The ideal specimen for conventional optical microscopy is two-dimensional. However, biological material is organized in three dimension. Using conventional fluorescence microscopy, all fluorescence generated over the depth of a biological specimen reaches the image plane. As a result, the material within the plane of focus is sharply imaged, and material outside of the focal plane produces contrast-reducing background. This problem is circumvented using confocal microscopy. With confocal microscopy, light originating from a laser-illuminated pinhole is focused on a certain point in an object. Fluorescence from the same point is subsequently imaged using a detector pinhole. Since the illumination pinhole and the back- projection of the detection pinhole have a common focus in the object, the only light that reaches the detector is that generated from the specimen layer, and virtually all out-of-focus fluorescence is eliminated from the image. Using confocal microscopy, we can resolve uptake of antisense DNA by cancer cells, organelle motility and inheritance during cell division, the effect of the tumor inducers on subcellular localization of proteins, and changes in cytoskeletal organization during establishment of cell polarization, cell migration and cell division. In January 1997, Dr. Liza Pon became the new director of the Cancer Center Confocal Microscopy Facility. She introduced a state-of-the-art Confocal Imaging System using funds obtained from a Shared Instrumentation Grant. The new Facility is now heavily used (ca. 6 hours/day) by Cancer Center members and other research scientists. The system consists of a Zeiss LSM 410 scanning laser confocal attachment mounted on a Zeiss Axiovert 100 TV inverted fluorescence microscope. Sample excitation and confocal image detection is accomplished using an argon-krypton laser and three highly sensitive photomultiplier detectors. This system is able to image up to three fluorophores simultaneously, and to obtain differential interference contrast (DIC) and phase contrast images. The software package for confocal image analysis offers three-dimensional reconstruction, stereoscopic display of three-dimensional images, time-lapse imaging, ratio imaging, photobleaching (e.g., FRAP), quantifying co-localization and morphometry. The combination of confocal microscopy with the inverted microscope and digital image analysis allows users to observe complex living or fixed system with greater accuracy and speed than ever before.
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