Adela Ben‐Yakar

Two-Photon Line Excitation Array Detection (2p-LEAD) is a novel, high-speed imaging method designed to overcome the speed limitations of traditional multi-photon microscopy. While conventional point-scanning is restricted to sub-30 Hz frame rates, 2p-LEAD can achieve 4 kHz frame rates at a 250 μm x 96 μm field-of-view. By coupling galvanometric line scanning with a parallelized 32-channel photomultiplier tube (PMT) array, we have constructed one of the fastest twophoton microscopes. Our system maintains the critical balance of subcellular resolution, kHz temporal resolution, and signal-to-noise ratio (SNR) through several key features. Temporal focusing confines the point-spread function (PSF) axially to reduce out-of-focus background, while the 32-channel PMT array enables highly efficient, parallelized photon collection. Additionally, optimizations of the optical configuration, excitation conditions, and detection hardware drastically improve the SNR for a given laser power, thereby mitigating the risk of phototoxicity and photodamage during sensitive, long-duration <i>in vivo</i> experiments. We demonstrate this advancement in capability by imaging the mammalian brain <i>in vivo</i>, resolving highly dynamic neurovascular events. The system's combination of high spatial resolution, temporal resolution, and SNR enables the quantitative measurement of fast-flowing red blood cells through cortical capillaries without motion artifacts. This demonstrated performance establishes a robust platform for future upgrades that may help enable comprehensive 4D investigation of functional and hemodynamic dynamics throughout large volumes of the mouse brain.

Two-photon laser scanning microscopy is widely used for deep-tissue imaging but is increasingly constrained by bulk tissue heating arising from linear absorption of excitation light. This volumetric heating accumulates with imaging depth, imposing conservative safe power limits that restrict achievable signal-to-noise ratio and imaging performance. While passive mitigation strategies such as objective are commonly employed, their effectiveness in suppressing bulk heating is limited. Here, we present a coupled numerical and experimental framework to quantify laser-induced bulk tissue heating during two-photon imaging and to evaluate active thermoelectric cooling strategies under realistic imaging conditions. Optical energy deposition is modeled using Monte Carlo simulations of photon transport and coupled to transient bioheat transfer calculations solved using an implicit finite difference scheme. The model is formulated in a two-dimensional domain and incorporates generalized thermal boundary conditions to represent localized thermoelectric cooling. The framework is validated against contact thermometry measurements performed in tissue-mimicking phantoms during 1035 nm point-scanning excitation at imaging depths of 300 μm and 1000 μm. Lateral temperature profiling demonstrates strong agreement between simulated and measured temperature rise, with an average error below 8%. Using a peak temperature rise criterion of 2&deg;C, depth-dependent safe average power thresholds are quantified in the absence of active cooling. Applying thermoelectric cooling under otherwise identical imaging conditions substantially relaxes these thermal constraints, enabling up to a 4× increase in deliverable average power at shallow imaging depths and a 3.67× increase at deeper imaging depths. These results demonstrate the potential of active thermoelectric cooling to mitigate bulk tissue heating and extend the operational envelope of high-power two-photon microscopy.

Ultrafast laser ablation offers unparalleled spatial and thermal confinement, making it a compelling candidate for high-precision spinal bone surgery. However, the inherently low ablation rates of ultrafast lasers and the challenges associated with probe miniaturization have significantly impeded their clinical translation. In this work, we present the design and prototyping of a new-generation, fiber-delivered ultrafast laser surgical scalpel optimized for both minimally invasive and open spinal procedures. The system integrates high-efficiency laser delivery via a Kagome hollow-core photonic crystal fiber, a piezoelectric fiber-scanning mechanism for compact beam steering, a high-demagnification miniaturized objective for enhanced ablation efficiency, and an opto-mechanical architecture enabling controlled depth-wise material removal and handheld operation. Prior efforts in parametric optimization informed the selection of pulse duration, spot size, and field-of-view to enable ablation rates exceeding 20 mm³/min under high-average-power operation. The optical design achieves a focused spot radius of 7.87 &mu;m with a working distance of 3.8 mm, suitable for deep spinal bone incisions. Preliminary handheld tests on bone and synthetic spine models demonstrate stable focal-plane ablation, ergonomic maneuverability, and access to confined anatomical regions. These results establish a scalable and clinically translatable platform for ultrafast laser–based spinal surgery and represent a significant step toward replacing conventional mechanical bone removal tools.

Ultrafast laser surgery presents a promising alternative to conventional surgical tools in procedures where high precision and thermal safety are paramount. However, clinical translation has been limited by low ablation speeds and challenges in system integration. In this work, we investigate the optimization of bone ablation performance using a compact ultrafast laser delivery benchtop system based on Kagome hollow‑core fiber and piezo‑enabled beam scanning. We systematically evaluate the influence of spot size, fluence, and pulse overlap on ablation efficiency. Our results reveal a strong dependence of threshold fluence on the number of overlapping pulses, with smaller spot sizes yielding higher ablation efficiencies. Ablation rates &gt;20 mm³/min has been achieved with 1 ps laser pulse at 11.8 W, highlighting a clear pathway toward achieving the clinically required rate of 1 mm³/s. Additionally, we report the first successful submerged ultrafast ablation of hard tissue using picosecond pulses and present initial thermal‑profiling data to assess heat accumulation during high‑power high-repetition rate ablation. Together, these findings provide key design considerations for the development of clinically viable, miniaturized ultrafast laser bone‑surgery probes.

Accurate modeling of optothermal transport in biological tissues is essential for applications such as two-photon brain imaging and laser microsurgery. Thermal transport physics in these processes are governed by the transient bioheat transfer partial differential equation (PDE), driven by complex, spatially varying laser source functions. Solving the bioheat transfer PDE across a range of beam parameters, such as numerical aperture, imaging depth, and field of view, is computationally intensive using conventional numerical solvers. To address this challenge, we introduce ResFouriONet, a neural operator architecture designed to learn the mapping from laser source functions to 2D spatiotemporal temperature fields. Built on the DeepONet framework, ResFouriONet combines a Fourier-based branch to capture global thermal diffusion with a residual convolutional branch for learning sharp, localized heating near the focal region. These outputs are fused using a self-attention mechanism, enabling dynamic feature selection across spatial scales. To reduce reliance on expensive labeled training data derived from Monte Carlo simulations, we propose a synthetic source generation scheme using parameterized Gaussian functions, ellipsoidal attenuation, and Gaussian random fields to emulate the complexity of realistic fluence profiles. ResFouriONet achieves strong generalization across both in-distribution and zero-shot test cases, with up accuracies close to 99% when compared with classical finite difference solvers. Our model remains accurate even when evaluated on realistic boundary conditions that are relevant in multiphoton imaging applications, as we demonstrate in this work. The ResFouriONet framework offers a promising surrogate model for rapid, simulation-free prediction of laser-induced heating in biomedical optics.

High-content imaging (HCI) and analysis are the keys for advancing our understanding of the science behind organogenesis. To this end, culturing adult stem cell-derived organoids (ASOs) in a platform that also enables live imaging, staining, immobilization, and fast high-resolution imaging is crucial. However, existing platforms only partially satisfy these requirements. In this study, we present the OrganoidChip+, an all-in-one microfluidic device designed to integrate both culturing and HCI of ASOs all within one platform. We previously developed the OrganoidChip as a robust imaging tool. Now, the OrganoidChip+ incorporates several additional features for culturing organoids in addition to fluorescence staining and imaging without the need for sample transfer. The organoids grown within a culture chamber are stained and then transferred to immobilization chambers for blur-free, high-resolution imaging at predetermined locations. We cultured adult stem cell-derived intestinal organoids in the chip for 7 days and tracked growth rates of each organoid using intermittent brightfield images, followed by multiple image-based assays, including viability assay using widefield fluorescence imaging, a redox ratio assay using label-free, two-color, two-photon microscopy, and immunofluorescence assays using confocal microscopy. These assays serve as proof-of-concept to showcase the chip's capabilities in HCI of ASOs. Organoids cultured in the chip exhibited superior average growth rates over those in traditional Matrigel dome cultures, off-chip. Viability and redox ratio measurements of on-chip organoids were comparable or slightly better than their off-chip counterparts. Confocal imaging further confirmed that the OrganoidChip+ supports robust organoid culture while enabling detailed, high-resolution analysis. This all-in-one platform holds great potential for advancing ASO-based research, offering a scalable and cost-effective solution for HCI and analysis in organogenesis, drug screening, and disease modeling.

Accurate modeling of laser-tissue interactions is essential for applications such as multiphoton brain imaging and laser-based therapeutics, where precise thermal control is critical. However, traditional numerical solvers for bioheat transfer, especially when paired with Monte Carlo (MC) simulations for light transport, are computationally prohibitive for real-time use. While operator learning offers a scalable alternative, existing architectures often struggle with extrapolatory tasks and rely on large, labeled datasets that are costly to generate. In this study, we introduce ResFouriONet, a residual deep operator network tailored for modeling 2D transient bioheat transfer with parametric laser sources. Our architecture features a dual-pathway branch network comprising a Fourier sub-branch for capturing global thermal patterns and a cascaded residual sub-branch for localized heating, fused via a self-attention mechanism. To reduce the reliance on expensive MC data, we introduce a synthetic laser source function generation scheme that combines parameterized Gaussian profiles, exponential decay, and random field perturbations to emulate the complexity of MC-derived fluence maps. ResFouriONet achieves a 5× reduction in prediction error compared to vanilla deep operator networks, despite having fewer parameters. It attains an average relative error of 0.93% on unseen synthetic data and 1.45% on 630 zero-shot MC-generated source functions. Together, the proposed architecture and data generation strategy enable accurate, real-time prediction of laser-induced heating, paving the way for simulation-free planning and control in biomedical imaging and laser-based therapies.

Development of relevant human induced pluripotent stem cell-derived cardiac organoids is essential to recapitulate myocardium physiology and functionality for the assessment of drug-induced toxicity evaluations. However, the optimal conditions for culturing self-aggregating multicellular cardiac organoids are not well-elucidated, particularly the impact of noncardiomyocytes. In this study, we generated cardiac organoids at varying seeding densities to formulate organoids that meet or exceed the biological diffusion limit. We assessed their morphology, gene expression profiles, beating functionality, viability, and mitochondrial activity over time. Our results show that organoid sizes stabilize by 7 days of culture, regardless of seeding density. However, organoids seeded with 20,000 cells retained a more optimal cardiac signature that promotes cardiac maturity and minimizes fibrotic tendencies, especially when cultured for longer than 7 days. While all organoid populations maintained their beating functionalities, those seeded with 80,000 cells exhibited greater cell shedding and increased apoptosis at long-term culture. In contrast, minimal apoptosis was observed in organoids seeded with 20,000 cells after 7 days. Mitochondrial staining further revealed that organoids seeded with 20,000 cells consistently demonstrated higher metabolic activity. Taken together, organoids seeded with 20,000 cells and cultured for 7 days yielded the healthiest morphology, transcriptional signature, and viability while maintaining robust beating kinetics. Importantly, the organoid model identified in this study demonstrated a selectivity index (SI) that is over an order of magnitude larger than that of two-dimensional cultures, showing improved sensitivity to clinically relevant doxorubicin-induced cardiotoxicity, enabling more accurate dose-response evaluations that better reflect therapeutic conditions.

Multiple scattering limits optical imaging in thick biological samples by scrambling sample-specific information. Physics-based inverse-scattering methods aim to computationally recover this information, often using non-convex optimization to reconstruct the scatter-corrected sample. However, this non-convexity can lead to inaccurate reconstructions, especially in highly scattering samples. Here, we show that various implementation strategies for even the same inverse-scattering method significantly affect reconstruction quality. We demonstrate this using multi-slice beam propagation (MSBP), a relatively simple nonconvex inverse-scattering method that reconstructs a scattering sample's 3D refractive-index (RI). By systematically conducting MSBP-based inverse-scattering on both phantoms and biological samples, we showed that an amplitude-only cost function in the inverse-solver, combined with angular and defocus diversity in the scattering measurements, enabled high-quality, fully-volumetric RI imaging. This approach achieved subcellular resolution and label-free 3D contrast across diverse, multiple-scattering samples. These results lay the groundwork for robust use of inverse-scattering techniques to achieve biologically interpretable 3D imaging in increasingly thick, multicellular samples, introducing a new paradigm for deep-tissue computational imaging.

High-content imaging (HCI) and analysis are the keys for advancing our understanding of the science behind organogenesis. To this end, culturing adult stem cell-derived organoids (ASOs) in a platform that also enables live imaging, staining, immobilization, and fast high-resolution imaging is crucial. However, existing platforms only partially satisfy these requirements. In this study, we present the OrganoidChip+, an all-in-one microfluidic device designed to integrate both culturing and HCI of ASOs all within one platform. We previously developed the OrganoidChip as a robust imaging tool. Now, the OrganoidChip+ incorporates several additional features for culturing organoids in addition to fluorescence staining and imaging without the need for sample transfer. The organoids grown within a culture chamber are stained and then transferred to immobilization chambers for blur-free, high-resolution imaging at predetermined locations. We cultured adult stem cell-derived intestinal organoids in the chip for 7 days and tracked growth rates of each organoid using intermittent brightfield images, followed by multiple image-based assays, including viability assay using widefield fluorescence imaging, a redox ratio assay using label-free, two-color, two-photon microscopy, and immunofluorescence assays using confocal microscopy. These assays serve as proof-of-concept to showcase the chip's capabilities in HCI of ASOs. Organoids cultured in the chip exhibited superior average growth rates over those in traditional Matrigel dome cultures, off-chip. Viability and redox ratio measurements of on-chip organoids were comparable or slightly better than their off-chip counterparts. Confocal imaging further confirmed that the OrganoidChip+ supports robust organoid culture while enabling detailed, high-resolution analysis. This all-in-one platform holds great potential for advancing ASO-based research, offering a scalable and cost-effective solution for HCI and analysis in organogenesis, drug screening, and disease modeling.

Development of relevant, human induced pluripotent stem cell-derived cardiac organoids is essential to recapitulate myocardium physiology and functionality for assessment of drug-induced toxicity evaluations. However, the optimal conditions for culturing self-aggregating multicellular cardiac organoids are not well-elucidated, particularly the impact of noncardiomyocytes. In this study, we generated cardiac organoids at varying seeding densities to formulate organoids that meet or exceed the biological diffusion limit. We assessed their morphology, gene expression profiles, beating functionality, viability, and mitochondrial activity over time. Our results show that organoid sizes stabilize by seven days of culture, regardless of seeding density. However, organoids seeded with 20,000 cells retained an optimal cardiac signature that promotes cardiac maturity and minimizes fibrotic tendencies, especially when culturing longer than seven days. While all organoid populations maintained their beating functionalities, those seeded with 80,000 cells exhibited greater cell shedding and increased apoptosis at long term culture. In contrast, minimal apoptosis was observed in organoids seeded with 20,000 cells after seven days. Mitochondrial staining further revealed that organoids seeded with 20,000 cells consistently demonstrated higher metabolic activity. Taken together, organoids seeded with 20,000 cells and cultured for seven days yielded the healthiest morphology, transcriptional signature, and viability, while maintaining robust beating kinetics. Importantly, compared to 2D cultures, these optimized organoids demonstrate improved sensitivity to clinically relevant doxorubicin-induced cardiotoxicity, enabling more accurate dose-response evaluations that better reflect therapeutic conditions. Impact Statement: This work highlights key tissue engineering considerations for generating self-assembling cardiac organoids suitable for scalable, high-throughput drug screening and discovery. Understanding the effect of seeding density and culture duration on organoid size and, consequently on gene expression, beating functionalities, apoptosis, and metabolic activity, has broader implications for establishing optimal organoid culture conditions. These insights enable the production of large quantities of cardiac organoids capable of modeling drug-induced toxicity effects on a clinically relevant timescale.

Recent advancements in ultrafast laser ablation technology redefine surgical precision while minimizing thermal damage, offering a promising alternative to traditional methods. However, the slow material removal rates (MRRs) have hindered clinical adoption. Addressing this challenge, we present a compact fiber-based laser delivery system, exhibiting an 82-fold increase in MRR compared to previous femtosecond laser probes. The system leverages a hollow-core Kagome fiber to deliver 10 ps laser pulses with high transmission efficiency and minimal nonlinear effects, even at high peak powers. The system distributes ultrashort pulses utilizing a piezo-scanned Lissajous-based beam steering mechanism onto the target surface over a larger field-of-view (FOV), enabling the scope for easy scalability of the system to a miniaturized probe. Drawing insights from our prior work, the focusing optics were carefully selected to deliver fluence three times the ablation threshold. An optimal combination of FOV size, translation speed, and repetition rate was identified, enabling clean ablations (devoid of carbonization) even at maximum laser power. We achieved a maximum MRR of 10.7 mm 3 /min with 8.8 W of laser power at 333 kHz, validating our hypothesis that high MRR can be achieved by ablating at high average powers over a large FOV with fast scanning of a large spot size. Numerical simulations further suggest that MRR up to 30 mm 3 /min can be achieved through increased repetition rates, expanded FOVs, and high translation speeds, defining an optimal parameter space for future probe designs. To validate the clinical relevance of high MRR, experiments were conducted to create deep bone incisions over a 3×3 mm 2 area within a clinically relevant timeframe. An ablation depth of ∼3 mm was achieved in ∼2 minutes without auxiliary cooling mechanisms. Scanning electron microscopy (SEM) of the deep incisions confirmed the preservation of healthy bone tissue, with clear evidence of canaliculi along the slopes and at the bottom surface of the ablated region. This study outlines a clear pathway toward developing a high-performance, miniaturized surgical probe with significant potential for spinal decompression surgery and other clinical applications, representing a transformative tool for future surgical precision.

To numerically characterize optothermal interactions at the laser-tissue interface during tissue imaging or surgery, the bioheat transfer partial differential equation (PDE) must be solved using a laser excitation source function. Given the range of possible experimental scenarios, the PDEs become parametric and must be solved for multiple optical and laser parameters. Due to constraints such as solver stability and domain size, classical numerical techniques used for performing such simulations are often computationally taxing and time consuming. To overcome these challenges, we propose to learn the mapping between infinite dimensional function spaces of the parameterized laser source functions and the solutions of the parametric bioheat transfer PDEs. In this work, we introduce Res-DeepONet, a new neural network architecture leveraging deep operator networks for rapidly accelerating PDE inferences. We train our operator network using synthetic laser heating source functions consisting of Gaussian beam profiles. The proposed neural network architecture demonstrates excellent generalization performance on unseen Gaussian source functions, achieving a 30× speedup over the classical finite difference method. With the introduction of Res-DeepONets, we aim to bring a paradigm shift in performing bioheat transfer simulations in the realm of laser-tissue interactions.

Capturing the fastest biological events in the mammalian brain, such as hemodynamic flow and neuronal signal propagation, requires high temporal resolution and imaging through scattering biological tissue. To fully resolve these functional dynamics, we present an improved version of 2-photon Line Excitation Array Detection (2p-LEAD) microscopy. Through shaping the 1035 nm excitation beam into a line, 2p-LEAD can operate at 4000 FPS, using a linear array of PMTs to collect the resulting fluorescence. With the replacement of the scanning mirror for an acousto-optic deflector, 2p-LEAD holds the potential of achieving kilohertz volumetric imaging over the visual cortex of the mice brain.

Developmental toxicity (DevTox) tests evaluate the adverse effects of chemical exposures on an organism's development. Although current testing primarily relies on large mammalian models, the emergence of new approach methodologies (NAMs) is encouraging industries and regulatory agencies to evaluate novel assays. C. elegans have emerged as NAMs for rapid toxicity testing because of its biological relevance and suitability to high throughput studies. However, current low-resolution and labor-intensive methodologies prohibit its application for sub-lethal DevTox studies at high throughputs. With the recent advent of the large-scale microfluidic device, vivoChip, we can now rapidly collect 3D high-resolution images of ~ 1000 C. elegans from 24 different populations. While data collection is rapid, analyzing thousands of images remains time-consuming. To address this challenge, we developed a machine-learning (ML)-based image analysis platform using a 2.5D U-Net architecture (vivoBodySeg) that accurately segments C. elegans in images obtained from vivoChip devices, achieving a Dice score of 97.80%. vivoBodySeg processes 36 GB data per device, phenotyping multiple body parameters within 35 min on a desktop PC. This analysis is ~ 140 × faster than the manual analysis. This ML approach delivers highly reproducible DevTox parameters (4-8% CV) to assess the toxicity of chemicals with high statistical power.

Developmental toxicity (DevTox) tests evaluate the adverse effects of chemical exposures on an organism’s development. While large animal tests are currently heavily relied on, the development of new approach methodologies (NAMs) is encouraging industries and regulatory agencies to evaluate these novel assays. Several practical advantages have made C. elegansa useful model for rapid toxicity testing and studying developmental biology. Although the potential to study DevTox is promising, current low-resolution and labor-intensive methodologies prohibit the use of C. elegans for sub-lethal DevTox studies at high throughputs. With the recent availability of a large-scale microfluidic device, vivoChip, we can now rapidly collect 3D high-resolution images of ~ 1,000 C. elegans from 24 different populations. In this paper, we demonstrate DevTox studies using a 2.5D U-Net architecture (vivoBodySeg) that can precisely segment C. elegans in images obtained from vivoChip devices, achieving an average Dice score of 97.80. The fully automated platform can analyze 36 GB data from each device to phenotype multiple body parameters within 35 min on a desktop PC at speeds ~ 140x faster than the manual analysis. Highly reproducible DevTox parameters (4–8% CV) and additional autofluorescence-based phenotypes allow us to assess the toxicity of chemicals with high statistical power.

Publisher's Note: This paper, originally published on 12 March 2024, was replaced with a corrected/revised version on 24 June 2024. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance.

In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.

We extend the resolution of our 2p autofluorescence imaging probe by incorporation of custom designed and fabricated optics. We will add an NA extender piece, inserted at excitation fiber tip to boost the beam divergence at the excitation optics input. The new custom designed miniaturized objective will achieve a resolution of 0.5 μm, without any changes to the diameter of the objective design. Custom design objective also will allow us to perform autofluorescence imaging at two colors. This will enable the optical redox ratio characterization of tissues, which is an important hallmark in diagnosis of cancers at early stages.

With the aim of advancing modern neuroscience and sampling neurons at up to 100 kHz frame rates, our group is developing a novel Two-photon Line Excitation Array Detection (2p-LEAD) imaging modality. Performing high resolution two-photon imaging at such high sampling rates necessitates the deposition of a large number of photons within the focal volume, which in turn warrants high laser powers. Consequently, the risk of heating and thermal damage limits the imaging speed and depth. In contrast to point-scan two-photon imaging, where safe average laser power values of 200 mW with conventional objective cooling have been established, there are no thermal characterization studies in the case of line-scan imaging modalities that could enable us in determining maximum laser powers to prevent tissue heating damage. We recently demonstrated through numerical investigations that enhanced cooling strategies of imparting laminar flow to the objective immersion water layer while implementing laser duty cycles could potentially increase safe power levels up to 600 mW of average surface power in the case of point scanning. A clear understanding of the effects of laser dosimetry on optical parameters of line-scan systems is essential to determine safe power values that would prevent thermal damage. In this work, we perform 3D MC-FDM numerical simulations at 1035 nm wavelength with a novel beam focusing framework over a parameter space spanning average powers and imaging depth to predict optothermal interactions. With experimental validation studies on tissue phantoms, our work would establish a much-needed power threshold in two-photon line scanning, which is an emerging modality of choice for high-speed volumetric imaging systems.

The major advancements in ultrafast laser ablation technology are revolutionizing surgical precision and minimizing thermal impact compared to traditional methods. However, the primary challenge hindering widespread clinical adoption has been the slow material removal rate (MRR). Towards this gap, a compact fiber-based laser delivery system has been developed, boasting an impressive 82-fold increase in MRR over the previous femtosecond laser surgical probes. This benchtop setup utilizes a hollow-core Kagome fiber (NA≈0.02) coupled to a high-power Yb-doped fiber laser (λ=1035 nm) to deliver laser pulses onto the sample. Employing a piezo-scanned Lissajous-based beam steering mechanism, the system achieves efficient distribution of ultrashort pulses onto the target surface. Remarkably, the system maintains a high transmission efficiency of 74% while operating at peak intensities, with no components exhibiting nonlinear behavior. For a FOV scan width of 550 µm, the logarithmic relationship between the ablation depth and laser fluence was determined for two different translational velocities. The system achieved material removal rates of ~10.7 mm3 /min for the maximum applied laser fluence of 9.3 J/cm², without initiating carbonization. Moreover, by fine-tuning laser parameters, the system can swiftly create clean-cut trenches of significant dimensions, 3 x 3 mm2 size and ~1 mm deep, mimicking conventional surgical procedures such as spinal decompression within a minute, all without carbonization or tissue damage. This remarkable achievement underscores the reliability and potential of ultrashort-laser ablation techniques for a wide array of surgical interventions.

In understanding the relationship between neuronal structure and functionality, we leverage calcium/voltage imaging to record the propagation of neuronal signals in vivo. To aptly capture these functional dynamics, we present 2-photon Line Excitation Array Detection (2p-LEAD) microscopy. Through shaping the 1035nm excitation beam into a line, 2p-LEAD can operate at 3000 FPS, using a linear array of PMT’s to collect the resulting fluorescence. With the replacement of the scanning mirror for an acousto-optic deflector, 2p-LEAD holds the potential of achieving kilohertz volumetric imaging over the visual cortex of the mice brain.

Two-photon excitation fluorescence microscopy has become an effective tool for tracking neural activity in the brain at high resolutions thanks to its intrinsic optical sectioning and deep penetration capabilities. However, advanced two-photon microscopy modalities enabling high-speed and/or deep-tissue imaging necessitate high average laser powers, thus increasing the susceptibility of tissue heating due to out-of-focus absorption. Despite cooling the cranial window by maintaining the objective at a fixed temperature, average laser powers exceeding 100-200 mW have been shown to exhibit the potential for altering physiological responses of the brain. This paper proposes an enhanced cooling technique for inducing a laminar flow to the objective immersion layer while implementing duty cycles. Through a numerical study, we analyze the efficacy of heat dissipation of the proposed method and compare it with that of the conventional, fixed-temperature objective cooling technique. The results show that improved cooling could be achieved by choosing appropriate flow rates and physiologically relevant immersion cooling temperatures, potentially increasing safe laser power levels by up to three times (3×). The proposed active cooling method can provide an opportunity for faster scan speeds and enhanced signals in nonlinear deep brain imaging.

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. The most efficient way of studying drug toxicity and efficacy requires high-resolution imaging of a large number of organoids acquired in the least amount of time. Currently missing are suitable platforms capable of fast-paced high-content imaging of organoids. To address this knowledge gap, we present the OrganoidChip, a microfluidic imaging platform that incorporates a unique design to immobilize organoids for endpoint, fast imaging. The chip contains six parallel trapping areas, each having a staging and immobilization chamber, that receives organoids transferred from their native culture plates and anchors them, respectively. We first demonstrate that the OrganoidChip can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. Next, we show the capability of our device in assessing the dose-dependent responses of organoids' viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-chip experiments. Importantly, the chip enables organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevents the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the OrganoidChip is a promising microfluidic platform that can serve as a building block for a multiwell plate format that can provide high-throughput and high-resolution imaging of organoids in the future.

Maternal RNAs are stored from minutes to decades in oocytes throughout meiosis I arrest in a transcriptionally quiescent state. Recent reports, however, propose a role for nascent transcription in arrested oocytes. Whether arrested oocytes launch nascent transcription in response to environmental or hormonal signals while maintaining the meiosis I arrest remains undetermined. We test this by integrating single-cell RNA sequencing, RNA velocity, and RNA fluorescence in situ hybridization on C. elegans meiosis I arrested oocytes. We identify transcripts that increase as the arrested meiosis I oocyte ages, but rule out extracellular signaling through ERK MAPK and nascent transcription as a mechanism for this increase. We report transcript acquisition from neighboring somatic cells as a mechanism of transcript increase during meiosis I arrest. These analyses provide a deeper view at single-cell resolution of the RNA landscape of a meiosis I arrested oocyte and as it prepares for oocyte maturation and fertilization.

BACKGROUND/OBJECTIVES: Tightly-focused ultrafast laser pulses (pulse widths of 100 fs-10 ps) provide high peak intensities to produce a spatially confined tissue ablation effect. The creation of sub-epithelial voids within scarred vocal folds (VFs) via ultrafast laser ablation may help to localize injectable biomaterials to treat VF scarring. Here, we demonstrate the feasibility of this technique in an animal model using a custom-designed endolaryngeal laser surgery probe. METHODS: in both healthy and scarred VFs. PEG-rhodamine was injected into these voids. Ex vivo optical imaging and histology were used to assess void morphology and biomaterial localization. RESULTS: Large sub-epithelial voids were observed in both healthy and scarred VFs immediately following in vivo laser treatment. Two-photon imaging and histology confirmed ~3-mm wide subsurface voids in healthy and scarred VFs of canine #2. Biomaterial localization within a void created in the scarred VF of canine #2 was confirmed with fluorescence imaging but was not visualized during follow-up two-photon imaging. As an alternative, the biomaterial was injected into the excised VF and could be observed to localize within the void. CONCLUSIONS: We demonstrated sub-epithelial void formation and the ability to inject biomaterials into voids in a chronic VF scarring model. This proof-of-concept study provides preliminary evidence towards the clinical feasibility of such an approach to treating VF scarring using injectable biomaterials. LEVEL OF EVIDENCES: N/A Laryngoscope, 133:3042-3048, 2023.

Ultrafast laser ablation supersedes conventional surgical techniques in terms of precision and thermal load generation. However, the main limiting criterion of the application of laser ablation techniques to surgeries has been the low material removal rate (MRR). In efforts to bridge the gap, a benchtop fiber-baser laser delivery system has been developed which demonstrated a MRR increase of ~15 times over the previously reported fs-laser surgical probes. The benchtop optical setup incorporates a hollow-core Kagome fiber (NA≈0.02) delivering high-power laser pulses from the Yb-doped fiber laser (λ=1035 nm) source to the sample. A Lissajous-based beam steering mechanism was employed to distribute the ultrashort laser pulses on the sample. The overall transmission efficiency of the system was 59%, with none of the components exhibiting any non-linear behavior at high peak intensities. For a FOV scan width of 250 &mu;m, the logarithmic relationship between the ablation depth and laser fluence was determined for two different translational velocities. The system achieved material removal rates of ~2 mm³/min for the maximum applied laser fluence of 18.9 J/cm², without initiating carbonization. Additionally, optimized laser parameters were implemented to achieve a clean-cut trench of 3 x 0.8 &mu;m² size and ~1.22 mm deep in under 3 minutes of laser exposure, which is within the surgical time bounds of a conventional spinal decompression technique. The fact that the trench is devoid of any carbonized section or unhealthy tissue, and was created without any irrigation setting only increases the reliability and viability of the ultrashort-laser ablation technique in surgical applications.

Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans . Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans . We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping. Femtosecond laser microdissection enables transcriptomic analyses of single neurons based on their phenotype.

The functional meaning associated with neuronal activity in the mammalian brain and sensory systems remains to be fully understood. Exploring this area of neuroscience requires high-speed 3D imaging operating at &gt;1 kHz volumetric scan rates with sub-cellular resolution, as neuronal signals propagate on sub-millisecond time scales. Additionally, since these studies must be performed in vivo, care must be taken to avoid invasive or damaging methods. Multi-photon imaging allows for non-invasive studies that deeply penetrate brain tissue, but has traditionally been limited to volumetric imaging between 10 to 100 Hz. We propose an improvement upon these systems with the novel imaging modality 2-photon Line Excitation and Array Detection (2p-LEAD) microscopy. 2p-LEAD is built on the main concept in our previous work where we developed single photon LEAD microscopy operating at 0.8 million FPS for 3D flow cytometry. In 2p-LEAD, we scan a 1035 nm excitation line of 2.4 μm x 220 μm (1/e² beam intensity diameter) at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a scanning mirror, we scan the line over a 140 μm x 160 μm FOV at 3,000 FPS, creating a frame of 16 x 320 pixels. Here we will present the design and imaging capabilities of our current 2p-LEAD instrument. This system lays the groundwork for higher speed imaging at 125 kHz frame rates with an acoustooptic deflector replacing the scanning mirror. When combined with vertical scanning, we will be able to volumetrically image at sub-millisecond time scales to allow for in vivo calcium imaging of the visual cortex.

I will present a new fluorescence imaging method called LEAD (line excitation array detection) microscopy, capable of providing 0.8 million frames per second. This method performs line-scanning of excitation laser beam using a chirped signal-driven longitudinal acousto-optic deflector to create a virtual light-sheet, and images the field-of-view with a linear photomultiplier tube array to generate a 66×14 pixel frame each scan cycle. I will present an implementation of the LEAD microscopy as a blur-free 3D imaging flow cytometer with 3.5-micron resolution and signal-to-background ratios >200. I will also present its conceptual implementation as an ultrafast two-photon LEAD microscopy (2p-LEAD).

Imaging modalities capable of detecting functional changes over small areas can increase sensitivity and specificity of early cancer detection. Label-free imaging of metabolic activity at cellular level resolution over full thickness of cervix epithelium is possible with 2p imaging. However, low probability of 2p excitation and scattering nature of tissues limit autofluorescence levels in 2p imaging. We present a 2p autofluorescence imaging endoscope system for detection of metabolic changes in cervix in a clinical setting, with an increased collection efficiency in scattering media. Collection of autofluorescence signals is done with a multitude of high NA fibers arranged around a miniaturized excitation objective. By cleaving the collection fibers at a specific angle, we increase the directivity of the collection and the collection efficiency per fiber. The endoscope performs imaging at 775 nm, which is capable of exciting NAD(P)H and FAD molecules. Laser pulses of 100 fs duration are delivered to the sample with an air core photonic bandgap fiber. Fiber is scanned in spiral pattern via a piezo actuator tube. Scanning at different tissue depths is possible with the axial actuation of the endoscope via a linear stepper motor. Benchtop tests indicate that the endoscope system has lateral and axial resolutions of 0.65 μm and 4.33 μm, respectively. Fluorescence images of pollen cores are presented to demonstrate the imaging quality of the endoscope system.

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and, therefore, can serve as in vitro miniaturized organ models for drug testing. The most effective way of studying drug toxicity and efficacy requires fast-paced and high-resolution imaging of organoids which is possible by first immobilizing the organoids. However, suitable platforms that are conducive to high-throughput imaging and can fully immobilize the organoids are missing. We propose a unique microfluidic device capable of hydrogel-free immobilization of cardiac organoids in predetermined locations for viability staining and calcium-transients imaging without compromising their morphology and behavior. In doing so, we treated cardiac organoids with Doxorubicin (DOX), and successfully immobilized and imaged them within our device to evaluate organoid viability upon treatment.

In an effort to better understand the functioning of the brain, a new two-photon imaging modality being developed by our group aims to achieve unprecedented imaging speeds at up to 100 kHz. The high powers required for facilitating the same pose the risk of brain heating beyond established thermal limits. This work explores various theoretical boundary conditions for evaluating the impact on resulting spatiotemporal thermal distribution for an input parameter space at 1035nm excitation wavelength using an MC-FDM coupled numerical model with an aim of further paving way towards devising cooling strategies for deep brain imaging.