High speed functional imaging with source localized multifocal two-photon microscopy

Abstract

Multifocal two-photon microscopy (MTPM) increases imaging speed over single-focus scanning by parallelizing fluorescence excitation. The imaged fluorescence’s susceptibility to crosstalk, however, severely degrades contrast in scattering tissue. Here we present a source-localized MTPM scheme optimized for high speed functional fluorescence imaging in scattering mammalian brain tissue. A rastered line array of beamlets excites fluorescence imaged with a complementary metal-oxide-semiconductor (CMOS) camera. We mitigate scattering-induced crosstalk by temporally oversampling the rastered image, generating grouped images with structured illumination, and applying Richardson-Lucy deconvolution to reassign scattered photons. Single images are then retrieved with a maximum intensity projection through the deconvolved image groups. This method increased image contrast at depths up to 112 μm in scattering brain tissue and reduced functional crosstalk between pixels during neuronal calcium imaging. Source-localization did not affect signal-to-noise ratio (SNR) in densely labeled tissue under our experimental conditions. SNR decreased at low frame rates in sparsely labeled tissue, with no effect at frame rates above 50 Hz. Our non-descanned source-localized MTPM system enables high SNR, 100 Hz capture of fluorescence transients in scattering brain, increasing the scope of MTPM to faster and smaller functional signals.

In vivo voltage-sensitive dye imaging of mouse cortical activity with mesoscopic optical tomography

“…Although we need to notice, there are emerging improvements for multi-photon microscopy in imaging depth, 75,76 large FoV, [77][78][79][80] and imaging speed. [81][82][83] At the macroscopic scale, fMRI and DOT have been applied to achieve 3D . The response in S1 shows a significantly lower value than that in L2/3 and L5 of M1.…”

3D Localization for Light-Field Microscopy via Convolutional Sparse Coding on Epipolar Images

“…However, this serial acquisition limits the imaging speed. Efforts to increase acquisition speed include engineered beam trajectories [7] – [10] , spatial and/or temporal multiplexing of multiple foci [6] , [11] – [17] , as well as sculpting fluoroscence excitation into an extended point-spread function [18] – [21] , either scanned or targeted statically onto neurons of interest.…”

Subcellular resolution three-dimensional light-field imaging with genetically encoded voltage indicators

“…This restricts Poisson-noise limited signal-to-noise ratio (SNR) to low levels, making point scanning voltage imaging applicable to a limited number of experimental paradigms. 12,[18][19][20] Fluorescence excitation parallelization with multiple spots, [21][22][23][24][25][26][27] spinning disks, 28,29 blobs, 30,31 lines, [32][33][34] sheets, [35][36][37][38][39][40][41][42] or specified patterns [43][44][45][46][47] increases the photon budget, enabling functional volumetric imaging or single-plane imaging at increased speeds. A small number of these have been applied to imaging voltage in two dimensions, 26,33,44,47 however, they are not able to image neuronal processes in 3D.…”

“…Fluorescence excitation parallelization with multiple spots, 21 – 27 spinning disks, 28 , 29 blobs, 30 , 31 lines, 32 – 34 sheets, 35 – 42 or specified patterns 43 – 47 increases the photon budget, enabling functional volumetric imaging or single-plane imaging at increased speeds. A small number of these have been applied to imaging voltage in two dimensions, 26 , 33 , 44 , 47 however, they are not able to image neuronal processes in 3D.…”