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.

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.

Design and fabrication of a miniature objective consisting of high refractive index zinc sulfide lenses for laser surgery.

A miniature laser ablation probe relying on an optical fiber to deliver light requires a high coupling efficiency objective with sufficient magnification in order to provide adequate power and field for surgery. A diffraction-limited optical design is presented that utilizes high refractive index zinc sulfide to meet specifications while reducing the miniature objective down to two lenses. The design has a hypercentric conjugate plane on the fiber side and is telecentric on the tissue end. Two versions of the objective were built on a diamond lathe-a traditional cylindrical design and a custom-tapered mount. Both received an antireflective coating. The objectives performed as designed in terms of observable resolution and field of view as measured by imaging a 1951 USAF resolution target. The slanted edge technique was used to find Strehl ratios of 0.75 and 0.78, respectively, indicating nearly diffraction-limited performance. Finally, preliminary ablation experiments indicated threshold fluence of gold film was comparable to similar reported probes.

Point-by-point near-field optical energy deposition around plasmonic nanospheres in absorbing media.

Here we investigate the effects of absorbing media on plasmon-enhanced near-field optical energy deposition. We find that increasing absorption by the medium results in increased particle scattering at the expense of particle absorption, and that much of this increased particle scattering is absorbed by the medium close to the particle surface. We present an analytical method for evaluating the spatial distribution of near-field enhanced absorption surrounding plasmonic metal nanospheres in absorbing media using a new point-by-point method. We propose criteria to define relevant near-field boundaries and calculate the properties of the local absorption enhancement, which redistributes absorption to the near-field and decays asymptotically as a function of the distance from the particle to background levels. Using this method, we performed a large-scale parametric study to understand the effect of particle size and wavelength on the near-field absorption for gold nanoparticles in aqueous media and silicon, and identified conditions that are relevant to enhanced local infrared absorption in silicon. The presented approach provides insight into the local energy transfer around plasmonic nanoparticles for predicting near-field effects for advanced concepts in optical sensing, thin-film solar cells, nonlinear imaging, and photochemical applications.

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.

vivoBodySeg: Machine learning-based analysis of C. elegans immobilized in vivoChip for automated developmental toxicity testing.

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.

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.

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.

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.

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.

Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons.

Functional neuronal recovery following injury arises when severed axons reconnect with their targets. In Caenorhabditis elegans following laser-induced axotomy, the axon still attached to the cell body is able to regrow and reconnect with its separated distal fragment. Here we show that reconnection of separated axon fragments during regeneration of C. elegans mechanosensory neurons occurs through a mechanism of axonal fusion, which prevents Wallerian degeneration of the distal fragment. Through electron microscopy analysis and imaging with the photoconvertible fluorescent protein Kaede, we show that the fusion process re-establishes membrane continuity and repristinates anterograde and retrograde cytoplasmic diffusion. We also provide evidence that axonal fusion occurs with a remarkable level of accuracy, with the proximal re-growing axon recognizing its own separated distal fragment. Thus, efficient axonal regeneration can occur by selective reconnection and fusion of separated axonal fragments beyond an injury site, with restoration of the damaged neuronal tract.

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 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.

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies.

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.

Optical design and imaging performance testing of a 9.6-mm diameter femtosecond laser microsurgery probe.

We present the optical design of a 9.6-mm diameter fiber-coupled probe for combined femtosecond laser microsurgery and nonlinear optical imaging. Towards enabling clinical use, we successfully reduced the dimensions of our earlier 18-mm microsurgery probe by half, while improving optical performance. We use analytical and computational models to optimize the miniaturized lens system for off-axis scanning aberrations. The optimization reveals that the optical system can be aberration-corrected using simple aspheric relay lenses to achieve diffraction-limited imaging resolution over a large field of view. Before moving forward with custom lenses, we have constructed the 9.6-mm probe using off-the-shelf spherical relay lenses and a 0.55 NA aspheric objective lens. In addition to reducing the diameter by nearly 50% and the total volume by 5 times, we also demonstrate improved lateral and axial resolutions of 1.27 µm and 13.5 µm, respectively, compared to 1.64 µm and 16.4 µm in our previous work. Using this probe, we can successfully image various tissue samples, such as rat tail tendon that required 2-3 × lower laser power than the current state-of-the-art. With further development, image-guided, femtosecond laser microsurgical probes such as this one can enable physicians to achieve the highest level of surgical precision anywhere inside the body.

Fast-updating and nonrepeating Lissajous image reconstruction method for capturing increased dynamic information.

We present a fast-updating Lissajous image reconstruction methodology that uses an increased image frame rate beyond the pattern repeat rate generally used in conventional Lissajous image reconstruction methods. The fast display rate provides increased dynamic information and reduced motion blur, as compared to conventional Lissajous reconstruction, at the cost of single-frame pixel density. Importantly, this method does not discard any information from the conventional Lissajous image reconstruction, and frames from the complete Lissajous pattern can be displayed simultaneously. We present the theoretical background for this image reconstruction methodology along with images and video taken using the algorithm in a custom-built miniaturized multiphoton microscopy system.

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.

Role of near-field enhancement in plasmonic laser nanoablation using gold nanorods on a silicon substrate.

We present experimental results for the plasmonic laser ablation of silicon with nanoscale features as small as 22 x 66 nm using single near-infrared, femtosecond laser pulses incident on gold nanorods. Near the ablation threshold, these features are photo-imprints of gold nanorod particles positioned on the surface of the silicon and have feature sizes similar to the nanorods. The single rod-shaped ablation pattern matches the enhancement patterns of the Poynting vector magnitude on the surface of silicon, implying that the ablation is a result of the plasmonic enhancement of the incident electromagnetic waves in the near-field of the particles. Interestingly, the ablation pattern is different from the two separated holes at the ends of the nanorod, as would be expected from the electric field--|E|(2) enhancement pattern. We measured the plasmonic ablation threshold fluence to be almost two orders of magnitude less than the femtosecond laser ablation threshold of silica, present in the thin native oxide layer on the surface of silicon. This value also agrees with the enhancement of the Poynting vector of a nanorod on silicon as calculated with electromagnetic simulations. We thus conclude that plasmonic ablation with plasmonic nanoparticles depends directly on the polarization and the value of the near-field enhancement of the Poynting vector and not the square of the electric field as previously suggested.

Neurosurgery: functional regeneration after laser axotomy.

Understanding how nerves regenerate is an important step towards developing treatments for human neurological disease, but investigation has so far been limited to complex organisms (mouse and zebrafish) in the absence of precision techniques for severing axons (axotomy). Here we use femtosecond laser surgery for axotomy in the roundworm Caenorhabditis elegans and show that these axons functionally regenerate after the operation. Application of this precise surgical technique should enable nerve regeneration to be studied in vivo in its most evolutionarily simple form.

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.