Transillumination Spatially Modulated Illumination Microscopy for Human Chromosome Imaging

摘要

Human chromosome analysis is an essential task in cytogenetics, especially in prenatal screening, genetic syndromediagnosis, cancer pathology research and mutagen dosimetry. Chromosomal analysis begins with the creation of akaryotype, which is a layout of chromosome images organized by decreasing size in pairs. Both manual and automaticclassification of chromosomes are limited by the resolution of the microscope and imaging system used. One way toimprove the results of classification and even detect subtleties now remaining undetected, is to enhance the resolution ofthe images. It is possible to achieve lateral resolution beyond the classical limit, by using spatially modulatedillumination (SMI) in a wide-field, non-confocal microscope. In this case, the sample is illuminated with spatiallymodulated light, which makes normally inaccessible high-resolution information visible in the observed image byshifting higher frequencies within the OTF limits of the microscope. Although, SMI microscopes have been reported inthe past, this manuscript reports the development of a transillumination microscope for opaque, non-fluorescentsamples. The illumination path consisted of a light source illuminating a ruled grating which was subsequently imagedon the sample. The grating was mounted on a rotating and translating stage so that the magnification and rotation of thepattern could be adjusted. The imaging lens was a 1.25 NA oil immersion objective. Test samples showed resolutionimprovement, as judged from a comparison of the experimentally obtained FWHM. Further studies using smaller fringedistance or laser interference pattern illumination will be evaluated to further optimize the SMI results.