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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.
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 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 ~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 μm2 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.
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.
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