Label-free neuroimaging in vivo using synchronous angular scanning microscopy with single-scattering accumulation algorithm

Moonseok Kim,Jin Hee Hong,Sungsam Kang,Seokchan Yoon,Kyung-Deok Song,Guang Hoon Kim,Yonghyeon Jo,Suhyun Kim,Wonshik Choi,Hae-Chul Park,Byunghak Lee

doi:10.1038/s41467-019-11040-z

Abstract

Label-free in vivo imaging is crucial for elucidating the underlying mechanisms of many important biological systems in their most native states. However, the applicability of existing modalities has been limited to either superficial layers or early developmental stages due to tissue turbidity. Here, we report a synchronous angular scanning microscope for the rapid interferometric recording of the time-gated reflection matrix, which is a unique matrix characterizing full light-specimen interaction. By applying single scattering accumulation algorithm to the recorded matrix, we removed both high-order sample-induced aberrations and multiple scattering noise with the effective aberration correction speed of 10,000 modes/s. We demonstrated in vivo imaging of whole neural network throughout the hindbrain of the larval zebrafish at a matured stage where physical dissection used to be required for conventional imaging. Our method will expand the scope of applications for optical imaging, where fully non-invasive interrogation of living specimens is critical.

Highlights

Label-free in vivo imaging is crucial for elucidating the underlying mechanisms of many important biological systems in their most native states
Typical adaptive optics (AO) microscopy for deep-tissue imaging has been designed to work for fluorescence imaging, and it has played a pivotal role in elucidating the underlying mechanisms of biological systems that could not otherwise be visualized
In the focus scanning microscope, the focused spot remains stationary at the detector during the scanning because the backscattered wave retro-reflected at the sample can be de-scanned by the scanning mirrors

Summary

Introduction

Label-free in vivo imaging is crucial for elucidating the underlying mechanisms of many important biological systems in their most native states. Typical AO microscopy for deep-tissue imaging has been designed to work for fluorescence imaging, and it has played a pivotal role in elucidating the underlying mechanisms of biological systems that could not otherwise be visualized It identifies the sample-induced aberration by either direct wavefront sensing[10,11,12] or feedback control of wavefront-shaping devices[13,14,15,16]. To overcome strong multiple scattering noise and sample-induced aberration, timegated reflection matrix approaches[31,32] have been investigated, which is composed of a set of wide-field and time-gated complex field maps of intrinsic elastic backscattering taken for various illumination angles This approach has been inapplicable for the in vivo imaging because a slow liquid-crystal spatial light modulator should be used for the recording of the timegated reflection matrix. In the conventional imaging modalities such as confocal fluorescence/reflectance microscopy, optical coherence microscopy and multi-photon microscopy, these structures have been inaccessible in the matured stage without the dissection of the specimens due to the severe sampleinduced aberrations

Methods

Results

Conclusion