10 May 1996 Novel fast laser scanning heterodyne differential-phase/fluorescence confocal microscopes for imaging live biological samples
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Abstract
Differential interference contrast imaging is frequently used to give improved contrast in studies of thin unstained live samples. Similarly, confocal microscopy is used to study stained live samples, because of its ability to optically section thick cells or tissues. We present two fast laser scanning heterodyne differential phase confocal microscopes, which combine the advantages of both these systems. The output of both systems is proportional to the sine of the phase gradient, which gives increased sensitivity when compared to DC interference systems. In one system the differentiation is performed in the image plane, with a single probe beam and a split detector, in the other system, the differentiation is performed in the object plane, using a split beam and a single detector. A direct comparison between the imaging performance of the two systems is made, and an optimized design has been developed from them. Resolution of better than 0.3 μm is achieved for both systems, fast beam scanning gives frame rates of approximately one second. The optimized microscope will also contain a fluorescence detection channel, the fluorescence image being obtained simultaneously with the reflection/differential phase image to ensure precise spatial and temporal alignment. The images obtained from these two detection techniques can be superimposed, and may be used to enhance each other, the differential phase image showing general structure. A number of novel imaging techniques will be investigated using the microscope.
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John R. Garside, John R. Garside, Michael G. Somekh, Michael G. Somekh, Chung W. See, Chung W. See, } "Novel fast laser scanning heterodyne differential-phase/fluorescence confocal microscopes for imaging live biological samples", Proc. SPIE 2678, Optical Diagnostics of Living Cells and Biofluids, (10 May 1996); doi: 10.1117/12.239548; https://doi.org/10.1117/12.239548
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