Evaluation of colour transparency films for photomicrography of fluorescent structures.
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Low-power bright field photomicrographs often suffer from insufficient sharpness, uneven illumination, and colour hues. Using a film scanner, commercially available and designed for digitizing 35-mm transparencies, we directly scanned microscopic slides that carried dye-labelled and stained sections. The digital images covered a field of up to 24 x 36 mm and revealed excellent sharpness, absolutely even illumination and superior colour reproduction as compared to conventional photomicrographs taken with binoculars, macro lenses, or microscopes. As the method requires neither specialized instrumentation nor expert knowledge of photomicrographic techniques, it reduces costs and saves time. The high-quality digital survey micrographs can easily be used for image processing, image analysis and morphometry. Thus, this new method is valuable not only for pathology, embryology, histochemistry, and the neurosciences, but also for the exchange of low-power micrographs via the internet and for computer media that are increasingly used in medical education.
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A FORTRAN program, called AUTOSCAN, is described. This program permits the collection of photomicrographic data from serial sections to be semiautomated. In essence the user defines a box around a microscopic field of interest. Then the program drives the stage incrementally in the x and y directions, taking photographs of contiguous subfields. The box is defined by the use of a joystick and the "return" key. That is, movements of a joystick cause the stage to translate in the x and y directions. When a corner of the object is reached, as defined by cross-hairs in the microscope eyepiece, the user hits the return key. Repetition of this process at each corner defines a "box" within which photographs are to be taken. AUTOSCAN then calculates the step size and the number of frames to be taken from the user-defined values for the magnification. The actual movements of the stage in the x and y directions and the photography are fully automated. Each frame of film has the x and y coordinates of the center of the subfield being photographed imprinted in one corner, along with other relevant data. The x and y coordinates permit the resultant information to be assembled correctly into a two-dimensional montage.
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Recent advances in microcomputers and high resolution digital video cameras provide pathologists the opportunity to combine precision optics with digital imaging technology and develop new educational and research tools. We review recent advances in virtual microscopy and describe techniques for viewing digital images using a microcomputer-based workstation to simulate light microscopic examination, including scanning at low power to select features of interest and zooming to increase magnification. Hardware and software components necessary to acquire digital images of histological and cytological slides, and closely simulate their examination under a light microscope are discussed. The workstation is composed of a MicroLumina digital scanning camera (Leaf Systems, Southborough, MA), light microscope (Olympus Optical Co., Lake Success, NY), Pentium (Intel Corp., Santa Clara, CA) 166 MHz microcomputer configured with 64 megabytes of random access memory (RAM), a MGA Millenium Powerdesk graphics card (Matrox Graphics, Inc., Montreal, Canada) and Photoshop software (Adobe Systems Inc., San Jose, CA) running in a Windows 95 (Microsoft Corp., Redmond, WA) environment. Images with spatial resolutions of up to 2700 x 3400 pixels in 36-bit color, can be displayed simultaneously as distinct images in a montage, or merged into a single composite image file to highlight significant features of a histological or cytological slide. These image files are saved in Joint Photographers Experts Group (JPEG) format using compression ratios of up to 80:1 without detectable visual degradation. The advantages and technical limitations of various workstation components are addressed and applications of this technology for pathology education, proficiency testing, telepathology, and database development are discussed.
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In summary, the production of high quality photomicrographs through the operating microscope requires considerable attention to detail. The author describes all the details which he has found essential for obtaining good photomicrographs for documentation and/or publication.
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Human sperm motility characteristics in 28 semen samples with sperm concentrations less than 40 x 10(6) ml-1, as determined by the World Health Organization manual analysis (WHO, 1987), were measured by the automated CellSoft semen analyser (Cryo Resources Ltd, New York, NY, USA) using different system parameter settings (Mortimer & Mortimer, 1988a). The results were compared with those obtained by time-exposure photomicrographic (TEP) analysis. It was found that the settings of the minimum video frame rate and the threshold velocity used to distinguish motile from immotile sperm by the automated CellSoft system had a significant influence on measurements of percentage motility but not on linear velocity. At the five different parameter settings used in the present study, the automated CellSoft system gave significantly lower mean values for percentage motility in comparison with the WHO manual and TEP analyses. Measurements for linear velocity between the automated CellSoft system and TEP analyses were found not to be significantly different in these defined semen samples.