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. 2016 Mar 2;22:203–212.

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Copyright © 2016 Molecular Vision.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, used for non-commercial purposes, and is not altered or transformed.

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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.