Optical design and spectral calibration of a laser scanning confocal microscopy spectral imaging system based on filters

Laser scanning confocal microscopy cannot distinguish fluorescence signals of different labels, and there are overlapping and interference of different fluorescence signals in the acquired images. In order to solve this problem, a laser scanning confocal microscopy spectral imaging system based on filters was designed, to realize spectral screening and detection of different fluorescence signals. Spectral screening of the optical signals from the laser scanning confocal microscopy was performed using dichroic filters and detected through the blue-violet channel, the green channel, the yellow channel and the red-orange channel, respectively. The spectral imaging system operates in the wavelength range of 400nm≤λ≤720nm. The green channel and the yellow channel jointly achieve signal detection in the 475nm≤λ≤625nm wavelength range. In addition, the green channel and the yellow channel can further screen the signal spectrum through the continuously variable filters. The minimum spectral screening resolution in the 475nm≤λ≤625nm wavelength range is better than 5nm. Spectral calibration light source is white LED, and the spectral analysis was performed by a fiber optic spectrometer. The spectral calibration results were tested using a low-pressure mercury lamp, and the experimental results verified the accuracy of the spectral calibration results. The spectral imaging system proposed in this paper, has the advantages of high spectral flexibility and high optical efficiency, meeting the requirements of engineering applications.