Background inhibited and speed-loss-free volumetric imaging in vivo based on structured-illumination Fourier light field microscopy

Abstract

Benefiting from its advantages in fast volumetric imaging for recording biodynamics, Fourier light field microscopy (FLFM) has a wide range of applications in biomedical research, especially in neuroscience. However, the imaging quality of the FLFM is always deteriorated by both the out-of-focus background and the strong scattering in biological samples. Here we propose a structured-illumination and interleaved-reconstruction based Fourier light field microscopy (SI-FLFM), in which we can filter out the background fluorescence in FLFM without sacrificing imaging speed. We demonstrate the superiority of our SI-FLFM in high-speed, background-inhibited volumetric imaging of various biodynamics in larval zebrafish and mice in vivo. The signal-to-background ratio (SBR) is improved by tens of times. And the volumetric imaging speed can be up to 40 Hz, avoiding artifacts caused by temporal under-sampling in conventional structured illumination microscopy. These suggest that our SI-FLFM is suitable for applications of weak fluorescence signals but high imaging speed requirements.

High-speed in vivo calcium recording using structured illumination with self-supervised denoising

“…One advancement presented in this work is the first implementation of interleaved pHiLo technique. This technique builds upon the already established interleaved OS-SIM and rSIM techniques by removing the need for squared subtractions to obtain an optically sectioned reconstruction [11][12][13]. While the pHiLo method shares similarities to interleaved rSIM, it is more resistant to artefacts caused by sample motion.…”

“…The interleaved reconstruction technique was developed to increase the temporal resolution of OS-SIM and rSIM [12,13]. Using this technique, temporal loss is reduced by reusing each frame for each phase of the grating pattern.…”

Compact, Hybrid Light-Sheet and Fourier Light-Field Microscopy with a Single Objective for High-Speed Volumetric Imaging In Vivo

“…Under the light-sheet illumination, the emitted light produced by the sample is collected by the FLFM imaging system, whose design is similar to our previous reports (see refs and ). The spatial frequency spectrum of the collected emission light is relayed to the microlens array (MLA, FEL-46S03-38.24PM, Sigma, 3 × 4 mm pitch, f = 38.24 mm) by the tube lens ( f = 200 mm, AC508-200-A, Thorlabs) and Fourier lens ( f = 300 mm, AC508-300-A, Thorlabs) behind the objective.…”

“…25−27 The background fluorescence not only severely reduces the image quality, but also introduces artifacts during the image reconstruction. 21,28 Thus, integrating optical sectioning methods, such as multiphoton excitation, 29 confocal detection, 27 light-sheet illumination, 25,30 or structured illumination, 28,31 into FLFM is effective to inhibit background fluorescence. Of which, light-sheet illumination has advantages in optimizing the axial coverage and suppressing the background from the out-of-DOF fluorescence, 32,33 proposed and demonstrated in a variety of in vivo experiments.…”

“…However, in practical imaging, FLFM is susceptible to image quality degradation caused by a strong background, which comes primarily from the out of depth-of-field (DOF) fluorescence excited by the epi-illumination light. − The background fluorescence not only severely reduces the image quality, but also introduces artifacts during the image reconstruction. , Thus, integrating optical sectioning methods, such as multiphoton excitation, confocal detection, light-sheet illumination, , or structured illumination, , into FLFM is effective to inhibit background fluorescence. Of which, light-sheet illumination has advantages in optimizing the axial coverage and suppressing the background from the out-of-DOF fluorescence, , as only samples in the regions of interest (ROIs) are excited.…”