This PDF file contains the front matter associated with SPIE Proceedings Volume 11652, including the Title Page, Copyright information, and Table of Contents.

Soil is a highly scattering media that inhibits imaging of plant-microbial-mineral interactions that are essential to plant health and soil carbon sequestration. In this work, we seek to overcome the fundamental challenges of imaging through soil minerals by developing a custom wavefront sensor-less adaptive optics (AO) system for a multiphoton microscope. We are using a combined experimental and modeling approach, characterizing mineral optical characteristics with scatterometry, modeling the wavefront distortion and the image quality degradation after imaging through the soil medium, simulating the image quality improvement with AO correction, and experimentally testing our models with a stand-alone AO testbed.

The data acquisition speed of point scanning microscopy techniques at sub-cellular resolution limits imaging of large samples, as sample stability and focus drift are becoming an issue. Therefore, light sheet fluorescent microscopy (LSFM) has become the method of choice for imaging cleared samples. However, this method still suffers from a trade-off between imaging depth and resolution, due to diffraction of the illuminating beam limiting the achievable field of view. After improvement of the latter with non-diffracting Bessel beams, lattice light sheet microscopy has substantially reduced the illumination point-spread-function (PSF). Here, we propose further improvement by generation of structured light sheets via phased arrays implemented as silicon nitride photonic integrated circuits (PICs). Beam generation with PICs results in much higher power efficiency than beam forming with conventional liquid crystal based spatial phase modulators, as it does not require the use of narrow blocking apertures. Moreover, this approach enables increased control over the generated field profile. Modeling of concrete PIC concepts indicates that sub-cellular resolution with mm scale imaging depths can be concomitantly achieved. Maintaining a small PSF along the axis perpendicular to the direction of light propagation is sacrificed in order to maintain it over increased imaging depth along the beam propagation axis. Rapid lateral scanning of the illumination beam inside the plane of the light sheet is then obtained by scanning of the laser wavelength within the excitation spectrum of the target fluorescent protein, allowing for a wide bidirectional field-of-view with high resolution.

Microendoscopy incorporating a gradient index (GRIN) lens has emerged as a powerful tool for in vivo imaging. The lack of optical sectioning capability of widefield microendoscopy and the intrinsic optical aberrations of the GRIN lens itself, however, limit the achievable image contrast and resolution in three-dimensional (3D) tissues. In this study, we applied HiLo to widefield microendoscopy for optical sectioning. We also utilized adaptive optics (AO) to measure and correct GRIN lens aberrations. Together, HiLo and AO enabled subcellular-resolution microendoscopy imaging with optical sectioning and allowed us to image fine neuronal processes and synapses in the mouse brain in vivo.

With sub-diffraction resolution in three dimensions and good optical sectioning capability, three-dimensional superresolution structured illumination microscopy (3D-SRSIM) can provide eight-fold more information than conventional widefield microscopy. However, the application of SR-SIM is limited to single cells due to optical aberrations in thick tissues. The destructive impacts of aberrations include the decrease in spatial resolution and signal-to-noise ratio (SNR), the distortion of sample morphology, and, even worse, the failure of SIM reconstruction. There are several adaptive optics (AO) methods to correct the optical aberration, including direct wavefront sensing using a Shack Hartmann wavefront sensor (SHWFS). The SHWFS possesses good wavefront measurement accuracy and high-speed response but works best with an isolated guide-star. Therefore, combining SHWFS with widefield microscopy poses difficulties and remains challenging. To effectively apply the direct wavefront sensing method, we built a reconfigurable microscopy system that can switch to a confocal setup for measuring the wavefront where the fluorescence light emitted from the confocal illumination spot is used as the “guide-star” for wavefront measurements. We experimentally demonstrate that the confocal illumination based direct wavefront sensing AO method can precisely correct the sample induced optical aberrations and help to improve the image quality and fidelity of 3D-SIM imaging in thick samples, exhibiting enormous potential for in vivo biomedical research.

Sensorless adaptive optics (AO) methods are widely used in microscopes, but are based upon different concepts, which makes comparison between methods challenging. We show that all such methods can be considered as part of the same framework, which permits side-by-side evaluation of effectiveness in different imaging scenarios. We describe these advances and the tools that have been developed for wider use. These tools include universal, adaptable schemes for image-based sensorless adaptive optics that have broad application across different microscopy modalities. Further developments include operating protocols, hardware designs and software algorithms that will support next generation AO microscope systems.

Multimodal imaging systems are designed to extract complementary multifaceted information from biological samples; sensorless adaptive optics (AO) seeks to extend their capabilities by estimating the wavefront from image-derived information. Typical sensorless AO techniques need the acquisition of several volumes for optimization. We present a novel single-shot closed-loop sensorless AO technique demonstrated on a label-free multimodal imaging system consisting of optical coherence, two-photon fast fluorescence lifetime imaging, and second harmonic generation microscopy. The wavefront is sensed by performing computational AO on an initial OCM volume that is translated to the deformable mirror to improve the image quality of all modalities in real-time.

Open-loop spatio-spectral control of broadband light transmission through complex media such as optical multimode fiber (MMFs) requires a priori knowledge of the multispectral transmission matrix (msTM). However, acquisition of msTMs generally requires dense sampling at multiple wavelengths over the operating spectrum. Here we report on a computational spectral memory effect in a 1m long MMF. We demonstrate that the spectral correlation length among the spectral channels of a msTM can be extended 50-fold using a constant correction matrix between adjacent channels. This insight may stimulate efficient multispectral calibration methods that mitigate physical measurement limitations.

Optical quality in microscopy has reached a point where it is only limited by aberrations arising from sample inhomogeneities or refractive index mismatch in the focusing path. In this scenario adaptive optics is playing an increasingly important role. Most common microscopy AO setups rely on sensor-less algorithms and are designed to optimize only for one field. We present a method capable of measuring aberrations in every point on the field-of-view. This combined with the use of two deformable lenses permits to correct for field dependent aberrations in a closed loop MCAO system with virtually no changes in the microscopy setup.

Adaptive optics (AO) is a powerful tool for correcting wavefront errors induced by complex structures of biological samples which significantly causes image degradation. A scene-based sensing technique is being popular in microscopic AO systems with Shack-Hartmann (SH) wavefront sensors. A problem in application of the technique is that the shapes of images observed on SAs vary dependently to their positions on the aperture, especially when using microscopic objectives with higher NAs. To mitigate this problem, a differential sensing technique is used that enables measuring image shifts with high correlations over the aperture. Experiments using an artificial testing target including fluorescent beads, which simulates the leaf of moss, were conducted to investigate imaging performances of the present AO system. Unbiased maximum ratios were measured from blurred and AO-corrected images, and then the Strehl ratios were derived from them. Resultant Strehl ratios were around 0.58.

Two-photon fluorescence microscopy has been widely applied to three-dimensional imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. We characterized the aberrations introduced by remote focusing and used adaptive optics to correct the remote-focusing-induced aberrations. Diffraction-limited focal quality over up to 800-µm axial range can be maintained. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in mouse brain.

Light Sheet Microscopy has many advantages for imaging living model organisms. Its optical sectioning capability and high volumetric imaging speed over a large field of view make it especially favorable for recording highly dynamic biological events, such as neural signaling. The combination of an electrical tunable lens (ETL) and a scanning light sheet allows us to record image stacks at high speed without moving the sample or the detection objective. The performance of the light sheet microscope is affected by aberrations from the sample mounting and the sample itself as well as aberrations introduced by The ETL which limit the usable field of view and focusing range of the system. Here, we present the development of a light sheet microscope optimized for volumetric imaging of zebrafish larvae with adaptive optics correction for extended focusing range and increased image quality at a speed of 0.6Hz over 400 × 400 × 100μm³ using an electrical tunable lens.

In order to study dynamic biological processes in-vivo in mammalian organisms techniques are required which enable non-invasive imaging at large tissue depth with sub-cellular resolution. However, optical aberrations and scattering in biological tissue lead to signal loss and a degradation of both spatial resolution and penetration depth. Here, we combine two powerful optical techniques, multi-photon microscopy and adaptive optics, to push the depth limit further while retaining diffraction limited resolution. We apply these techniques to open questions in the field of neuron and glia biology. By utilizing three-photon excitation at the 1300 nm spectral excitation window we achieve highresolution imaging of GFP-labeled neurons up to a depth of 1.2 mm in the in-vivo mouse brain. Furthermore, we have combined our approach with indirect modal-based wavefront correction and synchronization of our microscope to the animal’s heart pulsation. This allowed us to improve the resolution up to ~6-fold and achieve synaptic resolution throughout an entire cortical column. With adaptive optics correction, small structures such a dendritic branches thus become clearly visible at over 1mm depth in the hippocampus. Furthermore, our adaptive three-photon microscope system enabled, for the first time, to investigate calcium dynamics of fibrous astrocytes which reside in the corpus callosum and whose dynamics have previously not been possible to image.

We report a compact adaptive optics (AO) module with optimized optical design and photon budget, compatible with multiple wavelengths, and adaptable to most of existing Light-Sheet setups, enabling a 2 to 3-fold signal improvement on neuronal structures of the live, non-clarified drosophila brain (neurons, projections), at depths ranging from 50 to 100µm. We report similar signal improvement brought by AO on functional signals from neurons of the drosophila circadian clock network. The proposed setup paves the way to fully automatized AO Light-Sheet systems usable in routine by biologists.

We propose an imaging method for controlling the output of scattering media such as multimode fibers using machine learning. Arbitrary images can be projected with amplitude-only calibration (no phase measurement) and fidelities on par with conventional full-measurement methods.

There has been considerable interest in extending photoacoustic imaging techniques to endoscopic devices, which would enable a diverse range of applications, e.g. assessment of coronary artery disease or surgical guidance. However, the difficulty of miniaturising traditional piezoelectric sensors has mostly prevented tomography-mode endoscopic imaging, where an array of sensors is used to reconstruct the full ultrasound field to centimeter-scale depths. In this work we demonstrate how wavefront shaping through multimode fibres onto a Fabry-Perot optical ultrasound sensor can overcome this limitation, producing an endoscopic imaging system with a footprint an order of magnitude smaller than the state of the art.

Fluorescence imaging is indispensable to biomedical research, and yet it remains challenging to image through dynamic scattering samples. Wavefront shaping has enabled fluorescence imaging through scattering media. However, the translation of these techniques into in vivo applications has been hindered by the lack of high speed solutions to counter the fast speckle decorrelation of dynamic tissue. Here, we report an ultrasound enabled optical imaging method that instead leverages the dynamic nature to perform imaging. The method utilizes the correlation between dynamic speckle encoded fluorescence and ultrasound modulated light signal that originate from the same location within a sample. We successfully imaged fluorescent targets within a dynamic scattering medium.

In this paper we propose and simulate a design for an optical phased array that can produces spot sizes small enough to be used in optical trapping processes in a micro-scale. Furthermore we attempt to combat the large grating lobes that are characteristic of optical phased arrays via an optimized non-uniform spacing profile of emitter elements. Using this optimized spacing profile we are able to reduce the peak side lobe level of a 10×10 array to 60% of the central lobe. We simulate that the full width half max of the central lobe of our 10×10 non-uniformly spaced array is 23 microns which is on the order of the spot size necessary for optical trapping.