Non-invasive imaging of pathological scars using a portable handheld two-photon microscope

Background:Pathological scars are a disorder that can lead to various cosmetic,psychological,and functional problems,and no effective assessment methods are currently available.Assessment and treatment of pathological scars are based on cutaneous manifestations.A two-photon microscope(TPM)with the potential for real-time non-invasive assessment may help determine the under-surface pathophysiological conditions in vivo.This study used a portable handheld TPM to image epidermal cells and dermal collagen structures in pathological scars and normal skin in vivo to evaluate the effectiveness of treatment in scar patients.Methods:Fifteen patients with pathological scars and three healthy controls were recruited.Imaging was performed using a portable handheld TPM.Five indexes were extracted from two dimensional(2D)and three dimensional(3D)perspectives,including collagen depth,dermo-epidermal junction(DEJ)contour ratio,thickness,orientation,and occupation(proportion of collagen fibers in the field of view)of collagen.Two depth-dependent indexes were computed through the 3D second harmonic generation image and three morphology-related indexes from the 2D images.We assessed index differences between scar and normal skin and changes before and after treatment.Results:Pathological scars and normal skin differed markedly regarding the epidermal morphological structure and the spectral characteristics of collagen fibers.Five indexes were employed to distinguish between normal skin and scar tissue.Statistically significant differences were found in average depth(t=9.917,P<0.001),thickness(t=4.037,P<0.001),occupation(t=2.169,P<0.050),orientation of collagen(t=3.669,P<0.001),and the DEJ contour ratio(t=5.105,P<0.001).Conclusions:Use of portable handheld TPM can distinguish collagen from skin tissues;thus,it is more suitable for scar imaging than reflectance confocal microscopy.Thus,a TPM may be an auxiliary tool for scar treatment selection and assessing treatment efficacy.

Dual-objective two-photon microscope for volumetric imaging of dense scattering biological samples by bidirectional excitation and collection

Full view observation throughout entire specimens over a prolonged period is crucial when exploring the physiological functions and system-level behaviors.Multi-photon microscopy(MPM)has been widely employed for such purposes owing to its deep penetration ability.However,the current MPM struggles with balancing the imaging depth and quality while avoiding photodamage for the exponential increasement of excitation power with the imaging depth.Here,we present a dual-objective two-photon microscope(Duo-2P),characterized by bidirectional two-photon excitation and fluorescence collection,for long-duration volumetric imaging of dense scattering samples.Duo-2P effectively doubles the imaging depth,reduces the total excitation energy by an order of magnitude for samples with a thickness five times the scattering length,and enhances the signal-to-noise ratio up to 1.4 times.Leveraging these advantages,we acquired volumetric images of a 380-μm suprachiasmatic nucleus slice for continuous 4-h recording at a rate of 1.67 s/volume,visualized the calcium activities over 4000 neurons,and uncovered their state-switching behavior.We conclude that Duo-2P provides an elegant and powerful means to overcome the fundamental depth limit while mitigating photodamages for deep tissue volumetric imaging.