Figure 11

Effect of emission wavelengths on the Carmine Alum image. Orthogonal views of Carmine Alum images (from the experiment shown in Figures 4 whole mount and 8) are compared with the same TEB imaged using different settings. The left panels contain Carmine Alum images obtained using laser excitation set to 750 nm and emission wavelengths from 565–615 (BP 565–615 filter); the right panels contain Carmine Alum images obtained using laser excitation set to 860 nm and emission wavelengths from 650–710 (BP 650–710 filter). A. At left, Em 565–615 nm identifies primarily ECM/fibrillar layers and images the TEB epithelial cells poorly at increased depth (compare Z = 16 with Z = 52). At right, Em 650–710 nm identifies epithelial cells with improved imaging details at increasing z-depth. Dotted arrows identify the same position in the image. 3D movies are included in the Additional file (Additional file 6: Movies S4-5). C. Imaging orientation of the TEB is shown at low magnification (asterisk indicates the TEB imaged in A-B). A-B, Scale bars = 50 μm, C, Scale bars = 100 μm.