Purpose: In situ 2-photon excitation fluorescence (TPEF) of the human trabecular meshwork (TM) reveals beams of heterogeneous autofluorescence (AF) comprising high intensity fluorescent fibers (AF-high) on a background of lower intensity fluorescence (AF-low). To determine the sources of this AF heterogeneity, we imaged human TM to characterize AF, second harmonic generation (SHG) for collagen, and eosin-labeled fluorescence identifying elastin.
Methods: Corneoscleral rims retained after corneal transplantation were incubated with and without eosin, and imaged by TPEF. TPEF was collected through multiphoton bandpass filters to obtain AF, SHG (collagen bandwidth), and eosin-labeled fluorescence images. For qualitative comparisons, near-simultaneous image acquisition pairs of AF-SHG (+/-eosin coincubation), AF-eosin, and SHG-eosin were captured. For quantitative comparisons, multiple regions of interest (ROI) were defined in separate TM beam regions within the uveal and corneoscleral meshwork for image acquisition pairs of AF-SHG (without eosin coincubation) and SHG-eosin. We defined 18 ROI within each acquisition pair as the basis for Manders colocalization analysis. Perfect colocalization was defined as a Manders coefficient (Mcoeff) of 1.
Results: Qualitatively and quantitatively, AF-low colocalized with SHG (Mcoeff=1), but not SHG signal-voids. AF-high colocalized with SHG signal-voids (Mcoeff=1), but not the SHG signal. Like AF-high, eosin-labeled fluorescence qualitatively and quantitatively colocalized (Mcoeff=1) with SHG signal-voids, but not the SHG signal.
Conclusions: Heterogeneous AF in human TM is comprised of high intensity signal originating from elastin fibers in beam cores and lower intensity signal originating from collagen. These findings are relevant to interpreting structural extracellular matrix signals in AF images of the TM.
Keywords: autofluorescence; collagen; elastin; trabecular meshwork.