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- 摘要
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- 实验方案
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Slide-free imaging of hematoxylin-eosin stained whole-mount tissues using combined third-harmonic generation and three-photon fluorescence microscopy
摘要： Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time-consuming and labor-intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically-sectioned histopathological imaging of hematoxylin-eosin stained whole-mount skin tissues with a sub-femtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications.
关键词： hematoxylin-eosin,three photon microscopy,third harmonic generation microscopy,surgical border,margin assessment
更新于2025-09-23 15:23:52
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Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy
摘要： Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex > 1 mm in depth in awake mice with low (<20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.
关键词： subplate neurons,deep brain imaging,visual cortex,neuronal activity,three-photon microscopy,GCaMP6s
更新于2025-09-23 15:23:52