Figure 5. Effect of excitation wavelength on Hoechst 33342 and autofluorescence bleed-through. Bleed-through of fluorescence (AF) associated with Hoechst 33342-stained nuclei (nuclei bleed-through) and of AF from extracellular matrix (AF bleed-through) was collected through a near red filter channel (565–605 nm) at different 2-photon (2P) excitation wavelengths. Black columns: AF bleed-through intensity from fixed trabecular meshwork (TM). Hatched columns: AF bleed-through intensity from fixed TM colabeled with Hoechst 33342. White columns: Nuclear bleed-through. Nuclear bleed-through was an order higher than AF bleed-through. y-axis (mean gray value of fluorescence intensity) is set to a logarithmic (base 10) scale. Error bars=standard deviation.