Periodic Reporting for period 1 - DEEP3P (Three-photon fluorescence imaging deep inside scattering media)
Periodo di rendicontazione: 2020-07-01 al 2022-06-30
This action addresses the problem of imaging deep inside scattering samples using three-photon (3P) excited fluorescence signals. In biomedical imaging, multi-photon microscopy serves as a tool to investigate structures and processes inside living tissues non-invasively. In this context, 3P microscopy got a lot of traction recently, due to its superior performance at large depth. However, light scattering within the tissue still poses the main limitation to successfully image deep tissue layers. While seemingly random, these scattering processes are coherent and can be reversed via wavefront shaping of the excitation light. While some initial studies showed that this can be done for low-order large-scale distortion of the wavefront, at the time this action was conceived, scattering correcting wavefront shaping for 3P microscopy was out of reach.
Particularly in neuroscience, the enhanced performance at depth lead to a fast adaptation of 3P microscopy. In order to image deep layers of the brain of awake mice, for example, without removing or compromising superficial layers one has to find optical solutions. Wavefront shaping can provide this solution to push the boundaries of in-vivo imaging deeper into the brain and thereby open new ways to understand the intricate functioning of the mammalian brain.
The objective of this action was to develop tools for scattering correcting wavefront shaping in 3P microscopy. In proof-of-principle experiments, we demonstrated that wavefront optimisation based on the total 3P fluorescence signal can be achieved with a simple continuous optimisation algorithm, even when the initial point-spread-function is strongly scattered. This enables imaging in situations where no image could be formed before. Another conclusion of the action is that the higher non-linearity of the 3P process leads to larger signal enhancements during wavefront optimisation but is not required for converging to a focus within volumetric fluorescent samples. This refutes claims made in the literature that 2P fluorescent feedback is insufficient to focus inside homogeneously dyed volumes. Finally, the action concludes that there is a largely untapped potential to employ computational techniques in multi-photon microscopy. In two separate techniques we used random illuminations paired with a reconstruction algorithm to form an image purely computationally.
Particularly in neuroscience, the enhanced performance at depth lead to a fast adaptation of 3P microscopy. In order to image deep layers of the brain of awake mice, for example, without removing or compromising superficial layers one has to find optical solutions. Wavefront shaping can provide this solution to push the boundaries of in-vivo imaging deeper into the brain and thereby open new ways to understand the intricate functioning of the mammalian brain.
The objective of this action was to develop tools for scattering correcting wavefront shaping in 3P microscopy. In proof-of-principle experiments, we demonstrated that wavefront optimisation based on the total 3P fluorescence signal can be achieved with a simple continuous optimisation algorithm, even when the initial point-spread-function is strongly scattered. This enables imaging in situations where no image could be formed before. Another conclusion of the action is that the higher non-linearity of the 3P process leads to larger signal enhancements during wavefront optimisation but is not required for converging to a focus within volumetric fluorescent samples. This refutes claims made in the literature that 2P fluorescent feedback is insufficient to focus inside homogeneously dyed volumes. Finally, the action concludes that there is a largely untapped potential to employ computational techniques in multi-photon microscopy. In two separate techniques we used random illuminations paired with a reconstruction algorithm to form an image purely computationally.
For this project, we built a multi-photon fluorescence microscope equipped with a spatial light modulator (SLM) to control the wavefront of the excitation laser. We implemented a continuous wavefront optimisation algorithm that controls the phase front of the fluorescence exciting laser in order to maximise the integrated 3P fluorescence signal coming from the sample. It was shown that this algorithm allows focusing through a scattering sample that fully scramble the wavefront of the excitation laser. By applying additional phase tilts on the SLM, the researcher further showed imaging of fluorescent beads behind a scattering layer that was completely opaque to unshaped excitation light. Additionally, investigating the algorithm’s convergence behaviour in different sample geometries, the researcher could show that for volumetric samples three-photon processes are not strictly needed to converge to a focus as two-photon fluorescence feedback suffices.
The core results of the project are published in 4 publications, 3 of which appeared in peer-reviewed scientific journals and one is currently under review. Two additional publications on computational imaging techniques, covering work performed at the end of the project, are currently being drafted.
The core results of the project are published in 4 publications, 3 of which appeared in peer-reviewed scientific journals and one is currently under review. Two additional publications on computational imaging techniques, covering work performed at the end of the project, are currently being drafted.
The project demonstrated focusing through wavefront shaping under unprecedentedly strong scattering in a 3P excitation setting. While most previous works in this domain showed the optimisation of an aberrated focus, here, there was no ballistic component present at the beginning of the optimisation process. This demonstrated that wavefront shaping for multi-photon microscopy does not necessitate complicated techniques but can be achieved with a straightforward continuous optimisation algorithms. Moreover, the project demonstrated that for volumetric targets, 2P feedback suffices to converge to a focus, which was doubted in the recent literature. Furthermore, the project developed novel computational imaging techniques for multi-photon fluorescence microscopy. This is a rarely studied topic and bears the potential to inspire new research in this direction.
The results of the project all help to improve fluorescence imaging in scattering environments and are particularly relevant for neuroscience where multi-photon fluorescence imaging is used to study the brains of living and behaving organisms. There, imaging deep regions of the brain is often elusive due to scattering in the upper tissue layers. Wavefront shaping can compensate these scattering events and allow for deeper brain areas to be investigated. In the long run, this will improve our understanding of the mammalian brain.
The results of the project all help to improve fluorescence imaging in scattering environments and are particularly relevant for neuroscience where multi-photon fluorescence imaging is used to study the brains of living and behaving organisms. There, imaging deep regions of the brain is often elusive due to scattering in the upper tissue layers. Wavefront shaping can compensate these scattering events and allow for deeper brain areas to be investigated. In the long run, this will improve our understanding of the mammalian brain.