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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.


Related in: MedlinePlus

One-(1P) and two-photon (2P) imaging of Hoechst 33342, autofluorescence, and Alexa-568 in the human juxtacanalicular meshwork. 1P (A–C) and 2P (D–I) 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 (D–F) or 850 nm (G–I). 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.
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f1: One-(1P) and two-photon (2P) imaging of Hoechst 33342, autofluorescence, and Alexa-568 in the human juxtacanalicular meshwork. 1P (A–C) and 2P (D–I) 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 (D–F) or 850 nm (G–I). 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.

Mentions: Differences between TM AF, Hoechst 33342, and Alexa-568 fluorescence due to 1P excitation as detected through red (555–700 nm) and green filters (500–535 nm), and 2P excitation through red (565–605 nm) and green (500–550 nm) filters were evident, as shown in Figure 1. One-photon excitation at 543 nm and 488 nm revealed Alexa 568 phalloidin fluorescence in the red channel (Figure 1A; 555–700 nm) and autofluorescent trabecular beams in the green channel (Figure 1B; 500–535 nm), respectively. Hoechst 33342–labeled nuclear fluorescence was not observed in the 1P green (500–535 nm) or red (555–700 nm) channels (Figure 1A-) as it requires a shorter wavelength emission filter to be seen. Hoechst 33342 nuclear labeling was detected in the 2P channels (arrows point to Hoechst 33342–labeled nuclei captured with 2P (Figure 1D,E,H), primarily in the green channel (500–550 nm), with some bleed-through into the near-red channel (565–605 nm), especially with 750 nm excitation.


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

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

One-(1P) and two-photon (2P) imaging of Hoechst 33342, autofluorescence, and Alexa-568 in the human juxtacanalicular meshwork. 1P (A–C) and 2P (D–I) 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 (D–F) or 850 nm (G–I). 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: One-(1P) and two-photon (2P) imaging of Hoechst 33342, autofluorescence, and Alexa-568 in the human juxtacanalicular meshwork. 1P (A–C) and 2P (D–I) 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 (D–F) or 850 nm (G–I). 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.
Mentions: Differences between TM AF, Hoechst 33342, and Alexa-568 fluorescence due to 1P excitation as detected through red (555–700 nm) and green filters (500–535 nm), and 2P excitation through red (565–605 nm) and green (500–550 nm) filters were evident, as shown in Figure 1. One-photon excitation at 543 nm and 488 nm revealed Alexa 568 phalloidin fluorescence in the red channel (Figure 1A; 555–700 nm) and autofluorescent trabecular beams in the green channel (Figure 1B; 500–535 nm), respectively. Hoechst 33342–labeled nuclear fluorescence was not observed in the 1P green (500–535 nm) or red (555–700 nm) channels (Figure 1A-) as it requires a shorter wavelength emission filter to be seen. Hoechst 33342 nuclear labeling was detected in the 2P channels (arrows point to Hoechst 33342–labeled nuclei captured with 2P (Figure 1D,E,H), primarily in the green channel (500–550 nm), with some bleed-through into the near-red channel (565–605 nm), especially with 750 nm excitation.

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.


Related in: MedlinePlus