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

Gonzalez JM, Ammar MJ, Ko MK, Tan JC - Mol. Vis. (2016)

Bottom Line: Region-of-interest (ROI) image analysis provided fluorescence intensity values for each fluorophore.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.

View Article: PubMed Central - PubMed

Affiliation: Doheny Eye Institute; University of California, Los Angeles, Los Angeles, CA.

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.

No MeSH data available.


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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835224&req=5

f6: 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.

Mentions: Fluorescence intensity of Alexa-568-phalloidin (for F-actin) diminished with increasing 2P excitation wavelength from 800 nm to 850 nm to 900 nm. Alexa-568 pixel intensity was 18,348 for 800 nm (Figure 6C), 4,064 for 850 nm (Figure 6G), and 871 for 900 nm excitation (Figure 6K). Pixel intensity of Alexa-568 emission through near-red (565–605 nm) and far-red filters (590–680 nm) decreased with increasing 2P excitation wavelength, as did AF intensity, as shown in Figure 7. At 850 nm excitation, Alexa-568 emission intensity through the far-red filter was more than 200% that of Alexa-568 emission detected through the near-red filter or AF detected through the near-red or far-red filter.


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

Gonzalez JM, Ammar MJ, Ko MK, Tan JC - Mol. Vis. (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835224&req=5

f6: 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.
Mentions: Fluorescence intensity of Alexa-568-phalloidin (for F-actin) diminished with increasing 2P excitation wavelength from 800 nm to 850 nm to 900 nm. Alexa-568 pixel intensity was 18,348 for 800 nm (Figure 6C), 4,064 for 850 nm (Figure 6G), and 871 for 900 nm excitation (Figure 6K). Pixel intensity of Alexa-568 emission through near-red (565–605 nm) and far-red filters (590–680 nm) decreased with increasing 2P excitation wavelength, as did AF intensity, as shown in Figure 7. At 850 nm excitation, Alexa-568 emission intensity through the far-red filter was more than 200% that of Alexa-568 emission detected through the near-red filter or AF detected through the near-red or far-red filter.

Bottom Line: Region-of-interest (ROI) image analysis provided fluorescence intensity values for each fluorophore.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.

View Article: PubMed Central - PubMed

Affiliation: Doheny Eye Institute; University of California, Los Angeles, Los Angeles, CA.

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.

No MeSH data available.