Deep imaging with time-reversed light

Microscopy enabled the birth of modern neuroscience by allowing Ramón y Cajal to formulate the neuron doctrine. Since then, remarkable advances in optical resolution, speed and probe development allowed scientists to study the function of neuronal circuits with ever increasing detail – with one critical limitation: No conventional microscope can focus light deeper into intact tissue than a fraction of a mm. This leaves large parts of the brain inaccessible. As a result, the deepest layers of the neocortex and nearly all subcortical structures have long been outside the reach of non-invasive microscopy, representing a fundamental barrier towards further progress in understanding the brain.
Existing fluorescence microscopy techniques, such as confocal and two-photon microscopy, attempt to image deeper by rejecting scattered light or by selecting non-scattered (ballistic) photons for focusing. However, beyond depths of several hundred µm, hardly any ballistic photons remain.
The field of optical wavefront shaping recently achieved several breakthroughs by turning this strategy upside down and focussing with scattered light, rather than rejecting it. Still, fundamental challenges remain for in vivo imaging. The goal of this project is to break the depth barrier of microscopy and investigate previously unreachable areas of the live brain, by harnessing optical wavefront shaping and scattering correlations.

1: Characterisation of scattering properties of live tissue
To overcome the problem of tissue opacity, we treat scattering as a linear transformation of wavefronts from the tissue surface to a target plane deep within the tissue. Contrary to the common assumption that scattering is random, we recently discovered significant spatial correlations of this transformation in biological tissues. Our framework for explaining these correlations also predicted a way to further increase this effect: by selecting for forward-scattered photons, also called ‘snake-photons’, using broadband light sources. Previously, our theory only covered single wavelengths. During the first half of the project, we built on this prior work and added the dimension of wavelength, by characterising spatio-spectral transmission properties of biological tissue. We demonstrated that the range of translational correlations for the early arriving light is increased almost fourfold, paving the way for more efficient scattering compensation.
This work was published as Kadobianskyi M, Papadopoulos IN, Chaigne T, Horstmeyer R, Judkewitz B. Scattering correlations of time-gated light, Optica 2018, 5(4):389-94

2: Design and construction of a multi-photon microscope with adaptive optics capability: we designed and built a multi-photon microscope for deep imaging. As part of that effort we developed an approach for faster wavefront-shaping.
This work was published as Hoffmann M, Papadopoulos IN, Judkewitz B. Kilohertz binary phase modulator for pulsed laser sources using a digital micromirror device, Optics Letters 2017, 43(1):22-25

3: Deep imaging in layer 6b, the deepest layer of the mammalian neocortex, which has long been inaccessible to optical microscopy.
Technical work was published as Berlage C, Tantirigama MLS, Babot M, Di Battista, Whitmire C, Papadopoulos IN, Poulet JFA, Larkum M, and Judkewitz B: Deep tissue scattering compensation with three-photon F-SHARP, Optica (2021) 8:1613-19

Microscopy approaches and code have been disseminated via open access publications.

Overall, this work increased our physical understanding of scattering in biological tissue, leading to improved deep tissue adaptive optics microscopy, thus enabling images of layer 6b with unprecedented resolution and brightness.

Periodic Reporting for period 4 - DeepLight (Deep imaging with time-reversed light)

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