Optimizing two-photon multiple fluorophore imaging of the human trabecular meshwork.
OBJECTIVE
Advances in two-photon (2P) deep tissue imaging provide powerful options for simultaneously viewing multiple fluorophores within tissues. We determined imaging parameters for optimally visualizing three fluorophores in the human trabecular meshwork (TM) to simultaneously detect broad-spectrum autofluorescence and multiple fluorophores through a limited number of emission filters.


METHODS
2P imaging of viable human postmortem TM was conducted to detect Hoechst 33342-labeled nuclei, Alexa-568-conjugated phalloidin labeling of filamentous actin, and autofluorescence of the structural extracellular matrix (ECM). Emission detection through green (500-550 nm), near-red (565-605 nm), and far-red (590-680 nm) filters following 2P excitation at 750, 800, 850, and 900 nm was analyzed. Region-of-interest (ROI) image analysis provided fluorescence intensity values for each fluorophore.


RESULTS
Red-channel Alexa 568 fluorescence was of highest intensity with 2P 750 nm and 800 nm excitation. Alexa 568 was imperceptible with 900 nm excitation. With excitation at 750 nm and 800 nm, Hoechst 33,342 intensity swamped autofluorescence in the green channel, and marked bleed-through into red channels was seen. 850 nm excitation yielded balanced Hoechst 33342 and autofluorescence intensities, minimized their bleed-through into the far-red channel, and produced reasonable Alexa 568 intensities in the far-red channel.


CONCLUSIONS
2P excitation at 850 nm and long-wavelength emission detection in the far-red channel allowed simultaneous visualization of the specific mix of endogenous and exogenous fluorophores with reasonably balanced intensities while minimizing bleed-through when imaging the human TM.
Doheny Eye Institute; University of California, Los Angeles, Los Angeles, CA.
Jose M
JM
Gonzalez
Keck School of Medicine, University of Southern California, Los Angeles, CA.
Michael J
MJ
Ammar
Doheny Eye Institute; University of California, Los Angeles, Los Angeles, CA.
MinHee K
MK
Ko
Doheny Eye Institute; University of California, Los Angeles, Los Angeles, CA; Department of Ophthalmology, University of California, Los Angeles, Los Angeles, CA.
James C H
JC
Tan
D001562:Benzimidazoles / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D010590:Phalloidine; D013194:Staining and Labeling / Q000379:methods; D014129:Trabecular Meshwork / Q000033:anatomy & histology* / Q000378:metabolism
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; C000607448:alexa 568; D010590:Phalloidine; C017807:bisbenzimide ethoxide trihydrochloride