Second harmonic generation laser scanning microscopy with intrinsic first-order modulation

Abstract

The contrast or signal-to-noise ratio (SNR) in images is a crucial parameter that determines the quality of images in second harmonic generation laser scanning microscopy (SHG-LSM). With a better image contrast/SNR, SHG-LSM can be performed at a faster frame rate or deeper tissue locations without compromising image quality. In this work, we present a novel strategy based on the SHG signals of first-order modulation (1 M) to enhance the image contrast/SNR. Notably, a photodetector (i.e., photomultiplier tube) has a better signal SNR during the photon-to-electron conversion process at the frequency of 1 M, which is equivalent to the laser pulse repetition frequency. The improved signal SNR thus enhances the image quality, which agrees well with the results of the measured electrical spectra. A remarkable increment of image contrast by more than a factor of two has been obtained in images by using 1 M. In addition, the image acquisition time is also shortened by a factor of 2.5 compared to that for acquiring images with similar contrast taken without signal modulation. Furthermore, by analyzing the dependencies of image contrast/SNR on sample depth, we demonstrate that the images obtained with 1 M have better quality than those obtained without signal modulation at the same imaging depth.