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Neuenheimer Feld 280, 0-6900 Heidelberg, and tDivision of Applied Microscopy, Carl Zeiss, 0-7082 Oberkochen, Germany KEY WORDS. Confocal scanning laser microscopy, UV-excitation, multiple-parameter fluorescence, immunofluorescence, lampbrush chromosome loops, amphibian oocytes, chromatin organization, blastocyst outgrowths, mammalian cells. SUMMARYThe use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488,514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals.Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.

K E Y w ORDS. Kidney, mitochondria, fluorescence, fluorometry, cell culture, loop of Henle, collecting tubule. S U M M A R YThe study of distribution and quantitation of a fluorescent probe in living epithelia with the aid of an inverted microscope requires that individual cells can be analysed without optical interference from adjacent cells. This report describes the application of fluorescence microscopy and fluorometry to a recently developed in vitro culture system of renal epithelial cells.Epithelial cells derived from the mammalian renal cortical collecting tubule (CT) and the thick ascending loop of Henle (TAL) are cultivated as continuous monolayers in serum-free, hormone-supplemented media. A specific mitochondrial marker (DASPMI) is added to the medium and incorporated into the cytoplasm. The microscopic image reveals that the mitochondrial fluorescence distribution differs between C T and TAL cultures. The fluorometric quantitation shows a normally distributed histogram of medium-range intensity in TAL cell cultures while C T cultures exhibit a two-peak pattern of mitochondrial fluorescence distribution among epithelial cells.

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