SignificanceThe 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.AimWe 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.ApproachA 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.ResultsFocused 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.ConclusionsWe 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.
We present an ultrafast laser surgery probe for bone tissue microsurgery. A custom miniaturized CaF2 mitigated the strong multiphoton absorption observed in our previous ZnS-based design while providing tighter beam focusing over a larger ablation field-of-view. The objective produced a beam waist radius of 1.71 μm covering a 130×130 μm2 scan area, delivering fluences >8 J/cm2 at the tissue surface at 53% transmission efficiency. The entire opto-mechanical system, enclosed within a 14 mm diameter metal housing with a 2.6 mm probe tip, exhibited material removal rates >0.1 mm3/min in bovine cortical bone. We performed simulations when using a high-power fiber laser and found that material removal rates >40 mm3/min could be achieved through selection of optimal laser surgery parameters. The device can serve as a clinically viable solution for minimally invasive spinal surgery applications.
We present the development of an endoscopic ultrafast laser scalpel with improved miniaturized optics as a follow-up to our previous studies. We previously determined that the nonlinear susceptibility of ZnS crystal lenses at high pulse energies can limit the maximum energy reaching the tissue surface. Here, we improve the nonlinear properties of miniaturized optics using CaF2 in lieu of ZnS as a lens material to mitigate the problem of three-photon absorption at the high energies needed for rapid tissue ablation. We built and tested a miniaturized objective consisting of a CaF2 crystal lens pair to focus ultrashort laser pulses delivered through a large air core Kagome fiber to a 3.4 μm diffraction limited spot and scan the beam with a piezo-tube across a 100×100 μm^2 field of view (FOV). Negligible three-photon absorption and high transmission through the probe (>50%) allows for delivery of fluences >8.0 J/cm^2. The entire opto-mechanical system, enclosed within a 5-mm hypodermic tubing, can remove tissue at material removal rates (MRR) >0.5 mm^3/min in excised soft (porcine vocal folds) and hard (bovine rib bone) tissue samples. We found that MRRs could be increased by the optimized combination of piezo- and translation scanning parameters, providing clinically relevant tissue removal speeds. Further, we present the first ultrafast tissue ablation experiments in a live animal model (hamster cheek pouch) using a handheld surgical probe. Towards clinical acceptance, we designed an injection-molded enclosure to seal the 5-mm diameter opto-mechanical assembly and constituent wires into a sterilization-ready ergonomic handheld stylus tool.
We present the development of a 5 mm, piezo-actuated, ultrafast laser scalpel for microsurgery with a capability to deliver energies in excess of 1 μJ per pulse. Having previously established that the maximum energy deliverable was limited by cladding damage in photonic badgap fibers, we utilized a large, 35μm cored inhibited-coupling Kagome fiber that allowed the delivery of micro-Joule energy femtosecond pulses. To maintain diffraction limited performance over the entire scan range of the piezo-actuated fiber tip, special objective lenses were developed and manufactured out of a high-refractive index Zinc Sulfide (ZnS) crystal. The probe was packaged in hypodermic 304SS stainless steel with a form factor minimizing in-line configuration. The probe’s performance was tested via metal and tissue ablation studies, characterizing highspeed ablation parameters and uniformity of ablation over the scan area. Additionally, we studied the nonlinear performance of ZnS and Calcium Fluoride (CaF2) as materials for refractive optics and determined the maximum energy deliverable through our probe using these optical materials. The high energy delivery through the probe system should allow for fast and effective tissue ablation.
Background: Deep tissue ablation is the next challenge in ultrafast laser microsurgery. By focusing ultrafast pulses below the tissue surface one can create an ablation void confined to the focal volume. However, as the ablation depth increases in a scattering tissue, increase in the required power can trigger undesired nonlinear phenomena out of focus that restricts our ability to ablate beyond a maximum ablation depth of few scattering lengths. Optical clearing (OC) might reduce the intensity and increase the maximal ablation depth by lowering the refractive index mismatch, and therefore reducing scattering. Some efforts to ablate deeper showed out of focus damage, while others used brutal mechanical methods for clearing. Our clinical goal is to create voids in the scarred vocal folds and inject a biomaterial to bring back the tissue elasticity and restore phonation.
Materials and methods: Fresh porcine vocal folds were excised and applied a biocompatible OC agent (75% glycerol). Collimated transmittance was monitored. The tissue was optically cleared and put under the microscope for ablation threshold measurements at different depths.
Results: The time after which the tissue was optically cleared was roughly two hours. Fitting the threshold measurements to an exponential decay graph indicated that the scattering length of the tissue increased to 83±16 μm, which is more than doubling the known scattering length for normal tissue.
Conclusion: Optical clearing with Glycerol increases the tissue scattering length and therefore reduces the energy for ablation and increases the maximal ablation depth. This technique can potentially improve clinical microsurgery.
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.
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