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.

An optically powered CMOS tracking system for 3 T magnetic resonance environment.

In this work, a fully optical Complementary Metal Oxide Semiconductor (CMOS) based catheter tracking system designed for 3 T Magnetic Resonance Imaging (MRI) environment is presented. The system aims to solve the Radio Frequency (RF) induced heating problem present in conventional wired catheter tracking systems used in MRI. It is based on an integrated circuit, consisting of a receiver and an optical power supply unit. The optical power supply unit includes a single on-chip photodiode and a DC-DC converter that boosts the low photodiode voltage output to voltages greater than 1.5 V. Through an optically driven switch, the accumulated charge on an a storage capacitor is transferred to the rest of the system. This operation is novel in the way that it is fully optical and the switch control is done through modulation of the applied light. An on-chip local oscillator signal for the receiver is avoided by application of an RF signal that is generated by the MRI machine at the receiving period. The signals received by a micro-coil antenna are processed by the on-chip direct conversion receiver. The processed signal is then transferred, also optically, to the outside world for tracking purposes. The frequency encoding method is used for MRI tracking. Operation with various levels of external optical power does not generate noticeble temperature increase in the system. The overall system is successfully tested in a 3 T MRI machine to demonstrate its full operation.