The main obstacle for optical imaging deep inside biological tissues is light scattering. Recently, three-photon (3p) microscopy extended the accessible depth ranges of fluorescence imaging due to its enhanced nonlinearity. On a different front, advances in optical wavefront shaping showed that scattering can be compensated for, even in regimes where light entirely lost its directionality. Combining these two approaches, we demonstrate focusing and imaging behind scattering layers, enabled through wavefront shaping guided by 3p fluorescence in situations where no ballistic light reaches the sample. We analyse different sample geometries and compare these results to the case of 2p excitation.

Imaging through optical multimode fibers (MMFs) has the potential to enable hair-thin endoscopes that reduce the invasiveness of imaging deep inside tissues and organs. Current approaches predominantly require active wavefront shaping and fluorescent labeling, which limits their use to preclinical applications and frustrates imaging speed. Here we present a computational approach to reconstruct depth-gated confocal images from reflectance measurements in response to a proximally raster-scanned illumination. We evidence the potential of this approach by demonstrating quantitative phase, dark-field, and polarimetric imaging. Computational imaging through MMF opens a new pathway for minimally invasive imaging in medical diagnosis and biological investigations.

Multiphoton microscopy has become a powerful tool for imaging the intact mammalian brain, however, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we developed a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub-)cortical dendrites up to a depth of 1.4 mm in the mouse hippocampus, as well as deep-layer calcium imaging of astrocytes that reside in the highly scattering corpus callosum.

Deep Raman imaging in complex media has been elusive, due to scattering of the excitation light. Here, we exploit non-invasive wavefront shaping methods using a spectrally resolved speckle variance optimization algorithm in order to enhance the spontaneous Raman signals of a single particle. We demonstrate unprecedented signal enhancement (>5x) in a non-invasive manner for an epi geometry.

We present Dynamic Adaptive Scattering Compensation Holography (DASH) [1], a novel algorithmic approach for indirect wavefront sensing in nonlinear scanning microscopy that is suited for imaging into highly scattering media. DASH utilizes a single phase-only SLM for sculpting a continuously updated aberration corrected beam and a test beam with a particular wavefront shape. The test beam is phase-stepped to evaluate its importance and optimal phase and then added to the corrected. We demonstrate DASH for 2-photon fluorescence imaging of GFP expressing microglia cells in fixed and living mouse hippocampal brain tissue at over 500 µm depth. 1. May, Molly A., Nicolas Barré, Kai K. Kummer, Michaela Kress, Monika Ritsch-Marte, and Alexander Jesacher. "Fast holographic scattering compensation for deep tissue biological imaging." Nature Communications 12, no. 1 (2021): 1-8.

In Photoacoustic Remote Sensing (PARS) microscopy, a pulsed excitation beam is co-focused with a continuous-wave interrogation beam to acquire photoacoustic signals in an all-optical non-contact way. This study implements a resonant deformable mirror (DM) as the active focal control element in PARS. The surface of the DM is deformed by a time-varying actuation force tuned to match the resonance frequency of the defocus mode. In preliminary experiments using the DM as a focusing element, a 5cm focal shift was realized. The Adaptive Optics (AO)-PARS is a promising technology for axial scanning, multi-wavelength imaging, and aberration correction applications.

Light control in dynamic scattering media, like living tissues, is of paramount importance in biomedical imaging. Although iterative wavefront shaping algorithms can focus light through dynamic media, more complicated tasks like transmission of arbitrary fields and energy delivery require longer calibration procedures, typically involving the measurement of the transmission matrix. We report and showcase the performance of an optimization routine, based on a conventional wavefront shaping setup, allowing the online and recursive estimation of the transmission matrix of scattering media. Because it combines the benefits of iterative and transmission-matrix based algorithms, it enables full light control through dynamic and noisy environments.

Modern bio-imaging techniques such as light-sheet and PALM/STORM are now aiming to image more complex biological samples at larger depth and therefore face larger-amplitude and more complex aberrations. Here we will present our newly-developed deformable mirror which addresses these challenges. It is a continuous-membrane deformable mirror with 91 centro-symmetrically-arranged electromagnetic actuators, which is much more efficient for correction of both low and high-order aberrations compared to square layout, used in the past. Mirror has 99.7% linearity and almost no hysteresis, which ensures exceptional precision and speed in closed-loop mode as well as accuracy and unrivaled temporal stability in open-loop mode.

Optical imaging in complex media is a challenging task: due to multiple scattering, ballistic light is exponentially attenuated. This prevents conventional microscopy techniques from retrieving information beyond a millimeter inside biological tissues. We present an innovative way of focusing and imaging through scattering media using a model-based computational approach: a 2-layer neural network. This technique allows to retrieve transmission matrices of the system and thus reverse the scattering phenomenon. We are then able to retrieve the position of fluorescent beads through holographic diffusers. This approach is versatile and appliable to more challenging scenarios, like other scattering media or non-linear phenomena.

Soil is a highly scattering media that inhibits imaging of plant-microbial-mineral interactions that are essential to plant health and soil carbon sequestration. However, wavefront shaping can be used to focus light through or even deep inside highly scattering objects. In this work, we seek to overcome the fundamental challenges of imaging through soil minerals by developing a custom wavefront shaping method for a multiphoton microscope. We use the adaptive stochastic parallel gradient descent optimization algorithm combined with Hadamard basis to correct the aberration and the scattering in order to focus through the soil.

Non-line-of-sight (NLOS) imaging is a rapidly developing research direction that has significant applications in autonomous vehicles, remote sensing, etc. Existing NLOS methods primarily depend on time gated measurements and/or sophisticated signal processing to extract information from the scattered light. Here, we introduce a new method that directly manipulates the light to counter the wall’s scattering. This method operates by actively focusing light onto the target in a NLOS path using wavefront shaping. By raster scanning that focus, we can actively image the occluded object. The focus thus formed is near diffraction limited and can be substantially smaller than the object itself, thereby enabling us to perform NLOS imaging with unprecedented resolution. We demonstrate that a resolution of ∼ 0.6 mm at a distance of 0.55 m is achievable in our experiment.