Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions.

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.

Numerical study of a convective cooling strategy for increasing safe power levels in two-photon brain imaging.

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.

OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids.

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.

Spatial single-cell sequencing of meiosis I arrested oocytes indicates acquisition of maternal transcripts from the soma.

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.

Ultrafast Laser Microlaryngeal Surgery for In Vivo Subepithelial Void Creation in Canine Vocal Folds.

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: Unilateral VF mucosal injuries were created in two canines. Four months later, ultrashort laser pulses (5 ps pulses at 500 kHz) were delivered via the custom laser probe to create sub-epithelial voids of ~3 × 3-mm2 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.

Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing.

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.

Ultrafast laser surgery probe for sub-surface ablation to enable biomaterial injection in vocal folds.

Creation of sub-epithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable therapeutic biomaterials towards an improved treatment for vocal fold scarring. Several ultrafast laser surgery probes have been developed for precise ablation of surface tissues; however, these probes lack the tight beam focusing required for sub-surface ablation in highly scattering tissues such as vocal folds. Here, we present a miniaturized ultrafast laser surgery probe designed to perform sub-epithelial ablation in vocal folds. The requirement of high numerical aperture for sub-surface ablation, in addition to the small form factor and side-firing architecture required for clinical use, made for a challenging optical design. An Inhibited Coupling guiding Kagome hollow core photonic crystal fiber delivered micro-Joule level ultrashort pulses from a high repetition rate fiber laser towards a custom-built miniaturized objective, producing a 1/e2 focal beam radius of 1.12 ± 0.10 µm and covering a 46 × 46 µm2 scan area. The probe could deliver up to 3.8 µJ pulses to the tissue surface at 40% transmission efficiency through the entire system, providing significantly higher fluences at the focal plane than were required for sub-epithelial ablation. To assess surgical performance, we performed ablation studies on freshly excised porcine hemi-larynges and found that large area sub-epithelial voids could be created within vocal folds by mechanically translating the probe tip across the tissue surface using external stages. Finally, injection of a model biomaterial into a 1 × 2 mm2 void created 114 ± 30 µm beneath the vocal fold epithelium surface indicated improved localization when compared to direct injection into the tissue without a void, suggesting that our probe may be useful for pre-clinical evaluation of injectable therapeutic biomaterials for vocal fold scarring therapy. With future developments, the surgical system presented here may enable treatment of vocal fold scarring in a clinical setting.

In vivo hamster cheek pouch subepithelial ablation, biomaterial injection, and localization: pilot study.

SIGNIFICANCE: The creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring. AIM: We aim to prove that subepithelial voids can be created in a live animal model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids. APPROACH: A benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention. RESULTS: Focused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation. CONCLUSIONS: We successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live animal model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large animals will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.

Ultrafast laser surgery probe with a calcium fluoride miniaturized objective for bone ablation.

We present a miniaturized ultrafast laser surgery probe with improved miniaturized optics to deliver higher peak powers and enable higher surgical speeds than previously possible. A custom-built miniaturized CaF2 objective showed no evidence of the strong multiphoton absorption observed in our previous ZnS-based probe, enabling higher laser power delivery to the tissue surface for ablation. A Kagome fiber delivered ultrashort pulses from a high repetition rate fiber laser to the objective, producing a focal beam radius of 1.96 µm and covering a 90×90 µm2 scan area. The probe delivered the maximum available fiber laser power, providing fluences >6 J/cm2 at the tissue surface at 53% transmission efficiency. We characterized the probe's performance through a parametric ablation study on bovine cortical bone and defined optimal operating parameters for surgery using an experimental- and simulation-based approach. The entire opto-mechanical system, enclosed within a 5-mm diameter housing with a 2.6-mm diameter probe tip, achieved material removal rates >0.1 mm3/min, however removal rates were ultimately limited by the available laser power. Towards a next generation surgery probe, we simulated maximum material removal rates when using a higher power fiber laser and found that removal rates >2 mm3/min could be attained through appropriate selection of laser surgery parameters. With future development, the device presented here can serve as a precise surgical tool with clinically viable speeds for delicate applications such as spinal decompression surgeries.

Thermal lensing effects and nonlinear refractive indices of fluoride crystals induced by high-power ultrafast lasers.

Thermo-optical and nonlinear property characterization of refractive optical components is essential for endoscopic instrumentation that utilizes high-power, high-repetition-rate ultrafast lasers. For example, ytterbium-doped fiber lasers are well suited for ultrafast laser microsurgery applications; however, the thermo-optical responses of many common lens substrates are not well understood at 1035 nm wavelength. Using a z-scan technique, we first measured the nonlinear refractive indices of CaF2, MgF2, and BaF2 at 1035 nm and found values that match well with those from the literature at 1064 nm. To elucidate effects of thermal lensing, we performed z-scans at multiple laser repetition rates and multiple average powers. The results showed negligible thermal effects up to an average power of 1 W and at 10 W material-specific thermal lensing significantly altered z-scan measurements. Using a 2D temperature model, we could determine the source of the observed thermal lensing effects. Linear absorption was determined as the main source of heating in these crystals. On the other hand, inclusion of nonlinear absorption as an additional heat source in the simulations showed that thermal lensing in borosilicate glass was strongly influenced by nonlinear absorption. This method can potentially provide a sensitive method to measure small nonlinear absorption coefficients of transparent optical materials. These results can guide design of miniaturized optical systems for ultrafast laser surgery and deep-tissue imaging probes.

Femtosecond Plasmonic Laser Nanosurgery (fs-PLN) mediated by molecularly targeted gold nanospheres at ultra-low pulse fluences.

Plasmonic Laser Nanosurgery (PLN) is a novel photomodification technique that exploits the near-field enhancement of femtosecond (fs) laser pulses in the vicinity of gold nanoparticles. While prior studies have shown the advantages of fs-PLN to modify cells, further reduction in the pulse fluence needed to initiate photomodification is crucial to facilitate deep-tissue treatments. This work presents an in-depth study of fs-PLN at ultra-low pulse fluences using 47 nm gold nanoparticles, conjugated to antibodies that target the epithelial growth factor receptor and excited off-resonance using 760 nm, 270 fs laser pulses at 80 MHz repetition rate. We find that fs-PLN can optoporate cellular membranes with pulse fluences as low as 1.3 mJ/cm2, up to two orders of magnitude lower than those used at lower repetition rates. Our results, corroborated by simulations of free-electron generation by particle photoemission and photoionization of the surrounding water, shed light on the off-resonance fs-PLN mechanism. We suggest that photo-chemical pathways likely drive cellular optoporation and cell damage at these off-resonance, low fluence, and high repetition rate fs-laser pulses, with clusters acting as local concentrators of ROS generation. We believe that the low fluence and highly localized ROS-mediated fs-PLN approach will enable targeted therapeutics and cancer treatment.

Planar Laser Activated Neuronal Scanning (PLANS) System for in vivo Flow Cytometry.

We present a light-sheet flow cytometer for screening of C. elegans. A machine learning approach is utilized to enable real-time analysis of protein aggregation models.

An automated microfluidic device for time-lapse imaging of mouse embryonic stem cells.

Long-term, time-lapse imaging studies of embryonic stem cells (ESCs) require a controlled and stable culturing environment for high-resolution imaging. Microfluidics is well-suited for such studies, especially when the media composition needs to be rapidly and accurately altered without disrupting the imaging. Current studies in plates, which can only add molecules at the start of an experiment without any information on the levels of endogenous signaling before the exposure, are incompatible with continuous high-resolution imaging and cell-tracking. Here, we present a custom designed, fully automated microfluidic chip to overcome these challenges. A unique feature of our chip includes three-dimensional ports that can connect completely sealed on-chip valves for fluid control to individually addressable cell culture chambers with thin glass bottoms for high-resolution imaging. We developed a robust protocol for on-chip culturing of mouse ESCs for minimum of 3 days, to carry out experiments reliably and repeatedly. The on-chip ESC growth rate was similar to that on standard culture plates with same initial cell density. We tested the chips for high-resolution, time-lapse imaging of a sensitive reporter of ESC lineage priming, Nanog-GFP, and HHex-Venus with an H2B-mCherry nuclear marker for cell-tracking. Two color imaging of cells was possible over a 24-hr period while maintaining cell viability. Importantly, changing the media did not affect our ability to track individual cells. This system now enables long-term fluorescence imaging studies in a reliable and automated manner in a fully controlled microenvironment.

Scattering properties and femtosecond laser ablation thresholds of human and canine vocal folds at 776-nm wavelength.

Ultrafast laser ablation may provide a treatment for vocal fold (VF) scarring. Optical properties of VFs must be known prior to clinical implementation to select appropriate laser surgery conditions. We present scattering lengths of epithelium ℓs , ep, superficial lamina propria ℓs , SLP, and ablation thresholds Fth of human and canine VF tissues. Our experimental approach involves an image-guided, laser-ablation-based method that allows for simultaneous determination of ℓs and Fth in these multilayered tissues. Studying eight canine samples, we found ℓs , ep = 75.3 ± 5.7 µm, ℓs , SLP = 26.1 ± 1.2 µm, Fth , ep = 1.58 ± 0.06 J / cm2, and Fth , SLP = 1.55 ± 0.17 J / cm2. Studying five human samples, we found ℓs , ep = 42.8 ± 3.3 µm and Fth , ep = 1.66 ± 0.10 J / cm2. We studied the effects of cumulative pulse overlap on ablation threshold and found no significant variations beyond 12 overlapping pulses. Interestingly, our studies about the effect of sample storage on the scattering properties of porcine VF show a 60% increase in ℓs , ep for fresh porcine VF when compared to the same sample stored in isotonic solution. These results provide guidelines for clinical implementation by enabling selection of optimal laser surgery parameters for subsurface ablation of VF tissues.

High-Content and High-Throughput In Vivo Drug Screening Platforms Using Microfluidics.

The drug-discovery process is expensive and lengthy, and has been causing a rapid increase in the global health care cost. Despite extensive efforts, many human diseases still lack a cure. To improve the outcomes, there is a growing need to implement novel approaches into the early stages of the drug-discovery pipeline. A specific such effort has focused on the development of novel disease models such as cellular models (genetically modified cell lines, spheroids, and organoids) and whole-animal models (small animal models and genetically modified large animal models). The whole-animal screens are advantageous as they can provide system-level information, off-target effects, complete absorption, distribution, metabolism, excretion, and toxicity architectures, and early in vivo toxicity, which help to prioritize compounds before using them for human trials. Such multivariate analysis helps to improve the translational potential of drug compounds. Drug testing in large animals is expensive and time consuming. A solution is small animal models that have simplified biological system with intact physiology and sufficient homology with human genes. In recent times, many such models have constantly been developed and tested to identify new disease mechanisms. Caenorhabditis elegans is one such small animal model that has been considered for large-scale drug testing. In this review, we will discuss the current state-of-the-art technologies, including two platforms developed in my group that have enabled high-throughput and high-content screening using C. elegans disease models.

Line excitation array detection fluorescence microscopy at 0.8 million frames per second.

Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity. The frame rates of current imaging methods are limited to kHz by the photon budget, slow camera readout, and/or slow laser beam scanners. Here, we present line excitation array detection (LEAD) fluorescence microscopy, a high-speed imaging method capable of providing 0.8 million frames per second. The method performs 0.8 MHz line-scanning of an 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. We implement LEAD microscopy as a blur-free flow cytometer for Caenorhabditis elegans moving at 1 m s-1 with 3.5-µm resolution and signal-to-background ratios >200. Signal-to-noise measurements indicate future LEAD fluorescence microscopes can reach higher resolutions and pixels per frame without compromising frame rates.

High-Content Microfluidic Screening Platform Used To Identify σ2R/Tmem97 Binding Ligands that Reduce Age-Dependent Neurodegeneration in C. elegans SC_APP Model.

The nematode Caenorhabditis elegans, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently, we developed a single-copy model of amyloid precursor protein (SC_APP) induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (σ2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure-activity relationships for unexplored chemical space in our σ2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our C. elegans model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that σ2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases.

A multi-trap microfluidic chip enabling longitudinal studies of nerve regeneration in Caenorhabditis elegans.

Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only.

Determination of scattering properties and damage thresholds in tissue using ultrafast laser ablation.

Ultrafast laser surgery of tissue requires precise knowledge of the tissue's optical properties to control the extent of subsurface ablation. Here, we present a method to determine the scattering lengths, ?s, and fluence thresholds, Fth, in multilayered and turbid tissue by finding the input energies required to initiate ablation at various depths in each tissue layer. We validated the method using tissue-mimicking phantoms and applied it to porcine vocal folds, which consist of an epithelial (ep) layer and a superficial lamina propia (SLP) layer. Across five vocal fold samples, we found ?s,ep=51.0±3.9???m, Fth,ep=1.78±0.08??J/cm2, ?s,SLP=26.5±1.6???m, and Fth,SLP=1.14±0.12??J/cm2. Our method can enable personalized determination of tissue optical properties in a clinical setting, leading to less patient-to-patient variability and more favorable outcomes in operations, such as femto-LASIK surgery.

Kagome fiber based ultrafast laser microsurgery probe delivering micro-Joule pulse energies.

We present the development of a 5 mm, piezo-actuated, ultrafast laser scalpel for fast tissue microsurgery. Delivery of micro-Joules level energies to the tissue was made possible by a large, 31 µm, air-cored inhibited-coupling Kagome fiber. We overcome the fiber's low NA by using lenses made of high refractive index ZnS, which produced an optimal focusing condition with 0.23 NA objective. The optical design achieved a focused laser spot size of 4.5 µm diameter covering a 75 × 75 µm2 scan area in a miniaturized setting. The probe could deliver the maximum available laser power, achieving an average fluence of 7.8 J/cm2 on the tissue surface at 62% transmission efficiency. Such fluences could produce uninterrupted, 40 µm deep cuts at translational speeds of up to 5 mm/s along the tissue. We predicted that the best combination of speed and coverage exists at 8 mm/s for our conditions. The onset of nonlinear absorption in ZnS, however, limited the probe's energy delivery capabilities to 1.4 µJ for linear operation at 1.5 picosecond pulse-widths of our fiber laser. Alternatives like broadband CaF2 crystals should mitigate such nonlinear limiting behavior. Improved opto-mechanical design and appropriate material selection should allow substantially higher fluence delivery and propel such Kagome fiber-based scalpels towards clinical translation.