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.

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.

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.

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.

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.

Resolution-enhanced multi-core fiber imaging learned on a digital twin for cancer diagnosis.

Significance: Deep learning enables label-free all-optical biopsies and automated tissue classification. Endoscopic systems provide intraoperative diagnostics to deep tissue and speed up treatment without harmful tissue removal. However, conventional multi-core fiber (MCF) endoscopes suffer from low resolution and artifacts, which hinder tumor diagnostics. Aim: We introduce a method to enable unpixelated, high-resolution tumor imaging through a given MCF with a diameter of around 0.65 mm and arbitrary core arrangement and inhomogeneous transmissivity. Approach: Image reconstruction is based on deep learning and the digital twin concept of the single-reference-based simulation with inhomogeneous optical properties of MCF and transfer learning on a small experimental dataset of biological tissue. The reference provided physical information about the MCF during the training processes. Results: For the simulated data, hallucination caused by the MCF inhomogeneity was eliminated, and the averaged peak signal-to-noise ratio and structural similarity were increased from 11.2 dB and 0.20 to 23.4 dB and 0.74, respectively. By transfer learning, the metrics of independent test images experimentally acquired on glioblastoma tissue ex vivo can reach up to 31.6 dB and 0.97 with 14 fps computing speed. Conclusions: With the proposed approach, a single reference image was required in the pre-training stage and laborious acquisition of training data was bypassed. Validation on glioblastoma cryosections with transfer learning on only 50 image pairs showed the capability for high-resolution deep tissue retrieval and high clinical feasibility.

Displacement, distance, and shape measurements of fast-rotating rough objects by two mutually tilted interference fringe systems.

The precise distance measurement of fast-moving rough surfaces is important in several applications such as lathe monitoring. A nonincremental interferometer based on two mutually tilted interference fringe systems has been realized for this task. The distance is coded in the phase difference between the generated interference signals corresponding to the fringe systems. Large tilting angles between the interference fringe systems are necessary for a high sensitivity. However, due to the speckle effect at rough surfaces, different envelopes and phase jumps of the interference signals occur. At large tilting angles, these signals become dissimilar, resulting in a small correlation coefficient and a high measurement uncertainty. Based on a matching of illumination and receiving optics, the correlation coefficient and the phase difference estimation have been improved significantly. For axial displacement measurements of recurring rough surfaces, laterally moving with velocities of 5 m/s, an uncertainty of 110 nm has been attained. For nonrecurring surfaces, a distance measurement uncertainty of 830 nm has been achieved. Incorporating the additionally measured lateral velocity and the rotational speed, the two-dimensional shape of rotating objects results. Since the measurement uncertainty of the displacement, distance, and shape is nearly independent of the lateral surface velocity, this technique is predestined for fast-rotating objects, such as crankshafts, camshafts, vacuum pump shafts, or turning parts of lathes.

Securing Data in Multimode Fibers by Exploiting Mode-Dependent Light Propagation Effects.

Multimode fibers hold great promise to advance data rates in optical communications but come with the challenge to compensate for modal crosstalk and mode-dependent losses, resulting in strong distortions. The holographic measurement of the transmission matrix enables not only correcting distortions but also harnessing these effects for creating a confidential data connection between legitimate communication parties, Alice and Bob. The feasibility of this physical-layer-security-based approach is demonstrated experimentally for the first time on a multimode fiber link to which the eavesdropper Eve is physically coupled. Once the proper structured light field is launched at Alice's side, the message can be delivered to Bob, and, simultaneously, the decipherment for an illegitimate wiretapper Eve is destroyed. Within a real communication scenario, we implement wiretap codes and demonstrate confidentiality by quantifying the level of secrecy. Compared to an uncoded data transmission, the amount of securely exchanged data is enhanced by a factor of 538. The complex light transportation phenomena that have long been considered limiting and have restricted the widespread use of multimode fiber are exploited for opening new perspectives on information security in spatial multiplexing communication systems.

Rapid computational cell-rotation around arbitrary axes in 3D with multi-core fiber.

Optical trapping is a vital tool in biology, allowing precise optical manipulation of nanoparticles, micro-robots, and cells. Due to the low risk of photodamage and high trap stiffness, fiber-based dual-beam traps are widely used for optical manipulation of large cells. Besides trapping, advanced applications like 3D refractive index tomography need a rotation of cells, which requires precise control of the forces, for example, the acting-point of the forces and the intensities in the region of interest (ROI). A precise rotation of large cells in 3D about arbitrary axes has not been reported yet in dual-beam traps. We introduce a novel dual-beam optical trap in which a multi-core fiber (MCF) is transformed to a phased array, using wavefront shaping and computationally programmable light. The light-field distribution in the trapping region is holographically controlled within 0.1 s, which determines the orientation and the rotation axis of the cell with small retardation. We demonstrate real-time controlled rotation of HL60 cells about all 3D axes with a very high degree of freedom by holographic controlled light through an MCF with a resolution close to the diffraction limit. For the first time, the orientation of the cell can be precisely controlled about all 3D axes in a dual-beam trap. MCFs provide much higher flexibility beyond the bulky optics, enabling lab-on-a-chip applications and can be easily integrated for applications like contactless cell surgery, refractive index tomography, cell-elasticity measurement, which require precise 3D manipulation of cells.

Physical Layer Security in Multimode Fiber Optical Networks.

The light propagation through a multimode fiber is used to increase information security during data transmission without the need for cryptographic approaches. The use of an inverse precoding method in a multimode fiber-optic communication network is based on mode-dependent losses on the physical layer. This leads to an asymmetry between legitimate (Bob) and illegitimate (Eve) recipients of messages, resulting in significant SNR advantage for Bob. In combination with dynamic mode channel changes, there are defined hurdles for Eve to reconstruct a sent message even in a worst-case scenario in which she knows the channel completely. This is the first time that physical layer security has been investigated in a fiber optical network based on measured transmission matrices. The results show that messages can be sent securely using traditional communication techniques. The technology introduced is a step towards the development of cyber physical systems with increased security.

Diffraction-limited axial scanning in thick biological tissue with an aberration-correcting adaptive lens.

Diffraction-limited deep focusing into biological tissue is challenging due to aberrations that lead to a broadening of the focal spot. The diffraction limit can be restored by employing aberration correction for example with a deformable mirror. However, this results in a bulky setup due to the required beam folding. We propose a bi-actuator adaptive lens that simultaneously enables axial scanning and the correction of specimen-induced spherical aberrations with a compact setup. Using the bi-actuator lens in a confocal microscope, we show diffraction-limited axial scanning up to 340 µm deep inside a phantom specimen. The application of this technique to in vivo measurements of zebrafish embryos with reporter-gene-driven fluorescence in a thyroid gland reveals substructures of the thyroid follicles, indicating that the bi-actuator adaptive lens is a meaningful supplement to the existing adaptive optics toolset.

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping.

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.