Autofocus vs voxel projection for vertical tracking in scanning cytometry

Jeffrey H. Price M.D.

doi:10.1117/12.273650

2 May 1997 Autofocus vs voxel projection for vertical tracking in scanning cytometry

Jeffrey H. Price M.D.

Proceedings Volume 2982, Optical Diagnostics of Biological Fluids and Advanced Techniques in Analytical Cytology; (1997) https://doi.org/10.1117/12.273650
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

Abstract

Raster scanning a microscope slide to obtain morphometric and fluorometric parameters of cells requires a method for tracking the focus positions. The vertical position of each field is unpredictable because of the irregularity of glass surfaces, the flexibility of thin coverslips, the mechanical instability of microscope and the varying thickness of the tissue layers. The conventional method for vertical tracking is autofocus. For autofocus, the best focus is calculated from measures of sharpness computed on a set of images acquired at each of several vertical test positions. The focus is then set to this position for acquisition of a final image. The vertical position could also be tracked by voxel projection, with the set of test images at a microscope field treated as a single 3D image of voxels. A 2D projection is created from a 3D image by retaining only the in-focus voxels. For fluorescence confocal microscopy, a projection is usually formed by selecting the maximum intensity voxels. Another technique would be to use the maximum absolute value of the highpass filtered image. The highpass projection may have advantages for fluorescence images and, unlike the maximum projection, would also be applicable to transmission microscopy. The advantage of highpass projection is that the final image is a composite of the most sharply focused voxels. The advantages and disadvantages of these vertical positioning techniques are compared with images of DAPI-stained cell nuclei.

Citation Download Citation

Jeffrey H. Price M.D. "Autofocus vs voxel projection for vertical tracking in scanning cytometry", Proc. SPIE 2982, Optical Diagnostics of Biological Fluids and Advanced Techniques in Analytical Cytology, (2 May 1997); https://doi.org/10.1117/12.273650

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KEYWORDS

Confocal microscopy

Microscopy

Luminescence

3D image processing

Bandpass filters

Image filtering

Microscopes

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