Fully refractive telecentric f-theta microscope based on adaptive elements for 3D raster scanning of biological tissues.

Various techniques in microscopy are based on point-wise acquisition, which provides advantages in acquiring sectioned images, for example in confocal or two-photon microscopy. The advantages come along with the need to perform three-dimensional scanning, which is often realized by mechanical movement achieved by stage-scanning or piezo-based scanning in the axial direction. Lateral scanning often employs galvo-mirrors, leading to a reflective setup and hence to a folded beam path. In this paper, we introduce a fully refractive microscope capable of three-dimensional scanning, which employs the combination of an adaptive lens, an adaptive prism, and a tailored telecentric f-theta objective. Our results show that this microscope is capable to perform flexible three-dimensional scanning, with low scan-induced aberrations, at a uniform resolution over a large tuning range of X=Y=6300 µ m and Z=480 µ m with only transmissive components. We demonstrate the capabilities at the example of volumetric measurements on the transgenic fluorescence of the thyroid of a zebrafish embryo and mixed pollen grains. This is the first step towards flexible aberration-free volumetric smart microscopy of three-dimensional samples like embryos and organoids, which could be exploited for the demands in both lateral and axial dimensions in biomedical samples without compromising image quality.

Nonlinear microscopy using impulsive stimulated Brillouin scattering for high-speed elastography.

The impulsive stimulated Brillouin microscopy promises fast, non-contact measurements of the elastic properties of biological samples. The used pump-probe approach employs an ultra-short pulse laser and a cw laser to generate Brillouin signals. Modeling of the microscopy technique has already been carried out partially, but not for biomedical applications. The nonlinear relationship between pulse energy and Brillouin signal amplitude is proven with both simulations and experiments. Tayloring of the excitation parameters on the biologically relevant polyacrylamide hydrogels outline sub-ms temporal resolutions at a relative precision of <1%. Brillouin microscopy using the impulsive stimulated scattering therefore exhibits high potential for the measurements of viscoelastic properties of cells and tissues.

Surveillance of few-mode fiber-communication channels with a single hidden layer neural network.

Multi- and few-mode fibers (FMFs) promise to enhance the capacity of optical communication networks by orders of magnitude. The key for this evolution was the strong advancement of computational approaches that allowed inherent complex light transmission to be surpassed, learned, or controlled, reined in by modal crosstalk and mode-dependent losses. However, complex light transmission through FMFs can be learned by a single hidden layer neural network (NN). The emerging developments in NNs additionally allow the implementation of novel concepts for security enhancements in optical communication. Once the transmission characteristics of FMFs are learned, it is possible to survey the incoming and outgoing light fields via monitoring channels during data transmission. If an eavesdropper tries to gain unauthorized access to the FMF, its transmission properties are impaired through sensitive modal crosstalk. This process is registered by the NN and thus the eavesdropper is revealed. With our solution, the security of optical communication can be improved.

Distortion correction for particle image velocimetry using multiple-input deep convolutional neural network and Hartmann-Shack sensing.

Aberrations degrade the accuracy of quantitative, imaging-based measurements, like particle image velocimetry (PIV). Adaptive optical elements can in principle correct the wavefront distortions, but are limited by their technical specifications. Here we propose an actuator-free correction based on a multiple-input deep convolutional neural network which uses an additional input from a wavefront sensor to correct time-varying distortions. It is applied for imaging flow velocimetry to conduct measurements through a fluctuating air-water phase boundary. Dataset for neural network is generated by an experimental setup with a deformable mirror. Correction performance of trained model is estimated in terms of image quality, which is improved significantly, and flow measurement results, where the errors induced by the distortion from fluctuating phase boundary can be corrected by 82 %. The technique has the potential to replace classical closed-loop adaptive optical systems where the performance of the actuators is not sufficient.

Benchmarking analysis of computer generated holograms for complex wavefront shaping using pixelated phase modulators.

Wavefront shaping with spatial light modulators (SLMs) enables aberration correction, especially for light control through complex media, like biological tissues and multimode fibres. High-fidelity light field shaping is associated with the calculation of computer generated holograms (CGHs), of which there are a variety of algorithms. The achievable performance of CGH algorithms depends on various parameters. In this paper, four different algorithms for CGHs are presented and compared for complex light field generation. Two iterative, double constraint Gerchberg-Saxton and direct search, and the two analytical, superpixel and phase encoding, algorithms are investigated. For each algorithm, a parameter study is performed varying the modulator's pixel number and phase resolution. The analysis refers to mode field generation in multimode fibre endoscopes and communication. This enables generality by generating specific mode combinations according to certain spatial frequency power spectra. Thus, the algorithms are compared varying spatial frequencies applied to different implementation scenarios. Our results demonstrate that the choice of algorithms has a significant impact on the achievable performance. This comprehensive study provides the required guide for CGH algorithm selection, improving holographic systems towards multimode fibre endoscopy and communications.

Velocity measurements with structured light transmitted through a multimode optical fiber using digital optical phase conjugation.

Lensless fiber microendoscopes enable optical diagnostics and therapy with minimal invasiveness. Because of their small diameters, multimode fibers are ideal candidates, but mode scrambling hinders the transmission of structured light fields. We present the generation of a localized fringe system at variable distances from the distal fiber end by exploiting digital optical phase conjugation. The replayed fringe system was used for quantitative metrology. Velocity measurements of a microchannel flow in the immediate proximity of the fiber end without the use of any imaging lenses are shown. Lensless multimode fiber systems are of interest especially for biomedical imaging and stimulation as well as technical inspection and flow measurements.

Video-rate lensless endoscope with self-calibration using wavefront shaping.

Lensless fiber endoscopes are of great importance for keyhole imaging. Coherent fiber bundles (CFB) can be used in endoscopes as remote phased arrays to capture images. One challenge is to image at high speed while correcting aberrations induced by the CFB. We propose the combination of digital optical phase conjugation, using a spatial light modulator, with fast scanning, for which a 2D galvo scanner and an adaptive lens are employed. We achieve the transmission of laser and image scanning through the CFB. Video-rate imaging at 20 Hz in 2D with subcellular resolution is demonstrated in 3D with 1 Hz. The sub-millimeter-diameter scanning endoscope has a great potential in biomedicine, for manipulation, e.g., in optogenetics, as well as in imaging.

Diffraction-grating-based in situ displacement, tilt, and strain measurements on high-speed composite rotors.

Polymer composite rotors offer promising perspectives in high-speed applications such as turbomachinery. However, failure modeling is a challenge due to the material's anisotropy and heterogeneity, which makes high-speed in situ deformation measurements necessary. The challenge is to maintain precision and accuracy in the environment of fast rigid-body movement. A diffraction-grating-based sensor is used for spatio-temporally resolved displacement, tilt, and strain measurements at surface velocities up to 260 m/s with statistical strain uncertainties down to $16\,\,\unicode{x00B5}{\epsilon}$. As a line camera is used, vibrations in the kHz range are measurable in principle. Due to sensor calibration and the use of a novel scan-correlation analysis approach, the rigid-body-movement-induced uncertainties are reduced significantly. The measurement of strain fluctuations on a rotating composite disc show that the crack propagation can be tracked spatially resolved and as a function of the rotational speed, which makes an in situ quantification of the damage state of the rotor possible.

Mechanical Mapping of Spinal Cord Growth and Repair in Living Zebrafish Larvae by Brillouin Imaging.

The mechanical properties of biological tissues are increasingly recognized as important factors in developmental and pathological processes. Most existing mechanical measurement techniques either necessitate destruction of the tissue for access or provide insufficient spatial resolution. Here, we show for the first time to our knowledge a systematic application of confocal Brillouin microscopy to quantitatively map the mechanical properties of spinal cord tissues during biologically relevant processes in a contact-free and nondestructive manner. Living zebrafish larvae were mechanically imaged in all anatomical planes during development and after spinal cord injury. These experiments revealed that Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animal's natural development. The Brillouin shift within the spinal cord remained comparable during development and transiently decreased during the repair processes after spinal cord transection. By taking into account the refractive index distribution, we explicitly determined the apparent longitudinal modulus and viscosity of different larval zebrafish tissues. Importantly, mechanical properties differed between tissues in situ and in excised slices. The presented work constitutes the first step toward an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a methodical basis to identify key determinants of mechanical tissue properties, and allows us to test their relative importance in combination with biochemical and genetic factors during developmental and regenerative processes.

Self-calibration of lensless holographic endoscope using programmable guide stars.

Coherent fiber bundle (CFB)-based endoscopes enable optical keyhole access in applications such as biophotonics. In conjunction with objective lenses, CFBs allow imaging of intensity patterns. In contrast, digital optical phase conjugation enables lensless holographic endoscopes for the generation of pixelation-free arbitrary light patterns. For real-world applications, however, this requires a non-invasive in situ calibration of the complex optical transfer function of the CFB with only single-sided access. We show that after an initial calibration in a forward direction, a differential phase measurement of the back-reflected light allows for tracking and compensating of bending-induced phase distortions. Furthermore, we present a novel in situ calibration procedure based on a programmable guide star, which requires access to only one side of the fiber.

Adaptive laser-induced ultrasound generation using a micro-mirror array spatial light modulator.

Laser ultrasonics is a powerful technique for contactless investigation of important material parameters such as Young's modulus or thin layer thickness. However, the often employed Gaussian beams result in diverging sound fields of quickly decreasing intensity. Conventionally, changing the laser beam profile requires the slow movement or exchange of optical elements. We present a laser ultrasonics setup for the creation of arbitrary intensity distributions by holographic projection using a MEMS spatial light modulator. High-intensity ultrasound foci with a focus width of 1.6 mm are scanned axially in a sample into depths of up to 7.4 mm by projecting ring-shaped intensity distributions of varying diameter without any mechanical movements. This technique is promising for highly spatially resolved flaw detection or a fast scanning investigation of biological tissue.

Camera-based speckle noise reduction for 3-D absolute shape measurements.

Simultaneous position and velocity measurements enable absolute 3-D shape measurements of fast rotating objects for instance for monitoring the cutting process in a lathe. Laser Doppler distance sensors enable simultaneous position and velocity measurements with a single sensor head by evaluating the scattered light signals. The superposition of several speckles with equal Doppler frequency but random phase on the photo detector results in an increased velocity and shape uncertainty, however. In this paper, we present a novel image evaluation method that overcomes the uncertainty limitations due to the speckle effect. For this purpose, the scattered light is detected with a camera instead of single photo detectors. Thus, the Doppler frequency from each speckle can be evaluated separately and the velocity uncertainty decreases with the square root of the number of camera lines. A reduction of the velocity uncertainty by the order of one magnitude is verified by the numerical simulations and experimental results, respectively. As a result, the measurement uncertainty of the absolute shape is not limited by the speckle effect anymore.

Transmission of independent signals through a multimode fiber using digital optical phase conjugation.

Multimode fibers are attractive for a variety of applications such as communication engineering and biophotonics. However, a major hurdle for the optical transmission through multimode fibers is the inherent mode mixing. Although an image transmission was successfully accomplished using wavefront shaping, the image information was not transmitted individually for each of the independent pixels. We demonstrate a transmission of independent signals using individually shaped wavefronts employing a single segmented spatial light modulator for optical phase conjugation regarding each light signal. Our findings pave the way towards transferring independent signals through strongly scattering media.

Wavefront shaping for imaging-based flow velocity measurements through distortions using a Fresnel guide star.

Imaging-based flow measurement techniques, like particle image velocimetry (PIV), are vulnerable to time-varying distortions like refractive index inhomogeneities or fluctuating phase boundaries. Such distortions strongly increase the velocity error, as the position assignment of the tracer particles and the decrease of image contrast exhibit significant uncertainties. We demonstrate that wavefront shaping based on spatially distributed guide stars has the potential to significantly reduce the measurement uncertainty. Proof of concept experiments show an improvement by more than one order of magnitude. Possible applications for the wavefront shaping PIV range from measurements in jets and film flows to biomedical applications.

Spiral phase mask shadow-imaging for 3D-measurement of flow fields.

Particle tracking velocimetry (PTV) is a valuable tool for microfluidic analysis. Especially mixing processes and the environmental interaction of fluids on a microscopic scale are of particular importance for pharmaceutical and biomedical applications. However, currently applied techniques suffer from the lag of instantaneous depth information. Here we present a scan-free, shadow-imaging PTV-technique for 3D trajectory and velocity measurement of flow fields in micro-channels with 2 µm spatial resolution. By using an incoherent light source, one camera and a spatial light modulator (LCoS-SLM) that generates double-images of the seeding particle shadows, it is a simply applicable and highly scalable technique.

Volumetric HiLo microscopy employing an electrically tunable lens.

Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 µm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.

High-speed light field camera and frequency division multiplexing for fast multi-plane velocity measurements.

Non-intrusive fast 3d measurements of volumetric velocity fields are necessary for understanding complex flows. Using high-speed cameras and spectroscopic measurement principles, where the Doppler frequency of scattered light is evaluated within the illuminated plane, each pixel allows one measurement and, thus, planar measurements with high data rates are possible. While scanning is one standard technique to add the third dimension, the volumetric data is not acquired simultaneously. In order to overcome this drawback, a high-speed light field camera is proposed for obtaining volumetric data with each single frame. The high-speed light field camera approach is applied to a Doppler global velocimeter with sinusoidal laser frequency modulation. As a result, a frequency multiplexing technique is required in addition to the plenoptic refocusing for eliminating the crosstalk between the measurement planes. However, the plenoptic refocusing is still necessary in order to achieve a large refocusing range for a high numerical aperture that minimizes the measurement uncertainty. Finally, two spatially separated measurement planes with 25×25 pixels each are simultaneously acquired with a measurement rate of 0.5 kHz with a single high-speed camera.

Interferometric velocity measurements through a fluctuating phase boundary using two Fresnel guide stars.

Laser optical techniques are widely used for flow measurements as they offer a high spatial and velocity resolution. However, undisturbed optical access to the measurement volume is desired. In order to measure through a fluctuating phase boundary, we present the use of adaptive optics. In an experiment, we prove that the Fresnel reflex of a phase boundary can be used as a proper guide star for adaptive velocity measurements with a single optical access. Interferometric flow measurements through a fluctuating phase boundary have been accomplished by a Mach-Zehnder interferometer.

Optical dynamic deformation measurements at translucent materials.

Due to their high stiffness-to-weight ratio, glass fiber-reinforced polymers are an attractive material for rotors, e.g., in the aerospace industry. A fundamental understanding of the material behavior requires non-contact, in-situ dynamic deformation measurements. The high surface speeds and particularly the translucence of the material limit the usability of conventional optical measurement techniques. We demonstrate that the laser Doppler distance sensor provides a powerful and reliable tool for monitoring radial expansion at fast rotating translucent materials. We find that backscattering in material volume does not lead to secondary signals as surface scattering results in degradation of the measurement volume inside the translucent medium. This ensures that the acquired signal contains information of the rotor surface only, as long as the sample surface is rough enough. Dynamic deformation measurements of fast-rotating fiber-reinforced polymer composite rotors with surface speeds of more than 300 m/s underline the potential of the laser Doppler sensor.

Axial scanning in confocal microscopy employing adaptive lenses (CAL).

In this paper we analyze the capability of adaptive lenses to replace mechanical axial scanning in confocal microscopy. The adaptive approach promises to achieve high scan rates in a rather simple implementation. This may open up new applications in biomedical imaging or surface analysis in micro- and nanoelectronics, where currently the axial scan rates and the flexibility at the scan process are the limiting factors. The results show that fast and adaptive axial scanning is possible using electrically tunable lenses but the performance degrades during the scan. This is due to defocus and spherical aberrations introduced to the system by tuning of the adaptive lens. These detune the observation plane away from the best focus which strongly deteriorates the axial resolution by a factor of ~2.4. Introducing balancing aberrations allows addressing these influences. The presented approach is based on the employment of a second adaptive lens, located in the detection path. It enables shifting the observation plane back to the best focus position and thus creating axial scans with homogeneous axial resolution. We present simulated and experimental proof-of-principle results.