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. 2016 Mar 2;22:203-12.
eCollection 2016.

Optimizing Two-Photon Multiple Fluorophore Imaging of the Human Trabecular Meshwork

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Free PMC article

Optimizing Two-Photon Multiple Fluorophore Imaging of the Human Trabecular Meshwork

Jose M Gonzalez Jr et al. Mol Vis. .
Free PMC article

Abstract

Purpose: 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.

Figures

Figure 1
Figure 1
One-(1P) and two-photon (2P) imaging of Hoechst 33342, autofluorescence, and Alexa-568 in the human juxtacanalicular meshwork. 1P (AC) and 2P (DI) fluorescence excitation imaging, at varying excitation wavelengths, resulted in unique combinations of Hoechst 33342, autofluorescence (AF), and Alexa-568-conjugated phalloidin emission signals. Emission was captured through red (565–605 nm; A, D, G) and green (500–550 nm; B, E, H) filters. 1P excitation was at 543 nm for the red channel and 488 nm for the green channel. 2P excitation was at 750 nm (DF) or 850 nm (GI). Alexa-568-phalloidin fluorescence was similar in the red channel across all conditions. Hoechst 33342 nuclear fluorescence was visible in the green channel (500–550 nm) with 2P, but not 1P, excitation, in which it was brighter at shorter excitation wavelengths (E versus H). With 750 nm excitation (E), Hoechst 33342 was bright, but AF was almost imperceptible. With 850 nm excitation (H), AF and Hoechst 33342 intensities and visualization were more balanced. Arrows=Hoechst 33342–labeled nuclei. Asterisk=extracellular matrix-associated AF. SP mirror=tunable prism-based spectrophotometric detector. Bar=25 μm. Insets: 2X magnification of regions indicated by asterisks.
Figure 2
Figure 2
Autofluorescence intensity in the human trabecular meshwork. Extracellular matrix (ECM)-derived autofluorescence (AF) intensity (mean gray value) was determined for tissues from 4 human donors (A, B, C, and D). Each column represents mean pixel intensity for a donor human trabecular meshwork (TM) excited at a particular two-photon (2P) wavelength. Mean intensity of four donor tissues for each excitation wavelength is shown as an adjacent black column. Error bars=standard deviation.
Figure 3
Figure 3
Increasing two-photon (2P) excitation wavelength decreased Hoechst 33342 nuclear fluorescence intensity and increased autofluorescence (AF) intensity. Two tissue sections from the same donor were fixed and incubated without (A, C, E) or with Alexa-568-conjugated phalloidin (B, D, F) and imaged. Emission from the uveal meshwork following 2P excitation at 800 nm (A, B), 850 nm (C, D), and 900 nm (E, F) was captured through a green filter (500–550 nm). Region-of-interest (ROI) analysis of AF (green) and Hoechst 33342 nuclear fluorescence (blue) are illustrated. Bar=25 μm.
Figure 4
Figure 4
Effect of excitation wavelength on relative Hoechst 33342 fluorescence and autofluorescence intensities. Autofluorescence (AF) from the extracellular matrix (black columns) and Hoechst 33342 nuclear fluorescence (white columns) were collected in the green channel (500–550 nm) with varying two-photon (2P) excitation wavelengths. Fluorescence intensities (mean gray value) were measured within regions-of-interest illustrated in Figure 3. AF-to-Hoechst 33342 ratios increased sharply with increasing excitation wavelength (800 nm to 900 nm). The y-axis (mean gray value of fluorescence intensity) is set to a logarithmic (base 10) scale. Error bars=standard deviation.
Figure 5
Figure 5
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.
Figure 6
Figure 6
Effect of emission filter bandwidth on fluorescence intensities of Alexa-568, autofluorescence (AF), and Hoechst 33342 after two-photon (2P) excitation. Fluorescence from Alexa-568-phalloidin (red), Hoechst 33342 (green; 500–550 nm) and AF was collected from the uveal meshwork through near-red (565–605 nm; A, C, E, G, I, K) and far-red (590–680 nm; B, D, F, H, J, L) emission filters following 2P excitation at 800 nm (800 nm ex), 850 nm (850 nm ex), or 900 nm (900 nm ex). Boxes: region-of-interest (ROI) analysis of fluorescence intensity (red, green, or red/green ratio (yellow values)). Tissues were without (A, B, E, F, I, J) or with Alexa-568-phalloidin (C, D, G, H, K, L) label. Arrows: Hoechst 33342–labeled nuclei. Bar=25 μm.
Figure 7
Figure 7
Effect of red filter bandpass on two-photon (2P) red-to-green fluorescence intensity ratios representing the signal-to-noise ratio in the red channel. Pixel intensity (mean gray value) was calculated in five regions of interest (ROIs) positioned in each image frame (see Figure 6). In the absence of Alexa-568 (-Red Dye), red channel fluorescence was attributed to broad spectrum Hoechst 33342 and autofluorescence (AF) bleed-through. Alexa-568-to-AF bleed-through mean gray value ratios decreased with increasing excitation wavelength (800 to 900 nm) and were highest through a far-red filter (590–680 nm). For the ratio values, see Table 1.

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References

    1. Gonzalez JM, Jr, Hsu HY, Tan JC. Observing live actin in the human trabecular meshwork. Clin Experiment Ophthalmol. 2014;42:502–4. - PMC - PubMed
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