Fig. 2: Comparison of two-photon microscopy modalities and evaluation of modulation contrast for LiL-SIM imaging.
From: Super-resolution upgrade for deep tissue imaging featuring simple implementation
Comparison of signal-to-background and signal-to-noise ratio in two-photon fluorescence microscopy images acquired in Pinus radiata tissue with a photomultiplier tube (PMT), b camera with rolling shutter (RS) mode, and c camera with lightsheet shutter (LSS) mode. The images presented in (a) were acquired with a commercial PMT-based microscope system (PMT-2PM) at the same imaging depth (z = 10 μm) but at distinct lateral positions of the specimen. The signal-to-background ratio is increased when using lightsheet line-scanning two-photon microscopy (LiL-2PM) over widefield line-scanning two-photon microscopy (WiL-2PM). d Normalized two-photon excited raw LiL-2PM volume of zebrafish with penetration depths ranging from 0 to 80 μm. These raw images were all acquired with the same pattern angle and phase at a pattern spacing of 350 nm. e Fourier-transformed planes of the zebrafish stack acquired with LiL-2PM represent the strength of the modulation contrast of the excitation pattern dependent on the imaging depth. The curve visualizes the strength of the modulation peaks for WiL-2PM (red) and LiL-2PM (white). SIM images can be reconstructed with a modulation contrast higher than 0.1. This corresponds to an imaging depth of 56 μm for super-resolution reconstruction. Extracted planes from 40 μm acquired with f WiL-2PM and g LiL-2PM demonstrate the improved modulation contrast achieved with LiL-2PM. h, i Fourier transforms of the images shown in (f, g) visualize the superior modulation contrast achieved with LiL-2PM over WiL-2PM. White rings indicate the spatial frequency in k-space. Data shown in (a–c) are representative images taken out of volume stack measurements (N = 5 for each modality). The data and the corresponding contrast enhancement shown in (d–i) has been verified in (N = 3) zebrafish volume stacks taken at distinct locations. Scale bars: a–c 10 μm, insets 3 μm. d 10 μm, inset 5 μm. f, g 10 μm, inset 2 μm.
