Optimizing the performance of multiline-scanning confocal microscopy

Line-scanning confocal microscopy provides high imaging speed and moderate optical sectioning strength, which makes it a useful tool for imaging various biospecimens ranging from living cells to fixed tissues. Conventional line-scanning systems have only used a single excitation line and slit, and thus have not fully exploited benefits of parallelization. Here we investigate the optical performance of multi-line scanning confocal microscopy (mLS) by employing a digital micro-mirror that provides programmable patterns of the illumination beam and the detection slit. Through experimental results and optical simulations, we assess the depth discrimination of mLS under different optical parameters and compare it with multi-point systems such as scanning disk confocal microscopy (SDCM). Under the same illumination duty cycle, we find that mLS has better optical sectioning than SDCM at a high degree of parallelization. The optimized mLS provides a low photobleaching rate and video-rate imaging while its optical sectioning is similar to single line-scanning confocal microscopy.