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James C H Tan, Jose Miguel Gonzalez; Multiphoton excitation parameters for three-color fluorescence imaging of the human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3301.
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© ARVO (1962-2015); The Authors (2016-present)
2-photon excitation fluorescence imaging (TPEF) permits tissue-based subcellular visualization of the human trabecular meshwork (TM). Optimal excitation by this non-linear technique to simultaneously observe multiple tissue fluorophores in tissue is hard to predict and needs to be determined empirically.
Human corneoscleral donor tissue was fixed and stained with Hoechst 33342 for nuclei and Alexa 568-conjugated phalloidin for F-actin. Autofluorescence from the structural extracellular matrix (ECM) and Hoechst signals were captured with a green (525/50nm) filter. F-actin epifluorescence was captured with near-red (585/40nm) or far-red (635/90nm) emission filters. TPEF excitation wavelengths of 750nm, 800nm, 850nm and 900nm were tested.
Shorter excitation wavelengths yielded the highest emission intensities for all fluorophores. Autofluorescence was barely evident relative to Hoechst at 750nm and 800nm excitations. Hoechst-to-autofluorescence intensity ratios (reflecting signal balance) were: 1.0 at 900nm; 2.7 at 850nm; and 9.2 at 800nm. Bleed-through of green (Hoechst and autofluorescence) signals into the red emission channel increased with longer excitation wavelengths, but a far-red filter reduced this bleed-through. F-actin red intensity-to-green fluorescence ratios increased with longer excitation wavelength up to 900nm excitation.
850nm excitation provided the optimal balance in emission intensities for TPEF triple fluorophore visualization in the human TM. At 850nm, Hoechst 33342-nuclear labeling was clearly visible without overpowering the ECM’s autofluorecsent signal. A far-red filter permitted Alexa-568 epifluorescence with the highest signal-to-noise ratio.
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