This PDF file contains the front matter associated with SPIE Proceedings Volume 9335, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Two-photon excitation laser scanning microscopy has enabled us to visualize deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations, which degrade the spatial resolution and the fluorescent signal during observation, especially at deeper regions. Recently, we developed transmissive liquid crystal devices for correcting a certain spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. The devices were inserted in front of the objective lens and supplied with appropriate voltages according to the observation depth. In our previous study, while the devices actually recovered the axial resolution and the fluorescent signal, which were degraded by artificially induced aberrations, those performances were not sufficient for practical use. In this paper, in order to improve the imaging performance of the devices and the objective lens, we first performed more precise numerical calculations. Next, we modified the design of the devices and evaluated these performances by observing fluorescent beads in a single-photon excitation laser scanning microscope. For a 25x water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, these modifications recovered the spatial resolution, and the fluorescent signal degraded within ±0.6 mm depth. Finally, we introduced these modified devices to a conventional two-photon excitation laser scanning microscope and succeeded in improving the spatial resolution; additionally, the fluorescent signal degraded in the same region. Therefore, our devices are expected to be useful for observing much deeper regions within a biospecimen.

Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: wavefront sensor, wavefront corrector and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, e.g., lenslet arrays for sensing or multi-acuator deformable mirrors for correcting. We have previously introduced an alternate approach to adaptive optics based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile is possible not only with the conventional coherent type of digital holography, but also with a new type of digital holography using incoherent light: self-interference incoherent digital holography (SIDH). The SIDH generates complex – i.e. amplitude plus phase – hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using a guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. The adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.

High-resolution real-time tomography of biological tissues is important for many areas of biological investigations and medical applications. Cellular level optical tomography, however, has been challenging because of the compromise between transverse imaging resolution and depth-of-field, the system and sample aberrations that may be present, and the low imaging sensitivity deep in scattering tissues. The use of computed optical imaging techniques has the potential to address several of these long-standing limitations and challenges. Two related techniques are interferometric synthetic aperture microscopy (ISAM) and computational adaptive optics (CAO). Through three-dimensional Fourierdomain resampling, in combination with high-speed OCT, ISAM can be used to achieve high-resolution in vivo tomography with enhanced depth sensitivity over a depth-of-field extended by more than an order-of-magnitude, in realtime. Subsequently, aberration correction with CAO can be performed in a tomogram, rather than to the optical beam of a broadband optical interferometry system. Based on principles of Fourier optics, aberration correction with CAO is performed on a virtual pupil using Zernike polynomials, offering the potential to augment or even replace the more complicated and expensive adaptive optics hardware with algorithms implemented on a standard desktop computer. Interferometric tomographic reconstructions are characterized with tissue phantoms containing sub-resolution scattering particles, and in both ex vivo and in vivo biological tissue. This review will collectively establish the foundation for high-speed volumetric cellular-level optical interferometric tomography in living tissues.

We demonstrate a compact GPU accelerated digital holography system for beam-shaping applications at the end of multimode fibre. The complete control of phase and amplitude on phase-only SLM is achieved through GPU implemented Gerchberg-Saxton and Yang-Gu algorithms. The GPU implementation is two orders of magnitude faster than the CPU implementation which allows video-rate image control at the distal end of the fiber virtually free of interference effects. We discuss the implementation of the algorithms and possible applications of the technology for structured illumination imaging at the end of multimode fibre

The pseudospectral time-domain (PSTD) simulation technique is employed to obtain numerical solutions of Maxwell’s equations. Amplitude and phase of the outgoing light is recorded and later used to generate phase-conjugated light which back-propagates through the scattering medium. By changing the angular span of the region of phase-conjugated light, we can analyze back-propagation of light for different angular spans. The simulation results may help determine the optimal angular span for practical back-propagation of light.

Adaptive optics (AO) can potentially allow high resolution imaging deep inside living tissue, mitigating against the loss of resolution due to aberrations caused by overlying tissue. Closed-loop AO correction is particularly attractive for moving tissue and spatially varying aberrations, but this requires direct wavefront sensing, which in turn requires suitable "guide stars" for use as wavefront references. We present a novel method for generating an orthogonally illuminated guide star suitable for direct wavefront sensing in a wide range of fluorescent biological structures, along with results demonstrating its use for measuring time-varying aberrations, in vivo.

Wide-field fluorescence microscope techniques such as single/selective plane illumination microscope (SPIM) are typically configured to image large regions of a sample at once. Here the illumination beam provides uniform excitation of several biological features across the region, `sliced' to a thickness of between 5-10 microns. In this paper we propose a simple alteration to the optical configuration of a SPIM by switching the light-sheet- forming cylindrical lens with a spatial light modulator. This has the potential to adaptively reconfigure the light sheet geometry to improve the optical sectioning of specific biological features, rather than the thicker sectioning of several features at once across a larger observation field-of-view. We present a prototype version of such a system, referred to as an Adaptive-SPIM (A-SPIM) system. We then suggest that the direct recording of illumination beam shapes within the working microscope system can better facilitate the analysis and subsequent re-configuration of the illumination beam to a specific geometry, and summarise the design and operation of a device that we have developed specifically for this purpose. We finally present reconstructed quantitative three dimensional flux maps of illumination beams from three microscope configurations taken using this miniature high-dynamic range beam profiling device, comparing the beam geometry of a regular SPIM system with our prototype A-SPIM system, and suggesting future improvements.

The use of Adaptive Optics (AO) to correct for aberrations in a wavefront of propagating light has become customary for Astronomical applications and is now expanding to many other areas going from medical imaging to industrial applications. However, the propagation of light underwater has remained out of the main stream AO community for a variety of reasons, not least the shear difficulty of the situation. Our group has become a program that attempts to define under which circumstances such a correction could be envisioned. We take advantage of the NRL laboratory facility in Stennis, MS, where a large Plexiglas tank of water is equipped with heating and cooling plates that allow for a well measured thermal gradient that in turn generates different degrees of turbulence that can distort a propagating laser beam. In this paper we report on the preliminary findings of this ongoing program. The paper will describe the facility and the AO test-bed, the measurements made and some of the preliminary result.

A novel minimally invasive micro-endoscopes utilizing disordered light within a standard multimode optical fibre have been introduced recently. The two most important limitations of this exciting technology are (i) the lack of bending flexibility (transformation matrix is only valid as long as the fibre remains stationary) and (ii) high demands on computational power, making the performance of such systems slow. Here we discuss possible routes to address the later one: We introduce a GPU toolbox to make this powerful technique faster and more accessible to bio-medical researchers.

Wavefront shaping through multimode fibers has many interesting applications, such as micromanipulation and endoscopy, where the small diameter of fibers is an advantage in terms of miniaturization. However, pattern projection can suffer from deleterious interference effects depending on the measurement conditions. We experimentally demonstrate the projection of high quality patterns through a multimode fiber by correcting for phase drifts while measuring the transmission matrix, and by controlling both the amplitude and the phase of the wavefront sent to the fiber. We also use the matrix in the reverse direction by reconstructing a distal image from a proximally measured speckle field. This opens up new possibilities for imaging through a multimode fiber by decoding wavefronts travelling both ways along the fiber.

Multimode optical fibers potentially allow the transmission of larger amounts of information than their single mode counterparts because of their high number of supported modes. However, propagation of a light pulse through a multimode fiber suffers from spatial distortions due to the superposition of the various exited modes and from time broadening due to modal dispersion. We present a method based on digital phase conjugation to selectively excite in a multimode fiber specific optical fiber modes that follow similar optical paths as they travel through the fiber. The excited modes interfere constructively at the fiber output generating an ultrashort spatially focused pulse. The excitation of a limited number of modes following similar optical paths limits modal dispersion, allowing the transmission of the ultrashort pulse. We have experimentally demonstrated the delivery of a focused spot of pulse width equal to 500 fs through a 30 cm, 200 micrometer core step-index multimode fiber. The results of this study show that two-photon imaging capability can be added to ultra-thin lensless endoscopy using commercial multimode fibers.

Optical Aberrations are a major challenge in imaging biological samples. In particular, in single molecule localization (SML) microscopy techniques (STORM, PALM, etc.) a high Strehl ratio point spread function (PSF) is necessary to achieve sub-diffraction resolution. Distortions in the PSF shape directly reduce the resolution of SML microscopy. The system aberrations caused by the imperfections in the optics and instruments can be compensated using Adaptive Optics (AO) techniques prior to imaging. However, aberrations caused by the biological sample, both static and dynamic, have to be dealt with in real time. A challenge for wavefront correction in SML microscopy is a robust optimization approach in the presence of noise because of the naturally high fluctuations in photon emission from single molecules. Here we demonstrate particle swarm optimization for real time correction of the wavefront using an intensity independent metric. We show that the particle swarm algorithm converges faster than the genetic algorithm for bright fluorophores.

We describe the design and performance of a recently implemented retinal imaging system for the human eye that combines adaptive optics (AO) with spectral domain optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO). The AO-OCT-SLO system simultaneously acquires SLO frames and OCT B-scans at 60 Hz with an OCT volume acquisition scan rate of 0.24 Hz. The SLO images are used to correct for eye motion during the registration of OCT B-scans. Key optical design considerations are discussed including: minimizing system aberrations through the use of off-axis relay telescopes; choice of telescope magnification based on pupil plane requirements and restrictions; and the use of dichroic beam splitters to separate and re-combine OCT and SLO beams around the nonshared horizontal scanning mirrors. We include an analysis of closed-loop AO correction on a model eye and compare these findings with system performance in vivo. The 2D and 3D OCT scans included in this work demonstrate the ability of this system to laterally and axially resolve individual cone photoreceptors, while the corresponding SLO images show the en face mosaics at the photoreceptor layer showing rods and cones. Images from both healthy and diseased retina are presented.

We have implemented an extended depth of field optical system by wavefront coding with a micromachined membrane deformable mirror. This approach provides a versatile extension to standard wavefront coding based on fixed phase mask. First experimental results validate the feasibility of the use of adaptive optics for variable depth wavefront coding in imaging optical systems.

Binary amplitude modulation promises to allow rapid focusing through strongly scattering media with a large number of segments due to the faster update rates of digital micromirror devices (DMDs) compared to spatial light modulators (SLMs). While binary amplitude modulation has a lower theoretical enhancement than phase modulation, the faster update rate should more than compensate for the difference – a factor of π² /2. Here we present two new algorithms, a genetic algorithm and a transmission matrix algorithm, for optimizing the focus with binary amplitude modulation that achieve enhancements close to the theoretical maximum. Genetic algorithms have been shown to work well in noisy environments and we show that the genetic algorithm performs better than a stepwise algorithm. Transmission matrix algorithms allow complete characterization and control of the medium but require phase control either at the input or output. Here we introduce a transmission matrix algorithm that works with only binary amplitude control and intensity measurements. We apply these algorithms to binary amplitude modulation using a Texas Instruments Digital Micromirror Device. Here we report an enhancement of 152 with 1536 segments (9.90%×N) using a genetic algorithm with binary amplitude modulation and an enhancement of 136 with 1536 segments (8.9%×N) using an intensity-only transmission matrix algorithm.

Precise control of light propagation through highly scattering media is a much desired goal with major technological implications. Since interaction of light with turbid media results in partial or complete depletion of ballistic photons, it is in principle impossible to transmit images through distances longer than the extinction length. In biomedical optics, scattering is the dominant light extinction process accounting almost exclusively for the limited imaging depth range. In addition, most scattering media of interest are dynamic in the sense that the scatter centers continuously change their positions with time. In our work, we employ single-pixel systems, which can overcome the fundamental limitations imposed by multiple scattering even in the dynamically varying case. A sequence of microstructured light patterns codified onto a programmable spatial light modulator are used to sample an object and measurements are captured with a single-pixel detector. Acquisition time is reduced by using compressive sensing techniques. The patterns are used as generalized measurement modes where the object information is expressed. Contrary to the techniques based on the transmission matrix, our approach does not require any a-priori calibration process. The presence of a scattering medium between the object and the detector scrambles the light and mixes the information from all the regions of the sample. However, the object information that can be retrieved from the generalized modes is not destroyed. Furthermore, by using these techniques we have been able to tackle the general problem of imaging objects completely embedded in a scattering medium.

Light scattering is known for blurring images to the point of making them appear as a white halo. For this reason imaging through thick clouds or deep into biological tissues is difficult. Here we discuss in details a method we developed recently to retrieve the shape of an object hidden behind a diffusing screen.

We present a novel adaptive lens that can correct high order aberrations, and which has the potential to increase the diffusion of adaptive optics to many new applications by simplifying the integration of a wavefront corrector inside existing systems. The adaptive lens design that we present can correct for Zernike wavefront aberrations up to the 4th order. The adaptive lens performance are compared with the one of a membrane and bimorph deformable mirrors used together in a wide field microscope setup, using a Shack-Hartmann wavefront sensor for closed loop control. The adaptive lens was also integrated with Fourier Domain Optical Coherence Tomography for in-vivo imaging of mouse retinal structures using an image based wavefront sensorless control.