Deep tissue scattering compensation with three-photon F-SHARP

Abstract

Optical imaging techniques are widely used in biological research, but their penetration depth is limited by tissue scattering. Wavefront shaping techniques are able to overcome this problem in principle, but are often slow, and their performance depends on the sample. This greatly reduces their practicability for biological applications. Here we present a scattering compensation technique based on three-photon excitation, which converges faster than comparable two-photon (2P) techniques and works reliably even on densely labeled samples, where 2P approaches fail. To demonstrate its usability and advantages for biomedical imaging, we apply it to the imaging of dendritic spines on layer 5 neurons labeled with green fluorescent protein in an anesthetized mouse.

Wavefront shaping: A versatile tool to conquer multiple scattering in multidisciplinary fields

“…To increase the resolution and signal-to-noise ratio at depths in vivo , WFS has been extended to longer optical wavelength with multiphoton microscopy. 188 , 190 , 191 , 192 It must be clarified that thus far, many of these new WFS implementations have only been demonstrated in the proof-of-principle phase. Nevertheless, continuous progress in merging, modifying, and advancing these efforts toward robust performance in real in vivo experiments will likely boost the second wave of developments in WFS, which may change the landscape of biomedical optical research and practice at the tissue level.…”

Roadmap on wavefront shaping and deep imaging in complex media

“…Soon after the seminal work from Mosk's group, it was realized that a feedback signal for single-grain focusing within the scattering medium was far from simple. Hence, the field has drastically focused on nonlinear signals as a feedback mechanism due to its ability to converge to a single focus in wavefront shaping experiments, as seen for instance very recently [85]. However, nonlinear processes are less popular in science and engineering due to the cost associated with the hardware (mostly the laser source).…”

Recent advances in optical imaging through deep tissue: imaging probes and techniques

“… Direct wavefront sensing High sensing speed / Require wavefront sensors, Need fluorescent labeling, weak at scattering Oligodendrocytes and neuronal nuclei in a zebrafish brain in vivo [ 33 ] Neurons in a Thy1-YFPH mouse brain in vivo [ 34 ] mRuby2-labeled layer 5 pyramidal neurons of vS1 mouse brain cortex in vivo [ 35 ] A live human stem cell-derived organoid [ 36 ] The eye of a zebrafish embryo 24 hpf. [ 36 ] Cortical neurons of a Thy1-GFP mouse brain in vivo [ 37 ] C. elegans in vivo expressed by the adherens junction marker ajm-1::GFP [ 38 ] Indirect wavefront sensing Better suited to opaque tissues than direct wavefront sensing, Require no wavefront sensors / Slow sensing speed due to hardware feedback, Deal with low-order aberrations modes, Need fluorescent labeling The visual cortex and Hippocampus of mouse brain in vivo [ 39 ] Synaptic structures in the deep cortical region of a Thy1-GFP mouse brain [ 40 ] High and basal dendritic spines of a mouse V1 neuron in vivo [ 41 ] GFP expressed microtubule of Drosophila larval macrophage [ 42 ] GFP-expressed pyramidal neuron in a living mouse brain [ 43 ] Single microglia cell from hippocampus tissue of a mouse brain [ 44 ] Coherence-gating Label-free, High sensing speed / Mixed phase retardations of input and output paths, Deal with low-order aberration modes Olfactory bulb in transgenic zebrafish larvae [ 45 ] Time-gated reflection matrix Label-free, Better suited to opaque tissues, Numerical post-processing, Seperation of input and output aberrations, High- order aberration correction / Matrix acqusition time depending on scattering Hyphae of Aspergillus cells in a rabbit’s cornea [ ...…”