Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

摘要: Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial temporal information from the same specimen is fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into structural functional dynamics cells in their physiological environment. At nanoscale, complex ultrastructure cell's environment tissue can be reconstructed three dimensions (3D) using serial block face scanning electron (SBF-SEM). This provides snapshot pertaining to shape, organization, localization multiple subcellular structures at time. The pairing these two modalities relate to the ultrastructural Until recently, approaches relocate region interest (ROI) 2P for SBF-SEM have inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by laser multiphoton microscope create fiducial markers accurate correlation (EM) volumes. process quick user defined each sample. Here, increase efficiency ROI relocation, NIRB marks are used 3D target ultramicrotomy. A workflow described discussed obtain data set correlated light different preparations brain as examples.