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18ADAPTIVE OPTICAL BESSEL BEAM TWO-PHOTON FLUORESENCE MICROSCOPY FOR VOLUMETRIC IMAGING OF SYNAPES IN DEEP CORTEX
WEI CHEN1, RYAN NATAN1, YUHAN YANG1 AND NA JI1
Department of physics, UC Berkeley, USA wei. chen. cal@berkeley. edu
ABSTRACT
Deciphering the information processing within neuronal circuities demands a technique that is capable to record multitude neuronal inputs at synaptic resolution and the dynamic of neuron ensembles across a large brain volume. In favor of its non-diffractive propagation in space, Bessel beam has been demonstrated in two-photon microscopy as an approach to improve the volumetric imaging speed via an axially elongated focus [1, 2]. However, the impacts of phase turbulence, such as aberrations presented in the imaging system and biological samples, are usually overlooked and less studied for Bessel beam. In this study, we have shown that aberrated Bessel beam reduces the two-photon excitation efficiency and degrades the signal-to-noise ratio (SNR) especially for small neuronal structures in deep cortical layers.
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Figure 1: (a) System schematic diagram. (b) AO correction process for the Bessel focus and measured PSFs without and with the
system AO correction.
In order to correct the aberrations for Bessel beam, we developed an adaptive optical (AO) Bessel beam multiphoton microscopy, which was enabled by two spatial light modulators conjugated to the image plane (SLM1) and the pupil plane (SLM2) respectively (Fig. 1a). System and sample induced aberrations were first probed by the Gaussian beam at the pupil plane based on the pupil-segmentation methods [3]. The wavefront correction pattern was then confirmed by imaging a 0.1um fluorescence bead and yielded about threefold signal improvement for Gaussian beam (Figs. 1b, c). Given a small interactive area between the SLM2 and the Bessel beam profile at the pupil plane, the wavefront correction pattern was computationally propagated to the image plane (SLM1) to construct the aberration-corrected Bessel beam for a greater correction efficiency (Fig. 1e). Imaging of a 0.1um bead by aberration-corrected Bessel beam (blue curve in Fig. 1f) showed about twofold signal improvement (Figs. 1d, 1f) and a dramatic higher correction efficiency compared to the AO correction directly applied at the pupil plane (green curve in Fig. 1f).
Figure 2: In vivo volumetric imaging of synaptic activity in the deep cortical layers with AO corrected Bessel two-photon fluorescence
microscopy.
By sparsely expressing GCaMP6s in layer 2/3 (200um below pial) and GCaMP7s in layer 4/5 (400um below pial) in the mice primary visual cortex, we were able to measure the sample induced aberration using the bright soma body as a guide star. The wavefront correction not only improved the signal and resolution for Gaussian imaging (Figs. 2a, 2g), but also enhanced the contrasts and resolvability of small structures (e.g., spines) for Bessel imaging (Figs. 2d, 2e, 2j, 2k). With AO-corrected Bessel beam, more photons were effectively delivered to the desired region of interest. Therefore, more Ca2+ transients can be detected for spines (Fig. 2f) and the orientation selectivity of dendritic spines could be determined with more confidence owe to the improved signal-to-noise ratio (Figs. 2l, 2m).
REFERENCES
[1] Lu, R., et al., Video-rate volumetric functional imaging of the brain at synaptic resolution. Nature Neuroscience, 20: p. 620, 2017.
[2] Botcherby, E., R. Juskaitis, and T. Wilson, Scanning two photon fluorescence microscopy with extended depth of field. Optics communications, 268(2): p. 253-260, 2006.
[3] Ji, N., D.E. Milkie, and E. Betzig, Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat Methods, 7(2): p. 141-7, 2010.
