FLIM an SLIM analysis of mammary tumors. (a) Multiphoton spectral lifetime imaging microscopy (SLIM) analysis of the emission spectrum from endogenous fluorescence resulting from excitation at 890 nm. The emission signals were separated by 10 nm spectral steps over 16 channels (10 channels are displayed) and the photons collected in each channel used to generate fluorescence lifetime images and signals for each channel plotted with SLIM-Plotter (shown). Emission from collagen (at half of the input wavelength) showed a very strong and sharp signal with a no appreciable decay (lifetime) confirming the SHG nature of the collagen signal (top). Emission spectra of endogenous fluorescence from tumor and stromal cells showed that the only substantial emission signal is at 530 nm, indicating that the source of the autofluorescence signal is FAD, with lifetime values from the 530 channel matching values obtained with fluorescence lifetime imaging microscopy (FLIM). (b) Multiphoton intensity and FLIM images of the stroma near a tumor (top) and the tumor and stromal components (bottom) from wild-type tumors showing the utility of FLIM to image tumor cells, stromal cells, and extracellular matrix components. Note the increased intensity and fluorescent lifetimes of stromal cells (quantified in (c)) and the low lifetime of collagen (matching system response, that is, no actual lifetime/color mapping toward blue). The color map in (b) represents the weighted average of the two-term model components (τm = (a
1τ1 + a
1 + a
2)) using the equation shown in (c). (c) Quantitative analysis of fluorescent lifetime components from tumor and stromal (subscript s) cells using the equation shown. Note the increase in the second (long) component and weighted mean component (see the equation above) for stromal cells when compared with cells from the primary tumor mass. Note that at least 30 measurements per tumor image from 4 independent tumors were used to calculate lifetime values for tumor cells in the primary tumor mass while at least 6 measurements per tumor image from 4 independent tumors were used for stromal cells. *Indicates a statistically significant (p < 0.05) difference following analysis with one-way analysis of variance (ANOVA) with a post-hoc Tukey-Kramer test.