The capillary electrophoresis (CE) with laser induced fluorescence detection (LIFD) system was founded according to confocal theory. The 3-D adjustment of the exciting and collecting optical paths was realized. The photomultiplier tube (PMT) is used and the signals are processed by a software designed by ourselves. Under computer control, high voltage is applied to appropriate reservoirs and to inject and separate DNA samples respectively. Two fluorescent dyes Thiazole Orange (TO) and SYBR Green I were contrasted. With both of the dyes, high signals-to-noise images were obtained with the CE-LIFD system. The single-bases can be distinguished from the electrophoretogram and high resolution of DNA sample separation was obtained.
Laser-induced fluorescence (LIF) is widely used in biological detection system in characteristic of high sensitivity and selectivity, especially for microarray biochip readout and capillary electrophoresis detection. In these systems, fluorescence separation from background noise is necessary. In this paper, two methods of fluorescence separation were investigated. One adopts a total reflection mirror with a hole at the center; the other uses a dichroic mirror. For dichroic mirror system, fluorescence could transmit through the filter or be reflected by it. Signal to noise ratio depends on dichroic mirror transmitting spectra and reflecting spectra. For center hole mirror system, partial fluorescence loses during propagating through the center hole directly. Detected fluorescence is the part that reflected by the mirror outside the center hole. Size of the hole in the mirror must be changed in different systems. Performance of system with an f-theta lens as scanning lens for laser focus and fluorescence collecting was simulated. Collinear systems with above-mentioned two methods were set up and compared. Simulated results were verified by experiments.
In general, the high resolution of a microscopy could be acquired by increasing the NA (numerical aperture) of lens, and simultaneously increasing the luminous flux. But many experiments shows: in turbid media, the NA of the lens we use is larger, the radius of focal spot is larger and the resolution decreases on the contrary. Because of highly scattering, there are different characters of focusing and propagation in turbid media .We simulate the process of photons motion in turbid media by means of Monte Carlo method according to a known model with representative parameter: changing the NA of lens in the program, the gray-scale images of the light spot in various layer are obtained. We also plot two figures; (1) distribution of the normalized light intensity versus the transverse axis; (2) the radius of light spots as a function of depth. In the figures: the trends of distribution of the normalized light intensity are coincident and centralized; when lens with larger NA is used, the normalized light intensity curve is further from the axis; it is also found the size of the focal spot is bigger and light beams focus in turbid media deeper. All above expound: the NA of the lens is larger; the radius of focal spot is larger in turbid media. In addition, it also suggests various factors including scattering, absorption and scalar diffraction etc must be taken into account when we choose the lens to focus in turbid media.
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