Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope |
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Authors: | Kim Dong Uk Moon Sucbei Song Hoseong Kwon Hyuk-Sang Kim Dug Young |
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Affiliation: | School of Information and Mechatronics, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea. |
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Abstract: | High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail. |
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Keywords: | laser scanning microscope image acquisition frame grabber masked illumination |
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