We developed a computerized system for analyzing digital photos of pigmented skin lesions for melanoma screening. The system consisted of a digital camera equipped with a polarization cross filter and high-speed flash lightening. Images were analyzed in a microcomputer equipped with Photo Shop® and NIH Image® software. This system eliminates the reflection at the epidermal surface that presents an obstacle to detecting the color below the horny layer. Digitized photographs of the lesions were evaluated for color, expressed quantitatively as “color value”. Skin lesions were evaluated in 8 Japanese patients, 3 with malignant melanomas and 5 with benign pigmented nevi. We found that the color value was below 50 in the normal skin of these Japanese patients; that of pigmented areas exceeded 93. Areas of deeply pigmented macules exceeded the color value of 137. Accordingly, we evaluated the area of the whole lesion with a value ≥93 and the center of pigmented lesions with a value ≥137. The borders of the pigmented nevi were longer and more irregular than those of the malignant melanomas, while the latter showed areas of deeper pigmentation. Results suggest that this system may be useful for the screening of malignant melanomas prior to invasive histopathologic evaluation.

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Recent developments in confocal fluorescence microscopy have made 3-D observation of biological specimens possible, an epoch-making event in the long history of microscopy. However, most confocal microscopes currently used are insufficient for the observation of living cells and organs, which require real-time observations with minimal fluorescence staining. We have developed an innovative Nipkow-type scanner for a confocal microscope in which a microlens array is placed in front of a pinhole array and thus the optical efficiency of high-speed scanning is improved. Scanning speeds of 1,000 frames/sec are attained with 10—1000 times greater S/N than those of conventional confocal microscopes.

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We have modified the method of in situ nick translation (ISNT) for analysis of the 3-dimensional (3-D) progression of DNA cleavage during the course of apoptosis. HL-60 cells treated with all-trans retinoic acid (RA) were used. DNA strand breaks are visualized as the incorporation of biotin-16-dUTP by DNA polymerase I, detected with streptavidin-FITC. To analyze the spatial progression of apoptotic change by ISNT, two experimental conditions were stipulated: first, that the nick translation reaction takes place at an equal rate throughout the nucleus; and second, that the procedure for fixing DNA preserves the structure in vivo. To fulfill these prerequisites, cells were fixed with 4% paraformaldehyde, permeabilized by NP-40 and digested by Proteinase K, followed by the second fixation. After these treatments, ISNT was performed and the fluorescent signal was analyzed either by conventional fluorescence microscopy or by confocal laser scanning microscopy. The first change detected by ISNT in the process of apoptosis was the homogeneous labeling of the interior of the nucleus, with the concurrent occurrence of several highly labeled distinct foci. These foci appear to increase in size and intensity as apoptosis proceeds. Finally the foci change into discretely delineated spherical regions, becoming the canonical apoptotic bodies. Although temporal, these sequential morphological changes during the early stages of apoptosis are a novel observation, establishing the usefulness of this method.

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The fine structure of skeletal muscle heavy meromyosin (HMM) with two heads connected to a thin tail was clearly imaged with a high-resolution scanning electron microscope (SEM) by negative staining on a hydrophilic silicon surface. The single molecular image of HMM was analyzed. A structural change in the HMM head on binding of adenosine triphosphate (ATP) was detected. The HMM molecules with head(s) bent at 6-8 nm from the head-tail junction directed toward the end of the tail were found frequently in the presence of ATP. The estimated value for the bending angle was about 20° on average.

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When starfish oocytes were stained with an anti-α-tubulin antibody during polar body formation, the spindle as well as the asters of the mitotic apparatus (MA) were asymmetrical, whereas centrifugation made the MA symmetrical and inhibited polar body formation (Satoh, S.K. et al., Develop. Growth & Differ., 36: 557-565, 1994). Asymmetry in the mitotic spindle was induced by the attachment to the cell surface during maturation in the starfish oocyte. We analyzed fluorescence micrographs of MA quantitatively in both intact and centrifuged oocytes using NIH Image software. Digital subtraction of images of two corresponding half spindles revealed that the half spindles of the intact MA were different in fluorescence intensity, but those of the centrifuged MA were equal. In the intact MA, the peripheral half spindle was brighter by 18% and larger by 20% than the inner half spindle. Consequently, the average ratio of fluorescence intensity in the peripheral half spindle (near the egg surface) to that in the inner half was 1.37. When polar body formation was inhibited by centrifugation, the ratio dropped to 1.02, i.e., both half spindles fluoresced equally. These results demonstrate clearly that MA during polar body formation in the starfish oocyte is asymmetrical.

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Fungi possessing dark-brown pigments such as plant melanin are difficult to detect by fluorescence microscopic techniques. Pretreatment of the pigmented fungi with oxides or chlorides improved the detection efficiency by the fluorescence microscopic technique. Chemical bleaching was effective for spores of all the fungi studied. The fluorescence intensity of probes for cytoplasmic membranes, nucleic acids, and esterases stained to the spores increased more than ten times by this pretreatment, and became detectable not only with an electronic camera but also with the eye.

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