Three dimensional high resolution and high throughput nonlinear optical microscopy

Abstract

High throughput and high resolution two-photon fluorescence microscopy is an essential tool for functional and structural in vivo imaging of the brain. First, simultaneous, high-resolution functional imaging across a large number of synaptic and dendritic sites is critical for understanding how neurons receive and integrate signals. Yet, functional imaging that targets a large number of sub-micron sized synaptic and dendritic locations poses significant technical challenges. We demonstrate a new parallelized approach to address such questions, increasing the signal-to-noise ratio by an order of magnitude compared to previous approaches. This selective access multifocal multiphoton microscopy (saMMM) uses a spatial light modulator to generate multifocal excitation in three dimensions (3D) and a Gaussian-Laguerre phase plate to simultaneously detect fluorescence from these spots throughout the volume.

We test the performance of this system by simultaneously recording Ca2+ dynamics from cultured neurons at 98-118 locations distributed throughout a 3D volume. This is the first demonstration of 3D imaging in a "single shot" and permits synchronized monitoring of signal propagation across multiple different dendrites. Second, monitoring changes in dendritic and synaptic structures are important for understanding brain plasticity requiring high resolution and high sensitivity imaging of micron size structures over large volume of ~~ 500[mu]m3 . We have developed temporal focusing two-photon microscopy in vivo brain imaging with improved imaging speed over standard point scanning approach. However, the imaging depth of temporal focusing two-photon microscopy is severely limited by blurring due to scattering of emission photons. We have developed Multiline Orthogonal Scanning Temporal Focusing (mosTF) microscopy that enable reassignment of scattered photons back to the original position.

mosTF is able to overcome the scattering issue without a prior knowledge of the scattering media. We demonstrated mosTF by acquiring in vivo brain images from mice under anesthesia. mosTF is not only 10 times faster imaging speed than point-scanning two-photon microscopy; mosTF has a remarkable signal-to- background ratio improvement for in vivo brain imaging over typical temporal focusing approach.