HIGHLY SENSITIVE, SPECIFIC, AND STABLE NEW FLUORESCENT DNA STAINS FOR CONFOCAL LASER MICROSCOPY AND IMAGE-PROCESSING OF NORMAL PARAFFIN SECTIONS

The area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three-dimensional (3-D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO-1 iodide and YOYO-1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO-1 iodide and YOYO-1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO-1 iodide and YOYO-1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO-1 iodide and YOYO-1 iodide. Contrary to acriflavine, bleaching of TOTO-1 iodide and YOYO-1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation. Moreover, integrated TOTO-1 iodide and YOYO-1 iodide fluorescence shows good linearity with the DNA content of diploid, tetraploid, and octaploid liver nuclei. The sections stained with TOTO-1 iodide and YOYO-1 iodide are particularly suitable for two-dimensional morphometric and cytometric assessments of nuclei in sections, and also for the 3-D reconstruction and visualization of nuclei by confocal microscopy. The high intensity and stability of the fluorescent image of the nuclei and the low uniform intensity of the background will decrease the work needed in image segmentation of nuclei and help to increase the accuracy of cytometric assessments. (C) 1994 Wiley-Liss, Inc.