Figure 7. Effect of red filter bandpass on two-photon (2P) red-to-green fluorescence intensity ratios representing the signal-to-noise ratio in the red channel. Pixel intensity (mean gray value) was calculated in five regions of interest (ROIs) positioned in each image frame (see Figure 6). In the absence of Alexa-568 (-Red Dye), red channel fluorescence was attributed to broad spectrum Hoechst 33342 and autofluorescence (AF) bleed-through. Alexa-568-to-AF bleed-through mean gray value ratios decreased with increasing excitation wavelength (800 to 900 nm) and were highest through a far-red filter (590–680 nm). For the ratio values, see Table 1.