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For several centuries, far-field optical microscopy has remained a key instrument in many scientific disciplines, including physical, chemical, and biomedical research. Nonetheless, far-field imaging has many limitations: the spatial resolution is controlled by the diffraction of light, and the imaging speed follows the Nyquist–Shannon sampling theorem. The recent development of super-resolution techniques has pushed the limits of spatial resolution. However, these methods typically require complicated setups and long acquisition times and are still not applicable to deep-tissue bioimaging. Here, we report imaging through an ultra-thin fibre probe with a spatial resolution beyond the Abbe limit and a temporal resolution beyond the Nyquist limit simultaneously in a simple and compact setup. We use the random nature of mode coupling in a multimode fibre, the sparsity constraint and compressive sensing reconstruction. The new approach of super-resolution endo-microscopy does not use any specific properties of the fluorescent label, such as depletion or stochastic activation of the molecular fluorescent state, and therefore can be used for label-free imaging. We demonstrate a spatial resolution more than 2 times better than the diffraction limit and an imaging speed 20 times faster than the Nyquist limit. The proposed approach can significantly expand the realm of the application of nanoscopy for bioimaging., Microscopy: Bio-imaging beyond limits A new procedure for imaging living cells and tissues using an ultra-fine optical fibre to detect light from fluorescent dyes overcomes fundamental limits restricting existing methods. Lyubov Amitonova and Johannes de Boer developed and demonstrated the technology at the Free University of Amsterdam in the Netherlands. Optical imaging has previously been subject to constraints known as the Abbe and Nyquist limits. The Abbe limit sets the smallest distance that can be resolved at about half the wavelength of the light. The Nyquist limit determines the fastest rate at which a light signal can be sampled. The researchers have achieved fluorescence microscopy more than three times better than the Abbe limit and 20 times faster than the Nyquist limit. Their breakthrough is based on innovative light manipulation and sampling procedures in a simple and compact set-up.

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by the presence of extracellular deposits (plaques) of amyloid-beta peptide and intracellular aggregates of phosphorylated tau. In general, these hallmarks are studied by techniques requiring chemical pre-treatment and indirect labeling. Imaging techniques that require no labeling and could be performed on tissue in its native form could contribute to a better understanding of the disease. In this article a combination of label-free and non-invasive techniques is presented to study the biomolecular composition of AD human brain tissue. We build on previous research that already revealed the autofluorescence property of plaque, and the presence of carotenoids in cored plaques. Here, we present further results on cored plaques: showing blue and green autofluorescence emission coming from the same plaque location. Raman microscopy was used to confirm the presence of carotenoids in the plaque areas, with clear peaks around 1150 and 1514 cm−1. Carotenoid reference spectra were recorded in hexane solution, but also adsorbed on aggregated Aβ42 peptides; the latter agreed better with the Raman spectra observed in plaques. From the six single carotenoids measured, lycopene matched closest with the peak positions observed in the cored plaques. Lastly, stimulated Raman scattering (SRS) microscopy measurements were performed, targeting the shift of the beta-sheet Amide I peak observed in plaques. Employing SRS in the C–H stretch region we also looked for the presence of a lipid halo around plaque, as reported in the literature for transgenic AD mice, but such a halo was not observed in these human AD brain samples.

We present a new approach to Micropipette Aspiration (MPA), a pioneering method in mechanobiology, that introduces an all-optical readout to retrieve both applied stress and material response. Our technique, i.e. Hydraulic Force Spectroscopy, expands on the current MPA experimental possibilities, allowing for frequency-dependent complex moduli measurements of soft suspended bodies over a large bandwidth, with nanometric resolution. This is achieved by oscillating the pressure on the sample by tens of Pascals with a multifrequency signal, by means of a fast pump. Our goal is to use this technique to define new label-free biomarkers for different applications, e.g. embryo viability control.

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions., Protein structural dynamics can be studied by time-resolved crystallography (TRC) and ultrafast transient spectroscopic methods. Here, the authors perform electronic and vibrational transient absorption measurements to characterise the full photocycle of Photoactive Yellow Protein (PYP) both in the crystalline and solution state and find that the photocycle kinetics and structural intermediates of PYP deviate in the crystalline state, which must be taken into consideration when planning TRC experiments.

Here, we present the design and simulation of an ultrawide-bandwidth on-chip spectrometer that can be used in various applications, e.g. spectral tissue sensing. It covers 1200 nm wavelength range (400 nm-1600 nm) with 2 nm spectral resolution. The overall design size is only 3 × 3 cm2. The ultra-wide spectral range is made possible by using novel on-chip band-pass filters for the coarse wavelength division. The fine resolution is provided by the arrayed waveguide gratings. The band-pass filter is formed by using bend waveguides and adiabatic full-couplers. The additional loss caused by the band-pass filter is relatively small. The proposed spectrometer covers entire 400 nm-1600 nm range continuously with low crosstalk values. We envision that this design can be used in several different applications including food safety, agriculture, industrial inspection, optical imaging, and biomedical research.

The mechanical behavior of dermal tissues is unarguably recognized for its diagnostic ability and in the last decades received a steadily increasing interest in dermatology practices. Among the various methods to investigate the mechanics of skin in clinical environments, suction-based ones are especially noteworthy, thanks to their qualities of minimal invasiveness and relative simplicity of setups and data analysis. In such experiments, structural visualization of the sample is highly desirable, both in its own right and because it enables elastography. The latter is a technique that combines the knowledge of an applied mechanical stimulus and the visualization of the induced deformation to result in a spatially resolved map of the mechanical properties, which is particularly important for an inhomogeneous and layered material such as skin. We present a device, designed for clinical trials in dermatology practices, that uses a handheld probe to (1) deliver a suction-based, controlled mechanical stimulus and (2) visualize the subsurface structure via optical coherence tomography. We also present a device-agnostic data-analysis framework, consisting of a Python library, released in the public domain. We show the working principle of the setup on a polymeric model and on a volunteer's skin.

PurposeTo explore the short‐term vascular and structural changes of type 3 neovascularization using optical coherence tomography angiography (OCT‐A) when treated with a combination of photodynamic therapy (PDT) and intravitreal bevacizumab (IVB), and to evaluate the course of different sequences of the combined therapies.MethodsThirty eyes of 29 treatment‐naïve patients with a type 3 neovascularization were included in this prospective observational cohort study. They were all treated with PDT and IVB 2 weeks apart, starting either with PDT (PDT‐first group) or IVB (IVB‐first group). Optical coherence tomography angiography (OCT‐A) imaging was performed at week 0, 2, 4 and 18, and best corrected visual acuity (BCVA) at week 0 and 18. Vascular, structural and functional features were graded and analysed over time.ResultsIn all patients, at all follow‐up visits, vascular and structural features were significantly more often decreased or resolved than unchanged or increased. Best corrected visual acuity (BCVA) significantly improved at 18 weeks. Vascular, structural and functional outcomes were all slightly better in the PDT‐first group compared to the IVB‐first group, although not statistically significant.ConclusionCombined treatment of PDT and IVB is effective in short‐term for type 3 neovascularization based on vascular and structural features. Initial treatment with PDT tended to be more effective than with IVB.

Here, we present a compact, high-resolution, and ultrabroad-bandwidth arrayed waveguide grating (AWG) realized in a silicon nitride (Si3N4) platform. The AWG has a cascaded configuration with a 1×3 flat-passband AWG as the primary filter and three 1×70 AWGs as secondary filters (i.e. 210 output channels in total). The primary AWG has 0.5-dB bandwidth of 45 nm over 190 nm spectral range. The ultrabroad-bandwidth is achieved by using an innovative design that is based on a multiple-input multi-mode interference (MMI) coupler placed at the entrance of the first free propagation region of the primary AWG. The optical bandwidth of the cascaded AWG is 190 nm, and the spectral resolution is 1 nm. The overall device size is only 1.1 × 1.0 cm2. Optical loss at the central channel is 4 dB, which is 3 dB less than a conventional design with the same bandwidth and resolution values but using a primary filter with Gaussian transfer function. To the best of our knowledge, this is the first demonstration of an ultrabroad-bandwidth cascaded AWG on a small footprint. We also propose a novel low-loss (∼ 0.8 dB) design using a small AWG instead of an MMI coupler in the primary filter part, which can be used in applications where the light intensity is very weak, such as Raman spectroscopy.

Primary cilia, organelles protruding from the surface of eukaryotic cells, act as cellular antennae to detect and transmit signals from the extracellular environment. They are built and maintained by continuous cycles of intraflagellar transport (IFT), where ciliary proteins are transported between the ciliary base and tip. These proteins originate from the cell body because cilia lack protein synthesis machinery. How input from the cell body affects IFT and ciliary function is not well understood. Here, we use femtosecond-laser ablation to perturb the dendritic input of proteins to chemosensory cilia in living Caenorhabditis elegans. Using fluorescence microscopy, we visualize and quantify the real-time response of ciliary proteins to dendritic ablation. We find that the response occurs in three distinct stages. First, IFT dynein is activated within seconds, redistributing IFT components toward the ciliary base; second, the ciliary axoneme shortens and motors slow down; and third, motors leave the cilium. Depletion of ATP by adding azide also results in IFT slowdown and IFT components leaving the cilium, but not in activation of retrograde IFT. These results indicate that laser ablation triggers a specific mechanism important for IFT regulation that allows the cilium to rapidly adapt to changes in the outside environment.

Purpose: To compare the rates of clinically significant artifacts for two-dimensional peripapillary retinal nerve fiber layer (RNFL) thickness versus three-dimensional (3D) neuroretinal rim thickness using spectral-domain optical coherence tomography (SD-OCT). Methods: Only one eye per patient was used for analysis of 120 glaucoma patients and 114 normal patients. For RNFL scans and optic nerve scans, 15 artifact types were calculated per B-scan and per eye. Neuroretinal rim tissue was quantified by the minimum distance band (MDB). Global MDB neuroretinal rim thicknesses were calculated before and after manual deletion of B-scans with artifacts and subsequent automated interpo-lation. A clinically significant artifact was defined as one requiring manual correction or repeat scanning. Results: Among glaucomatous eyes, artifact rates per B-scan were significantly more common in RNFL scans (61.7%, 74 of 120) compared to B-scans in neuroretinal rim volume scans (20.9%, 1423 of 6820) (95% confidence interval [CI], 31.6–50.0; P < 0.0001). For clinically significant artifact rates per eye, optic nerve scans had significantly fewer artifacts (15.8% of glaucomatous eyes, 13.2% of normal eyes) compared to RNFL scans (61.7% of glaucomatous eyes, 25.4% of normal eyes) (glaucoma group: 95% CI, 34.1–57.5, P < 0.0001; normal group: 95% CI, 1.3–23.3, P = 0.03). Conclusions: Compared to the most commonly used RNFL thickness scans, optic nerve volume scans less frequently require manual correction or repeat scanning to obtain accurate measurements. Translational Relevance: This paper illustrates the potential for 3D OCT algorithms to improve in vivo imaging in glaucoma.

© 2023 The Royal Society of Chemistry.The rheological characterization of soft suspended bodies, such as cells, organoids, or synthetic microstructures, is particularly challenging, even with state-of-the-art methods (e.g. atomic force microscopy, AFM). Providing well-defined boundary conditions for modeling typically requires fixating the sample on a substrate, which is a delicate and time-consuming procedure. Moreover, it needs to be tuned for each chemistry and geometry. Here, we validate a novel technique, called hydraulic force spectroscopy (HFS), against AFM dynamic indentation taken as the gold standard. Combining experimental data with finite element modeling, we show that HFS gives results comparable to AFM microrheology over multiple decades, while obviating any sample preparation requirements.

We present a pump-probe experiment in which high-order harmonics are generated in a laser-produced plasma as a bright EUV photon source and as a tool for plasma characterization.

The aggressive reduction of semiconductor device dimensions drives many improvements in optical wafer metrology. Currently, chips with device feature sizes below 10 nm are in production which requires robust overlay metrology with sub-nm precision. We will present a compact dark-field Digital Holographic Microscope (df-DHM) that is able to measure overlay on small metrology targets from visible to infrared wavelengths. The coherent amplification and the aberration correction capabilities that is offered by df-DHM allow robust overlay metrology even on weak targets that are covered by absorbing layers. Measured data will be shown that demonstrate the capabilities of this metrology concept.

We perform high-resolution multi-spectral wavefront sensing on extreme ultraviolet sources produced by high-harmonic generation processes. Using ptychography, we show spectrally resolved complex-valued beam reconstructions for eight harmonics simultaneously, with a spatial resolution of 1 µm.

Optical coherence tomography (OCT) is a non-contact method for imaging the topological and internal microstructure of samples in three dimensions. OCT can be configured as a conventional microscope, as an ophthalmic scanner, or using endoscopes and small diameter catheters for accessing internal biological organs. In this Primer, we describe the principles underpinning the different instrument configurations that are tailored to distinct imaging applications and explain the origin of signal, based on light scattering and propagation. Although OCT has been used for imaging inanimate objects, we focus our discussion on biological and medical imaging. We examine the signal processing methods and algorithms that make OCT exquisitely sensitive to reflections as weak as just a few photons and that reveal functional information in addition to structure. Image processing, display and interpretation, which are all critical for effective biomedical imaging, are discussed in the context of specific applications. Finally, we consider image artifacts and limitations that commonly arise and reflect on future advances and opportunities.

TI-REX is a self-seeded nanosecond (5-20ns) parametric amplifier with wide wavelength tunability (1.4-4.4µm) and high-resolution (0.25ns) temporal shaping capability. It enables systematic studies on laser produced plasmas and the wavelength-dependence of extreme-ultraviolet generation efficiency.

We present dark-field digital holographic microscopy for diffraction-based overlay metrology in semiconductor layers with feature size of a few nanometers. We aim for higher accuracy and precision by calibrating the illumination profile on the targets.

Based on a simply-built setup, we demonstrated an inexpensive refractive index sensor. An unprecedented sensitivity value was obtained, which is the highest value reported so far among intensity modulated fiber refractometers.

Background: Integrated circuits are fabricated layer by layer. It is crucial to their performance that these layers are well aligned to each other, and any undesired translation of a layer is called overlay. Thus far, overlay measurements have been limited to visible wavelengths, but the use of materials that are opaque to visible wavelengths necessitates measurements using infrared light. Aim: We set out to demonstrate that an overlay sensor based on digital holographic microscopy can perform such overlay measurement at infrared wavelengths, while maintaining functionality at visible wavelengths. Approach: This was done by constructing a breadboard setup that is capable of measuring overlay at wavelengths ranging from 400 to 1100 nm. Results: Using the setup, we demonstrated good linearity between an applied amount of overlay and the measured amount. In addition, we demonstrated that the setup is only sensitive to structures at the top of the wafer. Measurements are therefore unaffected by the fact that Si is transparent at 1100 nm. Conclusions: These results demonstrate the viability of an overlay sensor that is sensitive to visible and infrared light, allowing more freedom in choice of materials for integrated circuits.

We generate multi-wavelength extreme-ultraviolet vortex beams via high-harmonic generation. We characterize the wavefronts of these high orbital angular mo-mentum beams using ptychography.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) is a clinically and pathologically heterogeneous disease. The distinct underlying pathologies include aggregation of TDP-43, tau and FUS. Currently, no ante-mortem biomarkers are available to distinguish between the different pathological subtypes, providing challenges for diagnosis and therapeutic interventions. As an extension of the brain, the retina displays similarities in terms of anatomy and functionality. The accessibility of the retina provides a 'window' into the brain and a great potential for noninvasive imaging of neurodegenerative changes in patients. The aim of this study is to assess the presence of TDP-43 in the retina. METHOD: Post-mortem retina tissue was obtained from donors with FTLD-TDP (n=6), of which 3 donors carried a C9orf72 repeat expansion, one progranulin (PRGN) mutation and two sporadic donors. We also included donors with FTLD-tau (n=5), FTLD-FUS (n=1) , AD with limbic TDP-43 depositions (n=2), and donors with ALS-TDP (n=2). Immunohistochemical stainings were performed for panTDP-43, phosphorylated TDP-43 (pTDP-43), p62, and dipeptides (polyGA and polyGP). RESULT: In C9orf72 and PRGN mutation carriers, as well as one sporadic FTLD-TDP donor, TDP-43 inclusions were observed in the outer plexiform layer of the retina. One sporadic donor showed granular TDP-43 deposits in amacrine cells. No TDP-43 inclusions were observed in FTLD-tau, ALS and AD donors. Interestingly, all C9orf72 mutation carriers showed abundant presence of p62 and dipeptide inclusions in the inner nuclear layer of the retina. CONCLUSION: Manifestations of TDP-43 and dipeptide pathology are present in the retina of patients with FTLD-TDP. No TDP-43 inclusions are present in the retina of ALS or AD patients with limbic TDP-43 pathology, suggesting that retinal TDP-43 pathology is specific for FTLD-TDP. With the advances in ocular imaging techniques these findings provide opportunities for non-invasive retinal imaging for diagnosis and monitoring progression of FTLD-TDP.

Here we demonstrate an inexpensive, simple, and ultra-sensitive refractive index sensor based on a tapered tip optical fiber combined with a straightforward image analysis method. The output profile of this fiber exhibits circular fringe patterns whose intensity distribution dramatically changes even with ultra-small refractive index variations in the surrounding medium. The sensitivity of the fiber sensor is measured using different concentrations of saline solutions with a transmission setup consisting of a single wavelength light source, a cuvette, an objective lens, and a camera. By analyzing the areal changes in the center of the fringe patterns for each saline solution, we obtain an unprecedented sensitivity value of 24,160 dB/RIU (refractive index unit), which is the highest value reported so far among intensity-modulated fiber refractometers. The resolution of the sensor is calculated to be 6.9 ×10−9. Moreover, we measure the sensitivity of the fiber tip in the backreflection mode using salt-water solutions and obtained a sensitivity value of 620 dB/RIU. This sensor is ultra-sensitive, simple, easy to fabricate, and low-cost, which makes it a promising tool for on-site measurements and point-of-care applications.

Overlay metrology measures pattern placement between two layers in a semiconductor chip. The continuous shrinking of device dimensions drives the need to explore novel optical overlay metrology concepts that can address many of the existing metrology challenges. We present a compact dark-field digital holographic microscope that uses only a single imaging lens. Our microscope offers several features that are beneficial for overlay metrology, like a large wavelength range. However, imaging with a single lens results in highly aberrated images. In this work, we present an aberration calibration and correction method using nano-sized point scatterers on a silicon substrate. Computational imaging techniques are used to recover the full wavefront error, and we use this to correct for the lens aberrations. We present measured data to verify the calibration method and we discuss potential calibration error sources that must be considered. A comparison with a ZEMAX calculation is also presented to evaluate the performance of the presented method.

This thesis deals with an advanced laser-based microscopy technique to detect micrometer-size objects with molecular specificity. Applications are shown from the aquatic environment and from the medical world. The final chapters describe an option to increase the penetration depth through scattering samples and simulation software to help optimize the measurement settings. One of the most prominent materials in modern life is plastic, but this also results in the large-scale production of plastic waste. A portion of this waste reaches the environment and is fragmented into small pieces, called microplastics. Microplastics pollution affects the environment and potentially our health in ways we are only beginning to understand. To study it, we need to have a solid measurement and monitoring platform, based on reliable microplastics detection. Detection of microplastics is difficult due to their small size and heterogeneity and they can be found in different types of matrices in the environment and even in the human body. A label-free microscopy imaging technique, called Stimulated Raman Scattering (SRS) microscopy, is able to create images of small particles, like microplastics, based on their molecular structure. SRS makes use of two synchronized pulsed lasers of different colors, of which the energy difference matches a specific vibration of the target molecule. In this thesis, we used SRS for identifying five polymer types. First, we tested the approach on an artificial mixture of plastic particles, and we identified polyethylene terephthalate particles extracted from nail polish, demonstrating also the thousand‐fold higher speed of mapping compared with conventional Raman. Furthermore, we found 12,000 plastic particles per kilogram dry weight in a Rhine estuary sediment sample. SRS was the fastest microplastics detection method at the time of publication. We concluded that SRS can be an efficient method for monitoring microplastics in the environment and potentially many other matrices of interest. Another application area that was studied with SRS is breast tissue from explanted breast implants. Implant failure occurs in approximately a tenth of patients within 10 years, and even without a major rupture silicone can still leak. We showed how SRS can detect silicone material in breast tissue slices, without additional sample treatment. SRS images revealed the distribution and quantity of silicone material. Twenty-two donor-matched capsules from eleven patients experiencing unilateral capsular contraction complaints were included in a clinical study after bilateral explantation surgery. This method showed the correlation between silicone presence and capsular contraction. Depth penetration of the light into the sample is an issue with any light based technique. We showed the use of a long wavelength SRS microscope system capable of greater depth imaging compared with the more common configuration with shorter wavelengths. It showed an improved depth penetration in polyethylene plastic material, in a silicone test sample with embedded polyethylene microbeads, and into subcutaneous fat tissue. In SRS imaging we have to consider multiple parameters that influence the imaging speed, image quality and the spatial resolution. In order to find the optimized imaging setup, we developed two simulation programs for SRS imaging systems with lock-in amplifier. One simulation program was used to find parameters optimized for either image quality or acquisition time. With the second program we evaluated SRS imaging; the simulations agreed very well with experimental SRS images. The same software was used to simulate multiplexed SRS imaging. of six channels, including the inter-channel crosstalk. These programs will be useful for operating an SRS imaging setup, as well as for designing novel setups.

There is increasing evidence of altered tissue mechanics in neurodegeneration. However, due to difficulties in mechanical testing procedures and the complexity of the brain, there is still little consensus on the role of mechanics in the onset and progression of neurodegenerative diseases. In the case of Alzheimer’s disease (AD), magnetic resonance elastography (MRE) studies have indicated viscoelastic differences in the brain tissue of AD and healthy patients. However, there is a lack of viscoelastic data from contact mechanical testing at higher spatial resolution. Therefore, we report viscoelastic maps of the hippocampus obtained by a dynamic indentation on brain slices from the APP/PS1 mouse model where individual brain regions are resolved. A comparison of viscoelastic parameters shows that regions in the hippocampus of the APP/PS1 mice are significantly stiffer than wild-type (WT) mice and have increased viscous dissipation. Furthermore, indentation mapping at the cellular scale directly on the plaques and their surroundings did not show local alterations in stiffness although overall mechanical heterogeneity of the tissue was high (SD~40%). Therefore, reported mechanical alterations at a tissue scale indicates global remodeling of the brain tissue structure.

Microscopy with coherent extreme-ultraviolet (EUV) and soft-X-ray radiation is a promising diagnostic tool for nanoscience. High-harmonic generation (HHG) sources are becoming a mature table-top source for EUV and soft-X-ray radiation. HHG sources naturally produce broad EUV and soft-X-ray spectra, making them intrinsically suited for spectrally-resolved microscopy. This is particularly interesting because many materials possess unique absorption characteristics in this short-wavelength range. However, focusing and shaping polychromatic EUV radiation for microscopy experiments remains challenging due to the fact that most elements across the periodic table exhibit high absorption and low refractive index contrast, which makes manufacturing good refractive optics such as lenses very difficult. A promising technique capable of simultaneous wavefront sensing and quantitative phase contrast microscopy is ptychography [1], as the large amount of redundant information available in ptychography allows for polychromatic reconstructions with monochromatic detectors [2]. This is particularly useful for imaging experiments with HHG sources, where polychromatic operation offers largely increased flux. Studies have indicated that a ptychography reconstruction benefits from structured illumination probes [3] - [5].

In a world where miniaturization leads technology advancement, extreme ultraviolet (EUV) light is becoming the tool for leading companies to increase the number of transistors on a chip. EUV light is generated via laser-produced plasma (LPP), that is currently driven by 10.6 µ m CO 2 lasers, due to their high demonstrated conversion efficiency (CE) from laser power to EUV. In terms of laser technology, solid-state lasers based on Nd:YAG, operating around 1 µm wavelength have significant advantages in terms of efficiency, stability and control over temporal pulse shape and contrast. However, so far they provide lower CE than CO 2 lasers [1] , even though advanced pulse temporal shaping seems advantageous in this regard. A promising avenue is the investigation of the intermediate wavelength regime: first data for 2 µ m lasers already indicates a CE improvement compared to 1 µm [2] , and simulations predict even better performances for wavelengths between 2 and 10 µ m .

The attenuation coefficient provides a quantitative parameter for tissue characterization and can be calculated from optical coherence tomography (OCT) data, but accurate determination requires compensation for the confocal function. We present extensive measurement series for extraction of the focal plane and the apparent Rayleigh length from the ratios of OCT images acquired with different focus depths and compare these results with two alternative approaches. By acquiring OCT images for a range of different focus depths the optimal focus plane difference is determined for intralipid and titanium oxide (TiO2) phantoms with different scatterer concentrations, which allows for calculation of the attenuation coefficient corrected for the confocal function. The attenuation coefficient is determined for homogeneous intralipid and TiO2 samples over a wide range of concentrations. We further demonstrate very good reproducibility of the determined attenuation coefficient of layers with identical scatter concentrations in a multi-layered phantom. Finally, this method is applied to in vivo retinal data.

We explore the deformation of tin microdroplets of various diameters induced by two consecutive laser pulses having pulse durations of 0.4 ns. Impact of laser pulses with this duration mainly leads to shock-wave-induced cavitation and spallation. The main result obtained in this work is the observation of a strong reduction of the spall velocity that depends on the time delay between the two pulses. This reduction reveals a complex interplay between plasma recoil pressure and shock-wave-driven deformation, and enables an estimation of the moment of spall formation and the average shock-wave propagation velocity. We find that the shock wave traverses the droplet with an average velocity ranging from 1.2 to 1.6 times the speed of sound. We study the effects of the energy of the second pulse on the deformation and qualitatively discuss the formation of microjets. Crucially, we demonstrate the ability to manipulate the microdroplet expansion and spallation with double-pulse sequences, thereby increasing the portfolio of obtainable target shapes for droplet-based extreme ultraviolet light sources.

A profound characteristic of field cancerization is alterations in chromatin packing. This study aimed to quantify these alterations using electron microscopy image analysis of buccal mucosa cells of laryngeal, esophageal, and lung cancer patients. Analysis was done on normal-appearing mucosa, believed to be within the cancerization field, and not tumor itself. Large-scale electron microscopy (nanotomy) images were acquired of cancer patients and controls. Within the nuclei, the chromatin packing of euchromatin and heterochromatin was characterized. Furthermore, the chromatin organization was quantified through chromatin packing density scaling. A significant difference was found between the cancer and control groups in the chromatin packing density scaling parameter for length scales below the optical diffraction limit (200 nm) in both the euchromatin (p = 0.002) and the heterochromatin (p = 0.006). The chromatin packing scaling analysis also indicated that the chromatin organization of cancer patients deviated significantly from the control group. They might allow for novel strategies for cancer risk stratification and diagnosis with high sensitivity. This could aid clinicians in personalizing screening strategies for high-risk patients and follow-up strategies for treated cancer patients.

The physiological processes of cells and tissues are regulated not only by biochemical and electrical signaling but also by mechanics. The mechanical microenvironment is involved in the progression of diseases and is one of the key elements in fabricating physiologically relevant 3D tissues. The complexity of biological tissue structure leads to complex mechanical behavior. However, our ability to characterize the mechanical properties of biomaterials is limited due to technical challenges. Mechanosensation of brain cells and the mechanical microenvironment of brain tissue are relevant for healthy functioning, neurodevelopment, neurodegenerative diseases, and regeneration. There are many challenges associated with the mechanical testing of brain tissue such as restricted access to the brain, lack of available material, difficult sample preparation procedures, and preservation of its viability. Furthermore, mechanical testing can be performed at various scales, from single cells to the whole organ and with various mechanical testing modalities. As seen from previous studies, variation of the experimental parameters contribute to the observed variability in mechanical data. Finally, understanding which structural components of tissues and cells give rise to certain mechanical behavior remains an unmet objective. To address above mentioned issues, three objectives were set for this thesis: Objective 1: Develop indentation setup and protocols to measure mechanical properties of the brain slices in a reproducible manner. Novel indentation protocols using ferrule-top indentation device are established in this thesis. Chapter 2 introduces contact mechanics theory. As there are many different models available, general guidelines are given for selecting indentation profiles for soft tissue measurements. Chapter 3 shows experimental observations when indenting on the brain slices such as the influence of the indentation-depth, indentation-speed, oscillatory-ramp, dynamic mechanical analysis, tissue mounting, degradation, swelling, and conditioning. Objective 2: Understand the relationship between the structure of the brain and its mechanical properties. With the established novel measurement protocols, viscoelastic maps of the hippocampus of the mouse brain are reported in Chapter 4. For the first time in the literature, clear differences between subregions are observed, which agrees with anatomical region boundaries. Surprisingly, high cell-density regions are softer than low-cell density regions. Chapter 5 shows viscoelastic maps of the hippocampus and cerebellum of the juvenile mouse, where mechanical contrast overlaps with anatomical regions. Comparison between juvenile and adult shows that adult hippocampus is stiffer than juvenile. Correlations are found between the amount of different brain components such as nuclei, myelin, astrocytes, and viscoelastic parameters, and a linear regression model is suggested. Finally, Chapter 6 shows that the hippocampus of the Alzheimer's disease mouse model is stiffer than healthy controls. In summary, progress has been made in understanding the mechanical brain microenvironment in terms of viscoelasticity and structural composition by introducing novel dynamic indentation protocols. Objective 3: Adapt indentation setup and methodology from tissue characterization to single cells with the aim to study astrocytes and microglia in an inflammatory environment. Indentation protocols are adapted to single cells. Chapter 7 compares astrocytes derived from gray matter (GM) and white matter (WM) regions where the latter are found to be softer. As a response to treatment with pro-inflammatory lipopolysaccharide (LPS), GM astrocytes become softer, where the F-actin network appears rearranged, whereas WM astrocytes preserve their initial features. Chapter 8 compares microglia derived from GM and WM regions where the latter are more viscoelastic. When treated with LPS, the increase in viscoelasticity in GM microglia is accompanied by an increase in Tnf-alpha mRNA and reorganization of F-actin which is absent in WM microglia which decreases viscoelasticity. Together, these both studies show that glial cells have region-dependent phenotypes which can be observed not only in their biochemical responses but also in biomechanical.

A novel, interferometry-based approach to micropipette aspiration overcomes the spatial resolution limit of current methods, simplifies data retrieval, and enables the study of the dynamic mechanical behavior of biomembranes at the nanoscale.

Background Recent studies have created awareness that facial features can be reconstructed from high-resolution MRI. Therefore, data sharing in neuroimaging requires special attention to protect participants’ privacy. Facial features removal (FFR) could alleviate these concerns. We assessed the impact of three FFR methods on subsequent automated image analysis to obtain clinically relevant outcome measurements in three clinical groups. Methods FFR was performed using QuickShear, FaceMasking, and Defacing. In 110 subjects of Alzheimer’s Disease Neuroimaging Initiative, normalized brain volumes (NBV) were measured by SIENAX. In 70 multiple sclerosis patients of the MAGNIMS Study Group, lesion volumes (WMLV) were measured by lesion prediction algorithm in lesion segmentation toolbox. In 84 glioblastoma patients of the PICTURE Study Group, tumor volumes (GBV) were measured by BraTumIA. Failed analyses on FFR-processed images were recorded. Only cases in which all image analyses completed successfully were analyzed. Differences between outcomes obtained from FFR-processed and full images were assessed, by quantifying the intra-class correlation coefficient (ICC) for absolute agreement and by testing for systematic differences using paired t tests. Results Automated analysis methods failed in 0–19% of cases in FFR-processed images versus 0–2% of cases in full images. ICC for absolute agreement ranged from 0.312 (GBV after FaceMasking) to 0.998 (WMLV after Defacing). FaceMasking yielded higher NBV (p = 0.003) and WMLV (p ≤ 0.001). GBV was lower after QuickShear and Defacing (both p Conclusions All three outcome measures were affected differently by FFR, including failure of analysis methods and both “random” variation and systematic differences. Further study is warranted to ensure high-quality neuroimaging research while protecting participants’ privacy. Key Points • Protecting participants’ privacy when sharing MRI data is important. • Impact of three facial features removal methods on subsequent analysis was assessed in three clinical groups. • Removing facial features degrades performance of image analysis methods.

Précis: Neuroretinal rim minimum distance band (MDB) thickness is significantly lower in older subjects and African Americans compared with whites. It is similar in both sexes. Purpose: To evaluate the relationship between age, race, and sex with the neuroretinal rim using high-density spectral-domain optical coherence tomography optic nerve volume scans of normal eyes. Methods: A total of 256 normal subjects underwent Spectralis spectral-domain optical coherence tomography optic nerve head volume scans. One eye was randomly selected and analyzed for each subject. Using custom-designed software, the neuroretinal rim MDB thickness was calculated from volume scans, and global and quadrant neuroretinal rim thickness values were determined. The MDB is a 3-dimensional neuroretinal rim band comprised of the shortest distance between the internal limiting membrane and the termination of the retinal pigment epithelium/Bruch’s membrane complex. Multiple linear regression analysis was performed to determine the associations of age, race, and sex with neuroretinal rim MDB measurements. Results: The population was 57% female and 69% white with a mean age of 58.4±15.3 years. The mean MDB thickness in the normal population was 278.4±47.5 µm. For this normal population, MDB thickness decreased by 0.84 µm annually (P

Quantitative velocity estimations in optical coherence tomography requires the estimation of the axial and lateral flow components. Optical coherence tomography measures the depth resolved complex field reflected from a sample. While the axial velocity component can be determined from the Doppler shift or phase shift between a pair of consecutive measurements at the same location, the estimation of the lateral component for in vivo applications is still challenging. One approach to determine lateral velocity is multiple simultaneous measurements at different angles. In another approach the lateral component can be retrieved through repeated measurements at (nearly) the same location by an analysis of the decorrelation over time. In this paper we follow the latter approach. We describe a model for the complex field changes between consecutive measurements and use it to predict the uncertainties for amplitude-based, phase-based and complex algorithms. The uncertainty of the flow estimations follows from a statistical analysis and is determined by the number of available measurements and the applied analysis method. The model is verified in phantom measurements and the dynamic range of velocity estimations is investigated. We demonstrate that phase-based and complex (phasor) based lateral flow estimation methods are superior to amplitude-based algorithms.

Homochirality is a generic and unique property of all biochemical life, and the fractional circular polarization of light it induces therefore constitutes a potentially unambiguous biosignature. However, while high-quality circular polarimetric spectra can be easily and quickly obtained in the laboratory, accurate measurements in the field are much more challenging due to large changes in illumination and target movement. In this study, we measured various targets in the field, up to distances of a few kilometers, using the dedicated circular spectropolarimeter TreePol. We show how photosynthetic life can readily be distinguished from abiotic matter. We underline the potential of circular polarization signals as a remotely accessible means to characterize and monitor terrestrial vegetation, for example, for agriculture and forestry. In addition, we discuss the potential of circular polarization for the remote detection of extraterrestrial life.

The investigation of the mechanical properties of embryos is expected to provide valuable information on the phenomenology of morphogenesis. It is thus believed that, by mapping the viscoelastic features of an embryo at different stages of growth, it may be possible to shed light on the role of mechanics in embryonic development. To contribute to this field, we present a new instrument that can determine spatiotemporal distributions of mechanical properties of embryos over a wide area and with unprecedented accuracy. The method relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with Optical Coherence Tomography, which can reveal changes in tissue morphology and help the user identify the indentation point. To prove the working principle, we have collected viscoelasticity maps of fixed and live HH11-HH12 chicken embryos. Our study shows that the instrument can reveal correlations between tissue morphology and mechanical behavior. Statement of Significance: Local mechanical properties of soft biological tissue play a crucial role in several biological processes, including cell differentiation, cell migration, and body formation; therefore, measuring tissue properties at high resolution is of great interest in biology and tissue engineering. To provide an efficient method for the biomechanical characterization of soft biological tissues, we introduce a new tool in which the combination of non-invasive Optical Coherence Tomography imaging and depth-controlled indentation measurements allows one to map the viscoelastic properties of biological tissue and investigate correlations between local mechanical features and tissue morphology with unprecedented resolution.

Mechanical forces are increasingly recognized as major regulators of several physiological processes at both the molecular and cellular level; therefore, a deep understanding of the sensing of these forces and their conversion into electrical signals are essential for studying the mechanosensitive properties of soft biological tissues. To contribute to this field, we present a dual-purpose device able to mechanically stimulate retinal tissue and to record the spiking activity of retinal ganglion cells (RGCs). This new instrument relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with high-density multi-electrode array (HD-MEAs) to simultaneously record the spontaneous activity of the retina. In this paper, we introduce this instrument, describe its technical characteristics, and present a proof-of-concept experiment that shows how RGC spiking activity of explanted mice retinas respond to mechanical micro-stimulations of their photoreceptor layer. The data suggest that, under specific conditions of indentation, the retina perceive the mechanical stimulation as modulation of the visual input, besides the longer time-scale of activation, and the increase in spiking activity is not only localized under the indentation probe, but it propagates across the retinal tissue.

Stimulated Raman scattering (SRS) imaging is a fast, label-free, and sensitive technique to map the distribution of a vibrational species in a microscopy setting. It has great potential for applications in many fields, such as lipid imaging in biomedicine. However, depth penetration of the light into the sample is an issue with any light-based technique, especially with multiphoton techniques such as SRS. Using longer wavelengths allows deeper penetration into densely scattering materials, but applying wavelengths above 1,500 nm is challenging technically. We have built a flexible SRS microscope system capable of imaging with a combination of 1,064 nm and wavelengths over 1,500 nm, using the idler output of an optical parametric oscillator (OPO). For comparison, the same system was also operated in the more common configuration, using 1,064 nm in combination with the OPO signal output around 800 nm. With the long-wavelength settings, we show improved depth penetration in polyethylene plastic material and in a silicone phantom with embedded polymer microbeads, and we report images of lipid structure in biological tissue. These results demonstrate the technical feasibility of using these long wavelengths for SRS imaging. Disadvantages such as poorer spatial resolution and lower signal strength are also discussed. The application of this new approach to SRS microscopy can allow greater insight into deep-lying structures in a non-invasive way.

Precis: Three-dimensional (3D) spectral domain optical coherence tomography (OCT) volume scans of the optic nerve head (ONH) and the peripapillary area are useful in the management of glaucoma in patients with a type I or II Boston Keratoprosthesis (KPro). Purpose: The purpose of this study was to report the use of spectral domain OCT in the management of glaucoma in patients with a type I or II Boston KPro. Materials and Methods: This study is an observational case series. Four consecutive patients with KPro implants were referred for glaucoma evaluation. A comprehensive eye examination was performed which included disc photography, visual field testing, and high-density spectral domain OCT volume scans of the ONH and the peripapillary area. 2D and 3D parameters were calculated using custom-designed segmentation algorithms developed for glaucoma management. Results: Spectral domain OCT parameters provided useful information in the diagnosis and management of 4 KPro patients. OCT parameters which can be used in KPro patients included 2D retinal nerve fiber layer (RNFL) thickness, 3D peripapillary RNFL volume, 3D peripapillary retinal thickness and volume, 3D cup volume, and 3D neuroretinal rim thickness and volume. In 3 of 4 cases where the traditional 2D RNFL thickness scan was limited by artifacts, 3D spectral domain OCT volume scans provided useful quantitative objective measurements of the ONH and peripapillary region. Therefore, 3D parameters derived from high-density volume scans as well as radial scans of the ONH can be used to overcome the limitations and artifacts associated with 2D RNFL thickness scans. Conclusions: Spectral domain OCT volume scans offer the possibility to enhance the evaluation of KPro patients with glaucoma by using both 2D and 3D diagnostic parameters that are easily obtained in a clinic setting.

One of the primary goals in space research is the search for signs of extant or extinct extraterrestrial life, and Raman spectroscopy can play a role in this field. Raman spectrometers are planned for future missions to Mars and possibly the Moon to identify the mineralogical surface composition and potentially existing organic compounds (especially on Mars). However, a major challenge in Raman spectroscopy, especially in the visible range, is the strong fluorescence background. Time‐resolved Raman spectroscopy (TRRS) can provide selective detection of Raman signals over the generally longer living fluorescence. This study investigates the potential of a TRRS system, using 3‐ps, 440‐nm laser pulses and time‐gated detection with an intensified charge‐coupled device (CCD) camera. Test samples were the lichen Xanthoria elegans as an extraterrestrial life analogue, and a lunar regolith analogue material (LRS) as a planetary surface analogue. The TRRS technique is evaluated by comparing gated to nongated Raman spectroscopy using different detectors but with otherwise the same instrument and identical measurement conditions. The gated spectra of X. elegans showed significant signal‐to‐noise ratio (SNR) improvements compared to the nongated spectra. The visible Raman lines could be assigned to the photoprotective pigment parietin. For the LRS sample, measurement spots with a good SNR in the nongated spectrum were not significantly improved by measuring in gated mode. However, spots dominated by fluorescence showed significant improvement in gated mode because of fluorescence suppression. Minerals such as plagioclase, diopside, olivine, apatite, and a carbonate mineral were detected. In most cases, TRRS provided better results compared to nongated measurements, demonstrating the suitability for future space‐exploration missions.

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study we have measured the circular polarizance of several multicellular green, red and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life., 25 pages, 9 figures

Significance Photosynthetic organisms utilize sunlight as a form of energy. Under low light, they maximize their capacity to harvest photons; however, under excess light, they dissipate part of the harvested energy to prevent photodamage, at the expense of light-use efficiency. Optimally balancing light harvesting and energy dissipation in natural (fluctuating light) conditions is considered a target for improving the productivity of both algae and plants. Here we have studied the energy dissipation process in the green alga Chlamydomonas reinhardtii in vivo. We found that it is remarkably different from that of higher plants, highlighting the need of developing tailor-made strategies to optimize the light harvesting–energy dissipation balance in different organisms., Sunlight drives photosynthesis but can also cause photodamage. To protect themselves, photosynthetic organisms dissipate the excess absorbed energy as heat, in a process known as nonphotochemical quenching (NPQ). In green algae, diatoms, and mosses, NPQ depends on the light-harvesting complex stress-related (LHCSR) proteins. Here we investigated NPQ in Chlamydomonas reinhardtii using an approach that maintains the cells in a stable quenched state. We show that in the presence of LHCSR3, all of the photosystem (PS) II complexes are quenched and the LHCs are the site of quenching, which occurs at a rate of ∼150 ps−1 and is not induced by LHCII aggregation. The effective light-harvesting capacity of PSII decreases upon NPQ, and the NPQ rate is independent of the redox state of the reaction center. Finally, we could measure the pH dependence of NPQ, showing that the luminal pH is always above 5.5 in vivo and highlighting the role of LHCSR3 as an ultrasensitive pH sensor.

Purpose: This prospective case series is aimed at exploring optical coherence tomographic angiography (OCT-A) as a treatment monitoring tool in patients treated for retinal angiomatous proliferation (RAP). Methods: Twelve treatment-naïve RAP patients were included, with a median age of 79 years (range 65–90). Patients were imaged with an experimental 1,040-nm swept-source phase-resolved OCT-A instrument before and after treatment. Treatment consisted of either intravitreal bevacizumab or triamcinolone injections with or without photodynamic therapy (PDT). Abnormal blood flow after treatment was graded as increased, unchanged, decreased, or resolved. Results: OCT-A images before and after treatment could be obtained in 9 patients. The median follow-up period was 10 weeks (range 5–19). After various treatments, the RAP lesion resolved in 7 patients, in 1 patient the OCT-A depicted decreased flow in the lesion, and 1 patient showed unchanged abnormal blood flow. Monotherapy with intravitreal bevacizumab injections resolved RAP in 1 out of 2 patients. Combined therapy of bevacizumab with PDT resolved RAP in 6 out of 7 patients. Conclusions: OCT-A visualized resolution of abnormal blood flow in 7 out of 9 RAP patients after various short-term treatment sequences. OCT-A may become an important noninvasive monitoring tool for optimizing treatment strategies in RAP patients.

Interleaved arrayed waveguide gratings (AWGs) have a great potential in providing large channel counts and narrower channel spacings for many applications, including optical communication, spectroscopy, and imaging. Here, a 75-channel silicon nitride based interleaved AWG was experimentally demonstrated. The design is comprised of a 3-channel primary AWG with 1 nm of resolution and three 25-channel secondary AWGs each with 3 nm of resolution. The final device has a spectral resolution of 1 nm over 75 nm bandwidth centered at 1550 nm. Its performance is compared with a conventional AWG spectrometer with 75 nm of bandwidth and 1 nm of resolution. The interleaved AWG demultiplexer showed lower crosstalk and better uniformity in addition to being two times smaller than the conventional design.

Polarization beam splitters (PBSs) are central elements for polarization handling. Here, we present the design of an ultra-broadband, low-loss, and easy-to-fabricate PBS based on a silicon nitride asymmetrical directional coupler for polarization-sensitive optical coherence tomography systems. The phase difference between transverse electric and transverse magnetic modes is introduced by using straight waveguides with different widths and an offset between them. A bent waveguide is placed close to the end of the through port in order to increase the operating bandwidth (i.e., more than 220 nm with greater than 15 dB of extinction). The overall device length is only 400 µm.

Alzheimer’s disease (AD) neuropathology is characterized by hyperphosphorylated tau containing neurofibrillary tangles and amyloid-beta (Aβ) plaques. Normally these hallmarks are studied by (immuno-) histological techniques requiring chemical pretreatment and indirect labelling. Label-free imaging enables one to visualize normal tissue and pathology in its native form. Therefore, these techniques could contribute to a better understanding of the disease. Here, we present a comprehensive study of high-resolution fluorescence imaging (before and after staining) and spectroscopic modalities (Raman mapping under pre-resonance conditions and stimulated Raman scattering (SRS)) of amyloid deposits in snap-frozen AD human brain tissue. We performed fluorescence and spectroscopic imaging and subsequent thioflavin-S staining of the same tissue slices to provide direct confirmation of plaque location and correlation of spectroscopic biomarkers with plaque morphology; differences were observed between cored and fibrillar plaques. The SRS results showed a protein peak shift towards the β-sheet structure in cored amyloid deposits. In the Raman maps recorded with 532 nm excitation we identified the presence of carotenoids as a unique marker to differentiate between a cored amyloid plaque area versus a non-plaque area without prior knowledge of their location. The observed presence of carotenoids suggests a distinct neuroinflammatory response to misfolded protein accumulations., Lochocki et al. report detection of amyloid plaques by label-free fluorescence and Raman imaging in snap-frozen frontal cortex tissue from Alzheimer’s disease cases. They identify presence of carotenoid in a cored amyloid plaque area, versus a non-plaque area, suggesting a distinct neuroinflammatory response to misfolded protein aggregations.

Ptychography is a robust computational imaging technique that can reconstruct complex light fields beyond conventional hardware limits. However, for many wide-field computational imaging techniques, including ptychography, depth sectioning remains a challenge. Here we demonstrate a high-resolution three-dimensional (3D) computational imaging approach, which combines ptychography with spectral-domain imaging, inspired by optical coherence tomography (OCT). This results in a flexible imaging system with the main advantages of OCT, such as depth-sectioning without sample rotation, decoupling of transverse and axial resolution, and a high axial resolution only determined by the source bandwidth. The interferometric reference needed in OCT is replaced by computational methods, simplifying hardware requirements. As ptychography is capable of deconvolving the illumination contributions in the observed signal, speckle-free images are obtained. We demonstrate the capabilities of ptychographic optical coherence tomography (POCT) by imaging an axially discrete lithographic structure and an axially continuous mouse brain sample.

Ultrafast laser-induced guided acoustic waves in thin, freely suspended films are important for many applications adopting the laser-ultrasonics technique. These waves show unique dispersion relations, leading to minimal propagation losses and acoustic energy confinement. While this principle has been known, the separation of various physical effects in the formation of measured signals involving these guided acoustic waves has not been clearly elaborated. Here, we present a combined experimental and theoretical study on all-optical excitation and detection of these waves in a thin, freestanding aluminum membrane. The acoustic dynamics is excited and measured by using a femtosecond time-resolved pump-probe technique with controlled probing position, enabling spatially resolved detection. The measured signals are compared with an advanced numerical model that we developed earlier [H. Zhang, Phys. Rev. Appl. 13, 014010 (2020)2331-701910.1103/PhysRevApplied.13.014010], showing excellent agreement. The combination of experiment and simulation allows us to decode various physical effects in the signal formation, including different acoustic field components. Unknown material properties, such as acoustic attenuation coefficients, and the two complex photoelastic constants are quantitatively retrieved by fitting the measured signals. Furthermore, we provide evidence of nonlinear propagation of the excited guided acoustic waves.