Figure 12

Imaging unstained whole mount tissue. A. An unstained mammary gland whole mount was imaged to a depth of 380 μm using SHG-B (Em 390-465 nm, red), autofluorescence (Em 500–530 nm, blue), and SHG-F (unfiltered ChD, green). B. Measurement of intensities from background subtracted and thresholded images demonstrate that SHG and autofluorescence were detected throughout the Z-stack and are an example of the imaging depth details obtained for tissue archived as unstained whole mounts. Thresholded average intensities of individual frames plotted against Z-depth are shown for the SHG-B (red), SHG-F (green) and autofluorescence (blue) channels. In C and D, orthogonal views, and in E, a vertical 3D “cut” view of the same sample, features are revealed at Z-depths directly below a TEB which is positioned near the gland surface. In A-E, red solid arrows indicate the vessel at medium Z-depth and dotted red arrows indicate SHG-B and SHG-F signals at a deeper Z-depth. The white arrows point out SHG-F and SHG-B signals directly below the unstained TEB. The SHG-F signal is brighter in association with vessels compared with TEBs. C-E. Scale bar = 20 μm.