This PDF file contains the front matter associated with SPIE Proceedings Volume 10502, including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Liquid crystalline (LC) materials are user friendly materials for electro optics applications [1], the most popular of which is the flat panel display [2]. Another interesting development, involving LCs, was the electrically variable lens that already had several commercial applications in DVD pick-up systems, Webcams and cell phones [3,4]. Their quality can be very high [5] and our team has recently demonstrated that they can be used in endoscopes for the study of deep regions of the brain [6]. We shall first describe various approaches explored to build electrically variable LC lenses. We shall then describe electrically variable LC lenses with segmented electrodes that enable almost adaptive optical capability, including the creation of a dynamic lens, prism, astigmatism and coma. This could be used to compensate various wavefront deformations in optical systems used to study various biological systems. We shall describe their advantages and drawbacks for the same application. References [1]. P.G. de Gennes and J. Prost, The Physics of Liquid Crystals, (Oxford University Press, 1995), 2nd Edition. [2]. Robert H. Chen, Liquid Crystal Displays: Fundamental Physics and Technology, Wiley, July 2011, ISBN: 978-0-470-93087-8. [3]. T. Galstian, Smart Mini-Cameras, CRC Press, Taylor & Francis group, Boca Raton, 2013. [4]. www.lensvector.com [5]. T. Galstian, K. Asatryan, V. Presniakov, A. Zohrabyan, A. Tork, A. Bagramyan, S. Careau, M. Thiboutot, M. Cotovanu, Optics Letters, Vol. 41, Issue 14, pp. 3265-3268 (2016),  doi: 10.1364/OL.41.003265. [6]. A. Bagramyan, T. Galstian and A. Saghatelyan, Journal of Biophotonics, 1–13 (2016) / DOI 10.1002/jbio.201500261.

In this study, a field programmable gate array (FPGA)-based Shack-Hartmann wavefront sensor (SHWS) programmed on LabVIEW can be highly integrated into customized applications such as adaptive optics system (AOS) for performing real-time wavefront measurement. Further, a Camera Link frame grabber embedded with FPGA is adopted to enhance the sensor speed reacting to variation considering its advantage of the highest data transmission bandwidth. Instead of waiting for a frame image to be captured by the FPGA, the Shack-Hartmann algorithm are implemented in parallel processing blocks design and let the image data transmission synchronize with the wavefront reconstruction. On the other hand, we design a mechanism to control the deformable mirror in the same FPGA and verify the Shack-Hartmann sensor speed by controlling the frequency of the deformable mirror dynamic surface deformation. Currently, this FPGAbead SHWS design can achieve a 266 Hz cyclic speed limited by the camera frame rate as well as leaves 40% logic slices for additionally flexible design.

Wavefront shaping devices such as deformable mirrors, liquid crystal spatial light modulators (SLMs), and active lenses are of considerable interest in microscopy for aberration correction, volumetric imaging, and programmable excitation. Liquid crystal SLMs are high resolution phase modulators capable of creating complex phase profiles to reshape, or redirect light within a three-dimensional (3D) volume. Recent advances in Meadowlark Optics (MLO) SLMs reduce losses by increasing fill factor from 83.4% to 96%, and improving resolution from 512 x 512 pixels to 1920 x 1152 pixels while maintaining a liquid crystal response time of 300 Hz at 1064 nm. This paper summarizes new SLM capabilities, and benefits for microscopy.

Since a decade, wavefront shaping techniques has allowed to coherently manipulate speckle patterns. It opens the possibility to focus light through complex media and ultimately to image in them, provided that the medium can be considered as stationary during the process. However, scattering by tissues evolves over millisecond timescales, creating a fast decorrelation of the speckle pattern, thus limiting the use of this technique for in vivo microscopy. Therefore, focusing through biological tissues requires fast wavefront shaping devices, sensors and algorithms. It has been demonstrated by Akemann et al that an Acousto-Optic Deflector (AOD) time locked on the output laser pulses of a regenerative amplifier can be used as an arbitrary 1D beam shaper: the locally modulated acousto-optic phase grating allows the spatial control of the laser pulse wavefront, with refresh rate of several tens up to several hundreds of kHz, limited by the size of the AOD aperture. We have investigated through simulations and experiments, the use of two crossed AODs to implement 2D spatial wavefront shaping, and perform focusing by optimization through a scattering media. We have used different algorithms adapted to this grating modulator and analyzed in each case the AOD bandwidth used, the speed of convergence and the maximum intensity enhancement. In particular, we have shown that two crossed 1D modulators provide larger enhancement than a single 2D wavefront shaper with the same number of pixels. We will present our latest results towards achieving the ultimate optimization, limited by the AOD speed of 40 kHz.

The presence of aberrations is a major concern when using fluorescence microscopy to image deep inside tissue. Aberrations due to refractive index mismatch and heterogeneity of the specimen under investigation cause severe reduction in the amount of fluorescence emission that is collected by the microscope. Furthermore, aberrations adversely affect the resolution, leading to loss of fine detail in the acquired images. These phenomena are particularly troublesome for super-resolution microscopy techniques such as isotropic stimulated-emission-depletion microscopy (isoSTED), which relies on accurate control of the shape and co-alignment of multiple excitation and depletion foci to operate as expected and to achieve the super-resolution effect.

Aberrations can be suppressed by implementing sensorless adaptive optics techniques, whereby aberration correction is achieved by maximising a certain image quality metric. In confocal microscopy for example, one can employ the total image brightness as an image quality metric. Aberration correction is subsequently achieved by iteratively changing the settings of a wavefront corrector device until the metric is maximised. This simplistic approach has limited applicability to isoSTED microscopy where, due to the complex interplay between the excitation and depletion foci, maximising the total image brightness can lead to introducing aberrations in the depletion foci. In this work we first consider the effects that different aberration modes have on isoSTED microscopes. We then propose an iterative, wavelet-based aberration correction algorithm and evaluate its benefits.

Recent advances in optical microscopy, in particular those based on light sheet imaging, have significantly advanced the field of long term in vivo imaging with minimal perturbation to the sample. This means that there is now an interest in studying the processes by which a body repairs damage, in particular during development. A genetically encoded protein, KillerRed, is now available that is phototoxic, killing the cells in which it is present when illuminated with light at 561nm. The presentation will report for the first time its use in zebrafish with localised cell ablation in a SPIM system. We report on ablation within the living sample either through the use of the light sheet within the microscope, but more precisely using a beam introduced through the imaging arm of a SPIM microscope controlled by adaptive optics to ensure localization of the activation. The beam profile is optimized to target individual cells within the kidney of the fish and the loss of fluorescence from the KillerRed is used to quantify the damage. The integration of the adaptive optics and opto-genetic encoding and activation of the KillerRed will be demonstrated to have the ability to ablate single cells deep within a living zebrafish with 100% survival of the fish. The presentation will illustrate how significant advances in the life sciences can be made through multidisciplinary research with optical expertise.

Adaptive Optics (AO) is of growing importance for understanding the impact of retinal and systemic diseases on the retina. While AO retinal imaging in healthy eyes is now routine, AO imaging in older eyes and eyes with optical changes to the anterior eye can be difficult and requires a control and an imaging system that is resilient when there is scattering and occlusion from the cornea and lens, as well as in the presence of irregular and small pupils. Our AO retinal imaging system combines evaluation of local image quality of the pupil, with spatially programmable detection. The wavefront control system uses a woofer tweeter approach, combining an electromagnetic mirror and a MEMS mirror and a single Shack Hartmann sensor. The SH sensor samples an 8 mm exit pupil and the subject is aligned to a region within this larger system pupil using a chin and forehead rest. A spot quality metric is calculated in real time for each lenslet. Individual lenslets that do not meet the quality metric are eliminated from the processing. Mirror shapes are smoothed outside the region of wavefront control when pupils are small. The system allows imaging even with smaller irregular pupils, however because the depth of field increases under these conditions, sectioning performance decreases. A retinal conjugate micromirror array selectively directs mid-range scatter to additional detectors. This improves detection of retinal capillaries even when the confocal image has poorer image quality that includes both photoreceptors and blood vessels.

Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.

The small correction volume for conventional wavefront shaping methods limits their application in biological imaging through scattering media. In this paper, we take advantage of conjugate adaptive optics (CAO) and remote focusing (CAORF) to achieve three-dimensional (3D) scanning through a scattering layer with a single correction. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We demonstrate two-photon imaging with CAORF through mouse skull. The fluorescent microspheres embedded under the scattering layers can be clearly observed after applying the correction.

Wavefront-shaping devices incorporated into optical microscopy systems are capable of correcting sample-induced aberrations and recovering diffraction-limited imaging performance. The widespread dissemination and application of adaptive optical techniques, however, requires easy integration of adaptive optical modules, both in terms of hardware and software, into existing microscopes. We built an adaptive optical module with reduced complexity and simplified integration by utilizing a novel segmented deformable mirror and a standalone control software program. We demonstrated its ability to improve image brightness and resolution at depth in the mouse, zebrafish, and fly brains in vivo.

Adaptive Optics (AO) is required to achieve cellular resolution at high numerical aperture in small animal eyes. Development of AO technology is required to lower the barriers of the technology translation into pre-clinical vision research environments. Aberration correction for retinal imaging has been demonstrated with great results by direct wavefront sensor (WFS) measurement. However, in some cases the performance of WFS-based AO can be limited by several factors including common path errors, wavefront reconstruction errors and an ill-defined reference plane on the retina. Image-based AO can avoid these issues and the cost of algorithmic execution time. We are investigating and evaluating image-based approaches to potentially provide improvements to compactness, accessibility, and performance of AO systems. Our experiments were performed on a AO-SLO system which relayed the mouse pupil to a continuous deformable mirror (DM) and Shack-Hartmann wavefront sensor on conjugate planes. The system allows for closed-loop AO as well as the ability measure aberrations during the image-based optimization. We characterized our DM with a WFS in order to use open-loop modal control. The image-based optimization searched the Zernike polynomial coordinate system using a sharpness quality metric. Our results demonstrate diffraction limited performance (according to the WFS) with both closed-loop and image-based methods. The number of iterations required for the image-based method is dependent on the aberrations present as well as the number of dimensions being corrected. Image-based optimization after closed-loop correction can provide further improvements. We are applying these results to improve image-based AO for small animal in-vivo applications.

Light microscopy has been a key tool for biological and medical research for centuries, but the limited penetration depth due to light scattering has restricted its in vivo imaging ability to superficial regions. Nowadays, adaptive optics and active wavefront shaping techniques are increasingly used to compensate sample-induced aberrations in nonlinear optical microscopy. However, in most cases, the wavefront control element, such as deformable mirror, is imaged onto the pupil plane of the microscope objective. This configuration limits the field of view over which spatially irregular aberrations can be corrected. A better choice is to place the wavefront control element, in a plane conjugate to the primary source of aberrations. Here we demonstrate a novel design of a variable-conjugation plane adaptive optics two-photon microscope for deep-tissue bioimaging and systematically investigate all the trade-offs in the design. We use a liquid crystals spatial light modulator for precise control of the initial wavefront. The design of the microscope allows not only to extend the corrected field of view but also to easily adjust the position of the conjugate plane for different imaging depths in a three-dimensional scattering sample. We demonstrate the feasibility of the microscope and the efficiency of aberration cancellation at different depths of up to more than 1 mm. The enhancement of the intensity in the focal spot over the whole volume has been carefully investigated for variety of samples.

Neural-machine interfaces using optogenetics are of interest due to their minimal invasiveness and potential for parallel read in and read out of activity. One possible biological target for such an interface is the peripheral nerve, where axonlevel imaging or stimulation could greatly improve interfacing with artificial limbs or enable neuron/fascicle level neuromodulation in the vagus nerve. Two-photon imaging has been successful in imaging brain activity using genetically encoded calcium or voltage indicators, but in the peripheral nerve, this is severely limited by scattering and aberrations from myelin. We employ a Shack-Hartman wavefront sensor and two-photon excitation guidestar to quantify optical scattering and aberrations in peripheral nerves and cortex. The sciatic and vagus nerves, and cortex from a ChAT-Cre ChR-eYFP transgenic mouse were excised and imaged directly. In peripheral nerves, defocus was the strongest aberration followed by astigmatism and coma. Peripheral nerve had orders of magnitude higher aberration compared with cortex. These results point to the potential of adaptive optics for increasing the depth of two-photon access into peripheral nerves.

To exploit photonics technologies for in vivo studies in life science and biomedicine, it is necessary to efficiently deliver light energy to the target objects embedded deep within complex biological tissues. However, light waves diffuse randomly inside complex media due to multiple scattering, and only a small fraction reaches the target object. Here we present a method to counteract the random diffusion and to focus ‘snake-like’ multiple-scattered waves to the embedded target. To realize this, we experimentally identified time-gated reflection eigenchannels that have extraordinarily large reflectance at a specific flight time where most of the multiple-scattered waves have interacted with the target object. By injecting light to these eigenchannels, we achieved more than 10-fold enhancement in light energy delivery compared to ordinary wave diffusion cases. This method works up to depths of approximately 2 times the transport mean free path at which target objects are completely invisible by ballistic optical imaging. This work will lay a foundation for enhancing the working depth of imaging, sensing, and light stimulation.

Focusing light deep inside and through thick biological tissue is critical to many applications. However, optical scattering prevents light from being focused through thick biological tissue, which restricts biophotonics to a limited depth of about 1 mm. To break this optical diffusion limit, digital optical phase conjugation (DOPC) based wavefront shaping techniques are being actively developed. Previous DOPC systems employed spatial light modulators that modulated either the phase or the amplitude of the conjugate light field. Here, we achieve optical focusing through scattering media by using polarization modulation based generalized DOPC. First, we describe an algorithm to extract the polarization map from the measured scattered field. Then, we validate the algorithm through numerical simulations, and find the focusing contrast achieved by polarization modulation is similar to that achieved by phase modulation, and is higher than those achieved by binary-phase and binary-amplitude modulations. Finally, we build a system using an inexpensive twisted nematic liquid crystal based spatial light modulator, and experimentally demonstrate light focusing through 3-mm thick chicken breast tissue. Since the polarization modulation based SLMs are widely used in displays and are having more and more pixel counts with the prevalence of 4K displays, these SLMs are inexpensive and valuable devices for wavefront shaping. Thus, we anticipate that polarization modulation based SLMs will gain their prevalence in the field of wavefront shaping.

In biological applications, optical focusing is limited by the diffusion of light, which prevents focusing at depths greater than ~1 mm in soft tissue. Wavefront shaping aims to extend the focusing depth by compensating for phase distortions induced by scattering. This allows for focusing light through biological tissue beyond the optical diffusion limit through constructive interference. However, due to random motion, scattering of light in tissue is deterministic only within a brief speckle correlation time. In in vivo tissue this speckle correlation time is on the order of milliseconds, thus it is vital to optimize the wavefront within the correlation time. The speed of wavefront shaping has typically been limited by the time required to measure and display the optimal phase pattern due to the low speeds of cameras, data transfer and processing, and spatial light modulators (SLM). While methods of binary-phase modulation requiring only two images for phase measurement have recently been reported, the majority of studies require a minimum of four frames for full-phase measurement. Here, we present a full-phase digital optical phase conjugation method based on off-axis holography for single-shot optical focusing through scattering media. By using off-axis holography in conjunction with graphics processing unit (GPU) based processing; we take advantage of single-shot full-phase measurement while using parallel computation to quickly reconstruct the phase map. Using this system, we are able to focus light through scattering media with a system latency of approximately 10 milliseconds, on the order of the in vivo speckle correlation time.

Propagation of light through scattering media such as ground glass or biological tissue limits the quality and intensity of focusing point. Wave front shaping technique which uses spatial light modulator (SLM) devices to reshape the field profile of incoming light, is considered as one of the most effective and convenient methods. Advanced biomedical or manufacturing applications require drawing various contours or shapes quickly and precisely. However, creating each shape behind the scattering medium needs different phase profiles, which are time consuming to optimize or measure. Here, we demonstrate a technique to draw various shapes or contours behind the scattering medium by swiftly moving the focus point without any mechanical movements. Our technique relies on the existence of speckle correlation property in scattering media, also known as optical memory effect. In our procedure, we first modulate the phase-only SLM to create the focus point on the other side of scattering medium. Then, we digitally shift the preoptimized phase profile on the SLM and ramp it to tilt the beam accordingly. Now, the incoming beam with identical phase profile shines on the same scattering region at a tilted angle to regenerate the focus point at the desired position due to memory effect. Moreover, with linear combination of different field patterns, we can generate a single phase profile on SLM to produce two, three or more focus points simultaneously on the other side of a turbid medium. Our method could provide a useful tool for prominent applications such as opto-genetic excitation, minimally invasive laser surgery and other related fields.

The central goal in optical wave control is to manipulate light fields so that they fulfil a certain function, such as for imaging, detection and efficient transmission across complex photonic media. To reach this goal, different techniques have been considered, either for shaping an incoming wavefront or for shaping the medium itself. This talk covers two novel insights for both of these two contrasting approaches and their application to disordered media. In the first part of this talk I will speak about how to control wave scattering by delicately designing the refractive index of a scattering medium. In particular, I will show how to completely eliminate the highly fluctuating intensity profile inside a disordered material by adding a tailored gain and loss profile to it. The resulting constant-intensity waves in such non-Hermitian scattering landscapes are free of any backscattering and feature perfect transmission even through highly disordered media [1]. In the second part of this talk, I will present a novel approach for shaping a wave incident on a disordered medium to achieve a focus deep inside of it. This approach is based on a prior measurement of the system's transmission matrix and its derivative with respect to a shift of the target one aims to focus on. I will explain the connection of this novel approach to the concept of "principal modes" and present an experimental realization in the microwave regime [2]. [1] Konstantinos G Makris, Andre Brandstötter, Philipp Ambichl, Ziad H Musslimani, and Stefan Rotter. Wave propagation through disordered media without backscattering and intensity variations. Light: Science & Applications 6, e17035 (2017); doi: 10.1038/lsa.2017.35 [2] Philipp Ambichl, Andre Brandstötter, Julian Böhm, Matthias Kühmayer, Ulrich Kuhl, and Stefan Rotter. Focusing inside disordered media with the generalized Wigner-Smith operator. Phys. Rev. Lett. accepted article; arXiv:1703.07250

Cataracts is a common ocular pathology that increases the amount of intraocular scattering. It degrades the quality of vision by both blur and contrast reduction of the retinal images. In this work, we propose a non-invasive method, based on wavefront shaping (WS), to minimize cataract effects. For the experimental demonstration of the method, a liquid crystal on silicon (LCoS) spatial light modulator was used for both reproduction and reduction of the realistic cataracts effects. The LCoS area was separated in two halves conjugated with the eye’s pupil by a telescope with unitary magnification. Thus, while the phase maps that induced programmable amounts of intraocular scattering (related to cataract severity) were displayed in a one half of the LCoS, sequentially testing wavefronts were displayed in the second one. Results of the imaging improvements were visually evaluated by subjects with no known ocular pathology seeing through the instrument. The diffracted intensity of exit pupil is analyzed for the feedback of the implemented algorithms in search for the optimum wavefront. Numerical and experimental results of the imaging improvements are presented and discussed.

Near infrared and infrared multi-photon imaging through or inside bone is an emerging field that promises to help answer many biological questions that require minimally invasive intravital imaging. Neuroscience researchers especially have begun to take advantage of long wavelength imaging to overcome multiple scattering and image deep inside the brain through intact or partially intact bone. Since the murine model is used in many biological experiments, here we investigate the optical aberrations caused by mouse cranial bone, and their effects on light propagation. We previously developed a ray tracing model that uses second harmonic generation in collagen fibers of bone to estimate the refractive index structure of the sample. This technique is able to rapidly provide initial information for a closed loop adaptive optics system. However, the ray tracing method does not account for refraction or scattering. Here, we extend our work to investigate the wavefront aberrations in bone using a full electromagnetic model. We used Finite-Difference Time-Domain modeling of light propagation in refractive index bone datasets acquired with second harmonic generation imaging. In this paper we show modeled wavefront phase from different originating points across the field of view.

Spatial and temporal properties of an ultrashort pulse of light are naturally scrambled upon propagation in thick scattering media. Significant progresses have been realized over the last decade to manipulate light propagation in scattering media, mostly using monochromatic light. However, applications that require a broadband ultrashort pulse of light remain limited, as the pulse gets temporally broadened because of scattering effects. A monochromatic optical transmission matrix does not allow temporal control of broadband light. Although measuring multiple transmission matrices with spectral resolution allows fine temporal control, it requires lengthy measurements, as well as stability of the medium. In this work, we show that a single linear operator that we named Broadband Transmission Matrix, can be straightforwardly measured for a broadband pulse with a co-propagating reference. We exploit this operator for focusing purposes, and we analyze its phase conjugation properties. While the operator naturally allows for spatial focusing, unexpectedly, the focus duration is on average shorter than the natural temporal broadening due to the medium. More precisely, we observe a two-fold temporal recompression at the focus that we fully explain theoretically. We also explore the spectral content at the focus, and demonstrate a narrowing of the spectrum. These results are particularly relevant for non-linear imaging techniques in biological tissues, at depth where an ultrashort excitation pulse is broadened.

Focusing of light within and beyond a strong scattering disordered medium via wavefront shaping (WFS) using a spatial light modulator (SLM) has been demonstrated for speckle-sized target regions over a decade ago, and has since been explored extensively with potential applications for imaging, phototherapy, communications and cryptography. The intensity enhancement scales linearly with the number, M1, of SLM pixels and is unaffected by any correlations in the speckle field of transmitted or reflected light. However, a fundamental question of great importance for applications is the possibility of focusing to regions much larger than a single speckle, where neglect of correlations would predict a decrease in the enhancement as 1/M2, where M2 is the number of speckles in the target region. It has been known for many years that speckle patterns from highly scattering media have long-range field correlations. We have developed a quantitative theory of the effect of these correlations on large scale focusing and demonstrated excellent agreement between the theory and experiments on the transmission of light through ZnO nanoparticles. The correlations between speckles allows much greater control via WFS of focusing to larger target regions than the simple prediction above. Specifically, for a given sample, each set of experimental illumination conditions defines a measurable dimensionless number, g, which controls focusing efficiency. When the dimensionless size of the target region, M2 > g, significantly enhanced focusing through WFS is possible due to correlations, with the maximal enhancement increasing by M2/g compared to the result in the absence of correlations.

Coherent light propagation in random scattering media such as biological tissue, fog, and turbulent atmosphere is dictated by the eigenchannels of transmission matrices. The spatial profiles of these channels can be exploited for tailoring light-matter interactions inside a turbid medium. While the spatial structures of transmission eigenchannels in diffusive waveguides are extensively studied, most scattering systems in practical applications have an open slab geometry. Here, we present experimental and numerical studies on the spatial profiles of transmission eigenchannels in disordered slabs of thickness much less than the width. We discover that all transmission eigenchannels are localized in the transverse direction (parallel to the slab). The lateral dimension of each channel increases linearly with the slab thickness and the transport mean free path. Such localization, which are absent for the transmission eigenchannels in quasi-one-dimensional samples, originate from spatial disorder, partial mixing of spatial channels, and non-local correlations of waves in the slab. Experimentally not all input channels can be controlled, and usually only the phase of incident beam is modulated. In this case, light injected to a high-transmission channel remains laterally localized, but the beam in a low-transmission eigenchannel expands laterally as it propagates through the slab. Our results provide physical insight to the transmission eigenchannels in open disordered systems, therefore paving the way for their applications in optical imaging and communication.

We present a novel imaging system that combines the principles of Fourier spatial filtering and single-pixel imaging in order to recover images of an object hidden behind a turbid medium by transillumination. We compare the performance of our single-pixel imaging setup with that of a conventional system. We conclude that the introduction of Fourier gating improves the contrast of images in both cases. Furthermore, we show that the combination of single-pixel imaging and Fourier spatial filtering techniques is particularly well adapted to provide images of objects transmitted through scattering media.

When imaging with classical waves, multiple scattering (MS) is often seen as an unavoidable obstacle. The diffraction-limited resolution obtainable with methods such as microscopy requires that single-scattering (SS) dominates; for depths where MS processes become important, such methods result in an image without any connection to the reflectivity of the medium. Conversely, techniques such as diffuse optical tomography take advantage of the diffuse nature of light, but their resolution power is limited. To do better, methods such as wavefront shaping and adaptive optics have been developed. Focussing through a thick diffusive layer was demonstrated using a transmission matrix approach consisting of the measurement of Green’s functions between each pixel of a spatial light modulator (SLM) and of a charge-coupled device (CCD) camera across the medium. To image inside a multiple-scattering medium, we present a matrix approach based on the experimental measurement of a reflection matrix from the medium. An analysis based on the geometric and statistical properties of this reflection matrix can enhance the SS contribution which would otherwise be swamped by MS at large depths, and correct the resulting image for aberration effects induced by the turbid medium itself. The correction does not require the presence of bright scatterers, does not rely on any feedback loop and works even at depths where the field-of-view contains several isoplanatic patches. Here we present the application of our reflection matrix approach to optical imaging in biological tissues. Compared to OCT and related methods, we demonstrate an extension of the current imaging-depth limit.

Wavefront shaping techniques are being actively developed to achieve optical focusing through and inside opaque scattering media. These techniques promise to revolutionize biophotonics by enabling deep-tissue non-invasive optical imaging, optogenetics, optical tweezing, and light-based therapy. Among the existing wavefront shaping techniques, optical time-reversal-based techniques determine the optimum wavefront globally based on the principle of time reversal, without the need to perform time-consuming iterations to optimize each mode in sequence. In all previous optical time-reversal-based wavefront shaping experiments, Nyquist sampling criterion was followed so that the scattered light field was well-sampled during wavefront measurement and wavefront reconstruction. In this work, we overturn this conventional practice by demonstrating that a high-quality optical focus can still be achieved even when the scattered light field is under-sampled. Even more strikingly, we show both theoretically and experimentally that the focus achieved by the under-sampling scheme can be one order of magnitude brighter than that achieved by the well-sampling schemes used in previous works, where 3×3 to 5×5 pixels sampled one speckle grain on average. Moreover, since neighboring pixels were uncorrelated in feedback-based wavefront shaping, introducing the concept of sub-Nyquist sampling in time-reversal-based wavefront shaping makes the optimal phase maps obtained using these two different methods consistent. We anticipate that this newly explored under-sampling scheme will transform the understanding of optical time reversal and boost the performance of optical imaging, manipulation, and communication through opaque scattering media.

The problem of optical scattering was long thought to fundamentally limit the depth at which light could be focused through turbid media such as fog or biological tissue. However, recent work in the field of wavefront shaping has demonstrated that by properly shaping the input light field, light can be noninvasively focused to desired locations deep inside scattering media. This has led to the development of several new techniques which have the potential to enhance the capabilities of existing optical tools in biomedicine. Unfortunately, extending these methods to living tissue has a number of challenges related to the requirements for noninvasive guidestar operation, speed, and focusing fidelity. Of existing wavefront shaping methods, time-reversed ultrasonically encoded (TRUE) focusing is well suited for applications in living tissue since it uses ultrasound as a guidestar which enables noninvasive operation and provides compatibility with optical phase conjugation for high-speed operation. In this paper, we will discuss the results of our recent work to apply TRUE focusing for optogenetic modulation, which enables enhanced optogenetic stimulation deep in tissue with a 4-fold spatial resolution improvement in 800-micron thick acute brain slices compared to conventional focusing, and summarize future directions to further extend the impact of wavefront shaping technologies in biomedicine.

Three dimensional (3D) fluorescence microscopy has been essential for biological studies. It allows interrogation of structure and function at spatial scales spanning the macromolecular, cellular, and tissue levels. Critical factors to consider in 3D microscopy include spatial resolution, signal-to-noise (SNR), signal-to-background (SBR), and temporal resolution. Maintaining high quality imaging becomes progressively more difficult at increasing depth (where optical aberrations, induced by inhomogeneities of refractive index in the sample, degrade resolution and SNR), and in thick or densely labeled samples (where out-of-focus background can swamp the valuable, in-focus-signal from each plane). In this report, we introduce our new instrumentation to address these problems. A multiphoton structured illumination microscope was simply modified to integrate an adpative optics system for optical aberrations correction. Firstly, the optical aberrations are determined using direct wavefront sensing with a nonlinear guide star and subsequently corrected using a deformable mirror, restoring super-resolution information. We demonstrate the flexibility of our adaptive optics approach on a variety of semi-transparent samples, including bead phantoms, cultured cells in collagen gels and biological tissues. The performance of our super-resolution microscope is improved in all of these samples, as peak intensity is increased (up to 40-fold) and resolution recovered (up to 176±10 nm laterally and 729±39 nm axially) at depths up to ~250 μm from the coverslip surface.

The light-sheet fluorescence microscopy is an excellent tool for the investigation of large three dimensional microscopy samples at the cellular level, however, the ability to resolve features is strongly affected by the presence of scattering and aberrations. These effects are two fold in light-sheet microscopy, as the illumination path providing the optical sectioning and the fluorescence detection path are both affected by the aberrations in different ways. To overcome these difficulties, we have developed hybrid adaptive optical and computational microscopy techniques to remove the effect of the aberrations in both the excitation and the fluorescence paths of these microscopes.

Research trends in endoscopy have been to reduce the dimension of the system for minimally invasive diagnostics and to improve spatial resolution to the microscopic level for the detailed investigation of specimens. In developing endoscopes that meet these needs, ultrathin imaging probes such as graded index lenses and fiber bundles have been widely used. And a single imaging probe is used for both illumination and detection to maintain the small diameter of the probe unit. However, this causes a fundamental problem, that is the back-reflection noise from the surface of the imaging probes. This back-reflection noise can overwhelm signals from target objects with weak contrast, which is the case for biological tissues, and degrade image contrast to such an extent that the objects remain unresolved. Here, we present an endomicroscope free from back-reflection noise generated at an ultrathin imaging probe and yet guaranteeing microscopic spatial resolution. In our method, we send illumination through single individual core fibers in the image fiber bundle, and detect signal light by the other core fibers. By blocking the back-reflection occurring only at the core used for the illumination, we remove the back-reflection noise before it reaches the detector sensor. The transmission matrix of the fiber bundle is measured and used to reconstruct a pixelation-free and high-resolution image from the raw images captured by the other fibers, which are blurred and pixelated. We demonstrated that the proposed imaging method improved 3.2 times on the signal to noise ratio produced by the conventional illumination-detection scheme.

Two-photon microscopy has become a powerful tool in neuroscience as it can image and manipulate neural circuits in vivo with cellular resolution. But in conventional two-photon microscopes, a single laser beam scans regions of interest on the sample within a two-dimensional plane. This serial scanning constrains the temporal resolution of imaging and photostimulation. One way to overcome this limit is to increase the number of beamlets on the sample. Here, we discuss our recent progress on holographic beam multiplexing in two-photon microscopy using spatial light modulators (SLMs). The SLM generates a 3D holographic excitation pattern, targeting different cells on the sample simultaneously. In transparent samples such as zebrafish larva where wide field detection can be used, groups of neurons in 3D volume can be imaged simultaneously, and their fluorescence signals can be recorded with extended depth of view techniques. In scattering samples such as mice cortex which needs laser scanning for imaging, multiple planes can be imaged simultaneously, with the signals from different planes being separated by novel statistical algorithms. Using this approach, we also recorded neural activity across multiplanes in moving Hydra. Besides imaging, 3D optogenetics can be performed. We demonstrate 3D patterned photoactivation of groups of target neurons on mice cortex in vivo, while simultaneously monitoring activity of the neural network. Furthermore, spatial light modulators can switch patterns in high speed, facilitating time-multiplexing. SLM-based two-photon microscopy is thus an all-optical platform to study neural circuits in 3D.

Recent progress in controlling light propagation in multimode fibers in the linear regime, opened new opportunities for multimode fiber endoscopy. However, nonlinear light propagation in multimode fibers comprises complex intermodal interactions and rich spatiotemporal dynamics. In this work, we demonstrate a wave-front shaping approach for controlling nonlinear phenomena in multimode fibers. Using a spatial light modulator at the fiber’s input and a genetic algorithm optimization, we control a highly nonlinear stimulated Raman scattering cascade and its interplay with four wave mixing via a flexible implicit control on the superposition of modes that are coupled into the fiber. We demonstrate versatile spectrum manipulations that could be used to generate a multi-wavelength, tunable source. The wavefront shaping control allows spectral shifting and modal tuning. A theoretical analysis of modal phase matching in graded index multi-mode fibers is presented and we suggest potential bio-imaging applications.

Coherent fiber bundles (CFB) are commonly used for endoscopic imaging, e.g. in biomedicine. Usually a CFB with several ten thousand cores is employed together with a lens system on its distal end. However, pixelation effects occur and the imaging plane can’t be scanned, limiting the field of application of CFBs. To circumvent these limitations, a spatial light modulator (SLM) is employed on the proximal side of a single-mode CFB. This enables creating arbitrary wave fronts at the distal fiber end, e.g. for instance for optical tweezers, endoscopes with tunable image plane or for exciting transgenetic nerve cells. However, of the shelf CFBs show phase distortions between individual cores (e.g. coupling between cores, speckle effect) which need to be calibrated and corrected at the proximal side. These distortions depend on the wavelength, temperature, polarization and most importantly on the bending of the CFB. Therefore an on-line calibration during bending variations is required. For this purpose a semitransparent mirror is employed at the distal fiber end, which allows to measure double the distortion at the proximal side by digital holography without the need for a guide star. For correcting the distortion the same SLM as above is employed. However, the distortion for a single transmission through the CFB commonly exceeds several 2 pi. Thus, an incremental phase measurement yields unambiguous results. To circumvent this problem, two approaches for on-line calibration are compared. 1st Multiple wavelength holography and 2nd initial calibration in transmission mode with subsequent tracking of distortion changes in reflecting mode.

Progress in the domain of complex photonics enabled a new generation of minimally invasive, high-resolution endoscopes by substitution of the Fourier-based image relays with a holographic control of light propagating through apparently randomizing multimode optical waveguides. This form of endo-microscopy became recently a very attractive way to provide minimally invasive insight into hard-to-access locations within living objects. Here, we review our fundamental and technological progression in this domain and introduce several applications of this concept in bio-medically relevant environments. By taking advantage of the cylindrical symmetry of the fibre and the known distribution of the refractive index, we show how to simplify measurement of the transmission matrix of such fibres and correct for the influences of bending deformations. Our newest addition is the employment of Graded-index fibres, which, based on our numerical model, and first experimental verifications, allow for much simpler compensation of bending deformation when compared to step-index fibres. Lastly we show the development and exploitation of highly specialized fiber probes for optical manipulation. We show that light control through these fibres allows sufficiently tight focusing for confinement and manipulation of large particle arrays, and their positioning with nanometric precision.

We demonstrate high power ultrashort pulse delivery through a commercially available multicore fiber (MCF) and a multimode graded-index fiber (GRINF) for imaging and laser ablation. Lensless focusing and digital scanning of ultrashort pulses through the optical fibers is realized using wavefront shaping. We compare the performance of the two systems in terms of focusing efficiency and peak power delivery. Furthermore, we investigate the limitations that nonlinearities induce when high peak power ultrashort pulses are launched in MCFs and GRIN fibers. Proximally-only controlled two-photon fluorescence imaging and laser ablation are demonstrated through both investigated systems.

Multimode fibers are a promising tool for high resolution, low-cost, minimally invasive endoscopic imaging. The fiber can be used both to illuminate the sample, which may be buried deep inside the tissue, and to collect the backreflected light. Except for the bare fiber, no other imaging optics have to be inserted, enabling a device with a very small diameter. However, light propagating through the fiber is scrambled before it hits the sample. This renders straightforward imaging impossible, but if this scrambling is known with high accuracy, for instance because the transmission matrix has been measured, the scrambling process can be compensated before the light enters the fiber. For step index multimode fibers, where the refractive index profile consists of a cylindrical core with a constant but higher refractive index than the cladding, it has been shown that the transmission matrix can be predicted for any fiber orientation. Graded index fibers (GIF), where the refractive index profile resembles a parabola, offer numerous advantages, most prominently they are much less sensitive to bending. We measured the transmission matrix of a large GIF and show that we can fully understand the transmission matrix in terms of guided fiber modes, and simultaneously acquire accurate knowledge of the refractive index profile. We also show that although the quality of a commercially available graded index fiber is not sufficient to perform the same analysis, imaging performance of a graded index fiber is much more resilient to bending than the imaging performance of a comparable step index fiber. This demonstrates the need for a graded-index fiber with a high quality refractive index profile.

In life sciences, interest in the microscopic imaging of increasingly complex three dimensional samples, such as cell spheroids, zebrafish embryos, and in vivo applications in small animals, is growing quickly. Due to the increasing complexity of samples, more and more life scientists are considering the implementation of adaptive optics in their experimental setups. While several approaches to adaptive optics in microscopy have been reported, it is often difficult and confusing for the microscopist to choose from the array of techniques and equipment. In this poster presentation we offer a small guide to adaptive optics providing general guidelines for successful adaptive optics implementation.

We present the combined use of large aperture adaptive lens with large optical power modulation with a multi actuator adaptive lens. The Multi-actuator Adaptive Lens (M-AL) can correct up to the 4th radial order of Zernike polynomials, without any obstructions (electrodes and actuators) placed inside its clear aperture.

We demonstrated that the use of both lenses together can lead to better image quality and to the correction of aberrations of adaptive optics optical systems.