Multiphoton Microscopy for Assessment of Tissue Structure

Abstract

Multiphoton microscopies (MPM), including two-photon excited fluorescence (TPEF) andsecond harmonic generation (SHG) microscopy, are robust methodologies for visualizing and assessing tissue in the context of injury and cancer. SHG, a nonlinear scattering phenomenon, offers sensitivity to structures lacking axial symmetry, with collagen—a crucial protein surrounding cells—being a prime example. Collagen plays a supporting role in wound healing and cancer progression, with alterations in its structure serving as indicative markers of both processes. This work delves into the versatility of MPM in analyzing collagen’s involvement in wound healing and disease development, while also exploring creative solutions to microscope design that enhance the viability of endoscopic implementation. Specifically, MPM with TPEF and SHG is used to visualize and quantify the steps in Achillestendon healing following mechanical and thermal injury in rats. Qualitative and quantitative MPM findings complemented traditional histology by providing additional information about collagen organization and infiltrative cell densities not readily identifiable in traditional histology. This indicates that MPM is a worthwhile way to visualize tendon wound healing from tendinopathy with and without ITU therapy. Also, the utilization of randomly sampled SHG measurements in colorectal cancer diagnosisis proposed for the first time, potentially streamlining endoscope design complexities related to the scanning mechanism. High-resolution SHG images of colorectal tumor, tumor-adjacent, and normal tissue were randomly point- and line-sampled to simulate no- and one-dimensional scanning. Results included significant differences between SHG intensity of given tissue types with only 1000 points or lines in a 1M+ pixel image. This indicates feasibility of a simpler SHG measurement system consisting of less or no scanning components. Further, the incorporation of multiple numerical aperture (NA) SHG measurements inassessing colon tissue holds promise in eliminating the necessity for paired normal and cancer-suspicious measurements in each patient. Collection apertures of different diameters were placed into the emission pathway of the microscope in order to measure the relationship between emission NA and colorectal tumor SHG intensity. SHG images were taken of tumor, tumor-adjacent, and normal tissue. Upon linear regression analysis, it was found that tumor tissue responded differently to NA as an independent variable. This indicates that multiple NAs could be used as a way to make an endoscope self-referencing, which could simplify the clinical workflow. While MPM is an established microscopy in research settings, this investigation into noveldevice designs and expanded applications points towards an intriguing new direction for this technology. With these advancements, MPM with SHG is moving closer to achieving simpler and more feasible clinical application.