Deep brain vision: 3D adaptive 2-photon microscopy

Optical microscopy has seen some incredible advancements in recent years, pushing the limits of what can be resolved optically way beyond what was believed to be unscalable limits. But optical imaging still has one drawback which poses a severe obstacle for turning it into a true in-depth tissue imaging modality: Absorption and scattering in the visible regime restrict optical imaging of untreated tissue samples to superficial layers, limiting it typically to a depth of a few tens of micrometers. The present proposal aims at increasing the achievable imaging depth in mouse brain tissue to enable research investigating the role microglia cells, the innate immune cells of the central nervous system, and their migration play in the physiological mechanisms underlying pain. Since unhampered cell migration necessarily requires relatively thick tissue layers and since brain tissue suffers from particularly strong scattering, it is very difficult to assess it optically in native (unmodified) tissue into a depth of about 0.3 mm or more. The major goal of the project is to identify, implement and then apply the optimal strategy of adaptive optics and wavefront shaping techniques in order to undo the optical degradation induced by the sample itself, which refracts and diffracts the light. Especially in strongly-scattering or turbid materials such as brain tissue, if no counter-measures are taken, the imaging information is effectively washed out after only a few cell layers. A key element of the adaptive optics approach is the fast and precise measurement of these aberrations, followed by a compensation of the wavefront deformations by a programmable wavefront shaper. Similar techniques are applied in astronomy to get rid of fluctuations induced by turbulence in the earths atmosphere. Combining cutting-edge adaptive optics techniques with fast axial refocussing based on an innovative lens system developed in Innsbruck we hope to create a system for volumetric optical imaging which will enable microglia migration studies requiring sub-cellular length scales in order to resolve microglia dendrites with sub-second time resolution for the fast actions of glia cells during STP- mediated cell migration. These are high demands on the imaging task, and therefore this investigation represents an ideal test application for the various techniques developed and compared in the project. The proposal is strongly interdisciplinary, applying optical concepts developed by the Biomedical Physics group to neuro-physiological research in the focus of research groups in Physiology, both at the Medical University in Innsbruck.

In our research project, new methods were developed to be able to look as deeply as possible into tissue using microscopes. In principle, light microscopy is an excellent way of diagnosing diseases or finding out more about the function of tissues at a cellular level. Unfortunately, however, light microscopes are only suitable to a limited extent for looking into living tissue (such as human skin) or even thin histological sections, as the tissue itself scatters light strongly. The penetration depth of conventional microscopes is therefore only limited to small fractions of a millimetre. In our project, we used a technology that is already successfully used in astronomy under the term 'adaptive optics' and which can shape light in practically any way. In principle, this technology could also be used to counteract the scattering effect of tissue. The idea is to prepare the light that is to be used to illuminate a thick sample in such a way that the tissue recovers the light beam inside it instead of scattering it further. If this could be achieved, it would probably be possible to look several millimetres deep into scattering tissue such as brain tissue, histological sections or skin tissue, which would enable breakthroughs in the diagnosis of diseases and a greater understanding of the biological processes within organs. The big question, however, is how to mould the light so that a clear image can be obtained in the depth of the tissue. We have addressed this question with this project. The main result of our research is a new measurement method that can be used to find optimal light shapes with comparatively few measurements that can be carried out in just a few seconds. We combined this method with non-linear scanning microscopy, a type of microscopy developed specifically for high penetration depths, and investigated the new method on various biological samples. Among other things, we were able to look more than half a mm deep into fixed mouse brain tissue and visualise individual cells and their substructures with greatly increased resolution and signal strength (20 times more signal). To summarise, we have come a good deal closer to our goal of using light microscopes to look deeper into tissue. In a follow-up project, we are now trying to extend the image corrections achieved to larger image fields.