Evaluation of the preservation of tissue metabolic function in fresh and fixed epithelium using label-free two-photon fluorescence microscopy.
Sánchez-Hernández, Adriana.
2022
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Label-free two-photon microscopy can provide functional metabolic information with high spatial resolution of the dynamic microenvironment of a cell. It has been suggested that these metabolic interpretations can be maintained in tissue samples post-fixation. This would be useful given the large availability of fixed tissues for a wide range of conditions. However, there are few studies that address ... read morethe impact of fixation in fresh tissue. We present a two-photon excited fluorescence-based evaluation of cellular metabolism in freshly excised murine oral epithelia compared to corresponding optical signatures within bulk fixed tissue. We acquire TPEF images of NAD(P)H and FAD at 755 and 860 nm excitation, with signal acquisition at emission bands of 460/40 and 525/50 nm, respectively, and fluorescence lifetime images of NAD(P)H. We collect 3 to 4 image stacks from each fresh specimen, which is then fixed in formalin and imaged with the same parameters. A method involving ink markings allow for imaging of the same optical sections. The data is processed to get pixel-wise redox ratio maps with the TPEF intensity measurements. Phasor analysis of the FLIM data is used to estimate the contributions of bound and free NAD(P)H and corresponding lifetimes . Significant structural differences in the images of fixed biological tissues highlight the immediate effect that fixation has on freshly excised samples. The intensity variations that can be attributed to metabolic heterogeneity are lost after fixation as well as the ability to visualize the interstitial space delineating individual cells. The fluorescence lifetime of bound NAD(P)H changes upon fixation in a manner that is not well correlated to that of freshly excised tissue. These studies indicate that images of fixed tissues in wavelength ranges where metabolic readouts are typically acquired cannot be used as a reliable representation of the specimens' metabolic state pre-fixation.
3-minute talk presented at the Tufts Graduate Student Council's 27th Graduate Student Research Symposium, April 20, 2022.read less - Sánchez-Hernández, Adriana. "Evaluation of the preservation of tissue metabolic function in fresh and fixed epithelium using label-free two-photon fluorescence microscopy." Presentation at the 27th Graduate Research Symposium, Tufts University, April 20, 2022.
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