Optimizing two-photon multiple fluorophore imaging of the human trabecular meshwork.

Area of Science:

• Biomedical Optics
• Cellular Imaging
• Ophthalmology Research

Background:

• Two-photon (2P) microscopy enables deep tissue imaging with multiple fluorophores.
• The human trabecular meshwork (TM) presents imaging challenges due to autofluorescence and specific cellular structures.

Purpose of the Study:

• To determine optimal 2P imaging parameters for simultaneously visualizing three fluorophores in the human TM.
• To detect autofluorescence, nuclear labels (Hoechst 33342), and filamentous actin (Alexa 568) within the TM.
• To achieve balanced signal intensities and minimize spectral bleed-through using limited emission filters.

Main Methods:

• 2P imaging of postmortem human TM samples.
• Utilized Hoechst 33342 for nuclei, Alexa 568 for actin, and detected endogenous autofluorescence.
• Analyzed emission detection across green, near-red, and far-red filters with 2P excitation at 750, 800, 850, and 900 nm.
• Quantified fluorescence intensity using region-of-interest (ROI) analysis.

Main Results:

• Alexa 568 fluorescence was strongest with 750/800 nm excitation but undetectable at 900 nm.
• 750/800 nm excitation caused Hoechst 33342 to overwhelm autofluorescence and bleed into red channels.
• 850 nm excitation provided balanced Hoechst 33342 and autofluorescence intensities.
• 850 nm excitation minimized bleed-through into the far-red channel and yielded good Alexa 568 signals.

Conclusions:

• 2P excitation at 850 nm combined with far-red emission detection is optimal for human TM imaging.
• This approach allows simultaneous visualization of endogenous and exogenous fluorophores with balanced intensities.
• Minimized spectral bleed-through enhances clarity for studying TM structure and function.