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.

The functional meaning associated with neuronal activity in the mammalian brain and sensory systems remains to be fully understood. Exploring this area of neuroscience requires high-speed 3D imaging operating at >1 kHz volumetric scan rates with sub-cellular resolution, as neuronal signals propagate on sub-millisecond time scales. Additionally, since these studies must be performed in vivo, care must be taken to avoid invasive or damaging methods. Multi-photon imaging allows for non-invasive studies that deeply penetrate brain tissue, but has traditionally been limited to volumetric imaging between 10 to 100 Hz. We propose an improvement upon these systems with the novel imaging modality 2-photon Line Excitation and Array Detection (2p-LEAD) microscopy. 2p-LEAD is built on the main concept in our previous work where we developed single photon LEAD microscopy operating at 0.8 million FPS for 3D flow cytometry. In 2p-LEAD, we scan a 1035 nm excitation line of 2.4 μm x 220 μm (1/e² beam intensity diameter) at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a scanning mirror, we scan the line over a 140 μm x 160 μm FOV at 3,000 FPS, creating a frame of 16 x 320 pixels. Here we will present the design and imaging capabilities of our current 2p-LEAD instrument. This system lays the groundwork for higher speed imaging at 125 kHz frame rates with an acoustooptic deflector replacing the scanning mirror. When combined with vertical scanning, we will be able to volumetrically image at sub-millisecond time scales to allow for in vivo calcium imaging of the visual cortex.

Background/ObjectivesTightly‐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.MethodsUnilateral 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.ResultsLarge 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.ConclusionsWe 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 EvidencesN/A Laryngoscope, 2023