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 Confocal Microscopy

The Eye Center has three in vivo real-time confocal microscopes. One is located in the clinical area and is dedicated to patient research. The remaining two include an optically identical instrument that is located in the image analysis laboratory on the fourth floor and is devoted to animal research, and a ConfoScan 4 non-contacting microscope, also on the 4th floor. Real-time images are displayed on a high-resolution Sony Medical monitor. Off-line image processing and analysis are carried out in our Image Analysis Laboratory. Also available to Eye Center faculty is the Neuroscience Center confocal microscopy facility, located on the eighth floor of the Lions Eye Center Building. Instrumentation includes a Noran Odyssey XL laser scanning confocal microscope with argon ion laser, three photomultiplier tubes, and transmitted light detector connected to a Nikon Diaphot 200 inverted microscope with epifluorescence attachment imaging.

Principles of Confocal Microscopy
The unique properties of the confocal microscope stem from its ability to focus the illuminating light and the focal plane of the microscope objective on precisely the same point. This is accomplished by the distinctive light path employed in the confocal microscope with its Nipkow disc.

The Nipkow disc is a thin wafer with hundreds of pinholes that are arranged in a spiral pattern. When a portion of the disc is placed in the internal light path of the confocal microscope, the spinning disc produces a scanning pattern of the subject. As the subject is inspected, light is reflected back through the microscope objective. The light, that was reflected from in front of or behind the focal plane of the objective approaches the disc at an angle rather than perpendicularly. The pinholes of the disc permit only perpendicularly oriented rays of light to penetrate. This enables the microscope to view a very thin optical section of tissue.

LSU Eye Center Confocal Microscope
The confocal microscopes that we constructed utilize a Nipkow disc with 40 micron pores, which allows a 2-dimensional resolution of 1.5 to 2 microns and a vertical resolution of 12 microns. The objective consists of a modified specular microscope objective. Light is provided by a remote mercury or xenon lamp. Thus, the microscope and the subject are isolated from the heat and vibration produced by the lamp assembly. Images are captured with a CCD video camera and stored on S-VHS video tape.

This arrangment provides 230X magnification, high resolution, optically sectioned, in vivo images.

 

This oblique view of the cornea shows the stromal keratocyte nuclei (left) adjacent to the endothelial cell layer (right). Research confocal image of the cornea
This patient presented with a corneal ulcer after trauma. Confocal microscopy revealed the fine filaments of Aspergillus fumigatus. Thirty minutes after an applied stimulus, leukocytes are seen marginating within and surrounding the featured corneal limbal vessel.