Figure 10

Spectral emission scans at selected multiphoton excitation and effects of emission wavelengths: effects upon visualization of Carmine Alum-stained TEBs. A. Whole mount mammary glands stained with Carmine Alum were imaged using 6 different multiphoton excitation wavelengths (Ex 750, 800, 830, 860, 890, and 950 nm) with laser power set at 0.1% in lambda mode to obtain 6 resulting spectral emission scans. The laser gain was altered to obtain good image intensity without saturation for each excitation wavelength. One major peak at Em 623 nm and a minor peak at Em 484 nm were observed. An image was generated from the Ex 860 nm lambda scan (Em 575–671 nm) including the peak emission at 623 nm. The white circle indicates the ROI used for the lambda scan at each excitation. B. Images were generated from Ex 750, 860, and 950 nm using two different emission wavelength ranges, Em 565–615 nm and Em 650–710 nm, representing the proximal and distal sides of the 623 nm peak (illustrated at left). At all excitation wavelengths, the images at the longer wavelength side of the peak have a better representation of epithelial tissue, at the expense of stroma. Arrows indicate differences in the images. Scale bars = 20 μm.