. We also have the Confocal Assistant software package available to interested users. For advanced image analysis, the Metamorph software package from Universal Imaging will be offered to users in consult with the directors of the confocal facility and the Bioinformatics Core.
There is a dedicated computer for the confocal microscope which is a Dell Power Edge 1400 SC computer with 267 megabytes of RAM and 18 gigabytes on a partitioned hard drive running on a windows 2000 platform. This computer will be predominantly for image capture. Image analysis will be performed on the individual users computer using the "off-line" version of Lasersharp 2000, NIH Image freeware, Zeiss LSM software, or software of the user's choice.
