Abstract
Label-free multiphoton microscopy is a powerful platform for biomedical imaging. Recent advancements have demonstrated the capabilities of transient absorption microscopy (TAM) for label-free quantification of hemoglobin and stimulated Raman scattering (SRS) microscopy for pathological assessment of label-free virtual histochemical staining. We propose the combination of TAM and SRS with two-photon excited fluorescence (TPEF) to characterize, quantify, and compare hemodynamics, vessel structure, cell density, and cell identity in vivo between age groups. In this study, we construct a simultaneous nonlinear absorption, Raman, and fluorescence (SNARF) microscope with the highest reported in vivo imaging depth for SRS and TAM at 250–280 μm to enable these multimodal measurements. Using machine learning, we predict capillary-lining cell identities with 90% accuracy based on nuclear morphology and capillary relationship. The microscope and methodology outlined herein provides an exciting route to study several research topics, including neurovascular coupling, blood-brain barrier, and neurodegenerative diseases.
Highlights
Label-free multiphoton microscopy is a powerful platform for biomedical imaging
stimulated Raman scattering (SRS) microscopy has been widely used to image the spatial distribution of lipids and proteins in various biological systems ranging from cells to tissue by probing two transitions, 2850 cm−1 (CH2 stretching for lipids) and 2930 cm−1 (CH3 stretching for lipids and proteins)[9]
two-photon excited fluorescence (TPEF) will continue to play a dominant role in high resolution in vivo brain imaging, these limitations highlight the need for alternative imaging methods, such as labelfree multiphoton microscopy
Summary
Label-free multiphoton microscopy is a powerful platform for biomedical imaging. Recent advancements have demonstrated the capabilities of transient absorption microscopy (TAM) for label-free quantification of hemoglobin and stimulated Raman scattering (SRS) microscopy for pathological assessment of label-free virtual histochemical staining. The requirement of labeling brings limitations: (1) TPEF emission spectra are typically broad (~50 nm), which limits the number of separable fluorescent contrasts, and information that can be collected from one animal, (2) generating animals with multiple transgenes can be challenging, time-consuming, and expensive[3], and (3) application of fluorescent dyes— topically or intravenously—is invasive which can alter the animal’s physiology[4] To circumvent these limitations, orthogonal label-free multiphoton imaging techniques can generate additional image contrasts. Ji et al reported an SRS imaging depth of ~100 μm when demonstrating in vivo pathological differentiation between tumor and nonneoplastic tissue in a xenografted mouse brain model[9] To address this issue, our group and others have worked to increase signal sizes and penetration depth, via pulse optimization[15], wavelength optimization[16], aberration correction[17], tissue clearing[18,19], and deep learning[20,21]. Transient absorption microscopy (TAM) is another label-free chemical imaging technique that has found utility in biomedical imaging through the quantification of endogenous heme proteins
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