Design and test of a rigid endomicroscopic system for multimodal imaging and femtosecond laser ablation

Abstract

.SignificanceConventional diagnosis of laryngeal cancer is normally made by a combination of endoscopic examination, a subsequent biopsy, and histopathology, but this requires several days and unnecessary biopsies can increase pathologist workload. Nonlinear imaging implemented through endoscopy can shorten this diagnosis time, and localize the margin of the cancerous area with high resolution.AimDevelop a rigid endomicroscope for the head and neck region, aiming for in-vivo multimodal imaging with a large field of view (FOV) and tissue ablation.ApproachThree nonlinear imaging modalities, which are coherent anti-Stokes Raman scattering, two-photon excitation fluorescence, and second harmonic generation, as well as the single photon fluorescence of indocyanine green, are applied for multimodal endomicroscopic imaging. High-energy femtosecond laser pulses are transmitted for tissue ablation.ResultsThis endomicroscopic system consists of two major parts, one is the rigid endomicroscopic tube 250 mm in length and 6 mm in diameter, and the other is the scan-head (10 × 12 × 6 cm3 in size) for quasi-static scanning imaging. The final multimodal image accomplishes a maximum FOV up to 650 μm, and a resolution of 1 μm is achieved over 560 μm FOV. The optics can easily guide sub-picosecond pulses for ablation.ConclusionsThe system exhibits large potential for helping real-time tissue diagnosis in surgery, by providing histological tissue information with a large FOV and high resolution, label-free. By guiding high-energy fs laser pulses, the system is even able to remove suspicious tissue areas, as has been shown for thin tissue sections in this study.

Feasibility studies of multimodal nonlinear endoscopy using multicore fiber bundles for remote scanning from tissue sections to bulk organs

“…The power of the laser beams at the sample site was about 50 mW for pump and 25 mW for Stokes in the probe measurement and 70 mW for pump, and 40 mW for Stokes in LSM recordings. The average laser power used for nonlinear endoscopic imaging provided sufficient signal generation from the presented samples without causing visible damage to any of them and aligns with the power scales utilized in comparable nonlinear endoscopic imaging applications 18 , 35 with use of picosecond laser pulses. A useful point of reference in this context is provided by Galli et al 36 who investigated photodamage under similar excitation conditions as a function of recorded frame repetitions.…”

“…S2 ) would, locally, slightly change that number. Although this resolution may appear less competitive compared to the micron and sub-micron resolutions reported in other studies 18 , 35 , 40 , 41 essential to note that this multicore fiber-based endoscope was conceptualized differently. Its primary objective is to facilitate nonlinear imaging at a tissue level without the need for electronic and moving parts in the endoscope.…”

“…For easier prototyping and more versatility in the testing phase, the current design does not include a fixed glass window and instead relies on a cover glass to be placed on the sample. The probe design is largely agnostic to the specific LSM used and could, for example, be coupled with a compact fiber-based laser system and scan head to be incorporated into a mobile station, as demonstrated recently for a rigid-body nonlinear endoscope 35 . An overview of key structural and performance characteristics of the probe is given in Table 1 .…”

Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions

“…1) Maintenance of collection efficiency over the entire depth of imaging in a highly scattering tissue. 2) Delivery of ultrashort pulses of high peak intensities to the tissue over a flexible path, namely through optical fibers, without initiating nonlinear effects and, thus, damaging the fiber [ 30 , 39 ]. 3) Compatibility with the 2p excitation wavelengths of endogenous intracellular fluorophores, that are considered important metabolic markers, such as NAD(P)H and FAD [ 19 , 40 – 43 ].…”

“…Additional care in laser coupling to fiber core is also needed due to the requirement to direct the collected signal coming from the second cladding in the proximal end of the fiber with dichroic mirror [ 34 ]. The second approach to autofluorescence collection is to couple the autofluorescence signal to another fiber dedicated to collection, using optical elements in front of or behind the objective, which can create additional design and alignment challenges [ 25 , 29 , 30 ].…”