Penn Dental Medicine houses a Radiance 2100 laser confocal microscope with argon, green He/Ne, red diode, and blue diode lasers available for use by researchers throughout the School as well as others inside and outside the University. Three photomultiplier tubes (PMTs) are available for capture of up to three distinct fluorescent emissions simultaneously. With this laser configuration, the microscope is equipped to capture most fluors with excitations ranging from 340 to 650 and emission from 400 to over 700, allowing co-localization of three fluorescent markers in a single image, with the capability of capturing up to six 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. 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.
The software used to run the confocal microscope is Lasersharp 2000, which automates the image collection process, eliminating the need 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 to any user, and the Confocal Assistant software package is also available to interested users. For advanced image analysis, the Metamorph software package from Universal Imaging may be available to users in consult with the director of the confocal facility.
There is a dedicated computer used for image capture from the confocal microscope; image analysis must be performed on the individual users’ computers.
Dr. Kelly Jordan-Sciutto, Director
312 Levy Building