Key Technologies Research In Confocal Laser Scanning Microscopy

Confocal laser scanning optical microscopy imaging (CLSM) is different fromconventional fluorescence microscopy imaging. A point detection pinhole, which isconjugated with the object point, is employed in the optical path. It plays the role ofspatial filter that can remove the interference of defocus fluorescence, and do theoptical section. In the way of point by point and layer by layer scanning, CLSMachieves the observation of the sample area in three dimensions. CLSM is a kind offar-field optical imaging technology without damage, and also it provides highlateral and axial resolution at the same time, which makes it has a high using valuein the biomedical filed, even applied in the materials science filed. CLSM itself hashigh research value, its image quality and performance can be further improved, alsoit provides a good expanded platform for the study of new technologies. For thesereasons, it is necessary to do some researches of the key technologies in confocallaser scanning microscopy, and to introduce these key technologies into thedevelopment of propotype of CLSM.A complete CLSM system is extremely complex, including a number of keytechnologies. Considering the professional features, in this paper several keytechnologies were studied as follow:1. Imaging point spread functions. Based on the Richards-Wolf vectordiffraction theory, optical confocal imaging theory was studied from the very beginning. The point spread functions (PSF) of lighting, detection and confocal werederived. Also the effects of numerical aperture, multi-media and pinholes factors onthe PSFs were discussed.2. Scanning technology. Applied with galvanometer scanning mirrors, westudied the scanning optical path and the control of galvanometer mirrors, proposedtwo different scanning correction methods, namely scanning lens correction and arctangent galvanometer drive signal correction.3. Multispectral imaging technology. It can be divided into two parts: first,multi-wavelength laser excitation, to achieve the combination and selection ofdifferent wave beams; second, multispectral fluorescence detection, to achieve thespectral detection of fluorescence.4. Stimulated emission depletion. We analyzed its theoretical principles, and thetangential polarized incident light was used as the depletion beam to shape thehollow focus spot. By optimizing the wavefront with the phase and amplitude, thesize of hollow focus spot could be further reduced, and the optical resolution whichhas broken the diffraction limit could be achieved.5. Imaging system built and imaging experiments. A complete CLSM imagingsystem has been built which has achieved high-resolution three-dimensional imaging.And the related factors that influenced the quality of imaging were analyzed. Thepropotype of CLSM was finished.Finally, the research contents were summarized and analyzed. Some prospectsfor the CLSM were introuduced.