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

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.

Laser scanning reflection-matrix microscopy for aberration-free imaging through intact mouse skull

“…The LS-RMM proposed here is different from previous reflection-matrix-based approaches in its illumination and detection configurations 27,28,33 . Our earlier studies used planar wave illuminations, while the present LS-RMM uses a focused illumination.…”

“…The processing step is the transformation of the measured reflection matrix taken for focused illuminations to that for planar illuminations. We then administered a unique algorithm referred to as the closed-loop accumulation of single scattering (CLASS), developed previously to selectively identify and computationally correct sampleinduced aberrations from background multiple-scattering noise [27][28][29] . In particular, we improved the algorithm to correct a large number of correction modes (~10,000 angular modes) at each local area down to 10 × 10 µm 2 .…”

Distortion matrix concept for deep optical imaging in scattering media

“…10) can easily be met in biological tissues since a strong aberration regime takes place beyond a few scattering mean free paths. Note also that, even when this condition is not fulfilled and far-field correlations dominate, the distortion matrix approach can still work but the FOI has to be beforehand subdivided into individual IPs (47,51). The ability of identifying multiple IPs will also be particularly promising to map the specimen-induced aberration and the SMR.…”

Deep optical imaging within complex scattering media

“…In initial studies, a drawback of CLASS microscopy was the slow acquisition rate for recording the timegated reflection matrix owing to the use of a SLM for multi-angular illuminations. To resolve this issue, adaptive optical synchronous angular scanning microscopy (AO-SASM) 127 was proposed, in which a pair of scanning mirrors is used to increase the scanning speed up to 10,000 modes per second. The high-speed recording of R(τ 0 ) enabled aberration-free imaging of a living larval zebrafish over the entire volume of the hindbrain.…”

“…Optical-scale imaging using acousto-optic interactions and the selective detection of single optical speckles is advancing towards realistic conditions in which the speckle grain size reaches half of the wavelength 121,124 . High-resolution imaging based on a time-gated reflection matrix is also starting to gain control of MS waves in reconstructing the object image 126,127,139 . Additionally, improvements in the sensitivity and speed of cameras and the increase in the number of pixels and control speed of the SLM will help to resolve some of the practical issues.…”