Deep brain vision: 3D adaptive two-photon microscopy

Wider Research Context: Functional imaging of cellular and sub-cellular processes deep inside the living brain is one of the most desired capabilities in the neurosciences, but faces significant challenges. To address this need, nonlinear optical microscopies have been developed which provide sub-cellular resolution at high imaging speeds, but aberrations and scattering limit the depth at which these can be applied. The imaging depth can be increased by using even longer wavelengths and/or adaptive optics (AO) that correct wavefront aberrations. However, despite significant progress toward increasing the imaging depth, realistic implementations of AO in strongly scattering samples still do not exist because of two major challenges: (i) fast and accurate measurements of wavefront aberrations (ii) extending corrections over larger fields of view (FOV). Objectives: We propose to develop a novel AO approach for nonlinear scanning microscopy that will enable imaging with sub-cellular resolution over an expanded FOV through strongly scattering tissues while still achieving the imaging speed necessary to record the dynamics in living tissues. 3D imaging capabilities will be provided using a novel remote focusing scheme. Further, we will evaluate the effectiveness of our approach using numerical simulations and experiments on relevant biological samples such as brain and skin tissues. Methods and Originality: As in traditional AO, we will correct for aberrations that persist over the entire FOV with a deformable mirror. Additionally, we will correct for strong, field- dependent scattering using a high pixel count liquid crystal spatial light modulator (SLM) imaged into the sample volume (conjugated AO). To maximize the speed of the correction, we will combine conjugated AO with a recently introduced technique called F-SHARP, which allows for fast corrections even with slow SLMs. The power of correcting field-dependent turbulence with Millions of Pixels in real time will be novel and potentially groundbreaking for deep tissue imaging. We will perform rigorous numerical simulations to identify optimal parameters and experimentally evaluate the effectiveness of our technique for different tissue types and imaging modalities. We will introduce a new remote focusing method based on diffractive rotatable elements, which are compatible with high NA optics and provide an intrinsic correction of spherical aberrations. Finally, we will investigate the application of conjugated AO to second harmonic generation (SHG) imaging in biological tissues, which has not been previously considered. Primary Researchers: The applicant (Dr. Molly May) will carry out the numerical simulations, construction, and operation of the proposed microscope. Assoc. Prof. Alexander Jesacher and Prof. Monika Ritsch-Marte will supervise the activities, and Dr. Michiel Langeslag and Assoc. Prof. Sandrine Moreno-Dubrac will collaborate on the biological investigations and provide samples.

Understanding the structure and function of the living brain is one of the most important problems of our time. To truly understand the neural circuits underlying the brain`s activity, we need to resolve individual neurons with cell bodies as small as 10 micrometers without disturbing the tissue, which is not possible with existing techniques. The most promising approach to imaging the activity of individual neurons is to modify the neurons so that fluoresce, when active. This fluorescence can be imaged in a microscope to map out the neural connections. However, brain tissue is not transparent - it scatters the light which makes it difficult to look deeper than the topmost layers of the brain and impossible to look through the skull with normal fluorescence imaging. To address this issue, scientists can use different colors of light in the infrare d wavelengths that are less sensitive to scattering by the tissue using a technique called two photon fluorescence microscopy (TPEF). This technique can nearly double the achievable imaging depth. Recently, another approach has been developed which creates an `inverse` of the scattering that happens on a specific path through brain tissue and cancels the effect of the scattering. There is huge potential for this scattering compensation technique to increase the optical imaging depth in brain tissue, but it is difficult to determine the correct inverse scattering profile, which can change at every position in the sample. Here, we developed an approach to quickly determine this inverse scatter ing profile in just a few seconds. We combined the scattering compensation technique with TPEF to image microglia cells over 0.5 mm deep in mouse brain tissue. With our scattering correction, the signal brightness can be increased by up to 20x and cell structures can be imaged with 300 nm spatial resolution. We also began a technology transfer which would duplicate our novel microscope in the laboratory of our collaborators in the Physiology In stitute. There, we will work together to apply the technique to image living tissues and brain activity in living mice.