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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.


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.
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f2: 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.

Mentions: AF intensity varied between tissues from different individuals, as shown in Figure 2A-D (four donors). Pixel fluorescence intensity in autofluorescent beams decreased with 2P excitation wavelength; at 750 nm excitation, mean intensity was 2495±1331 (mean ± standard deviation (SD); maximum range: 1–65355); at 800 nm, mean intensity was 2448±1200; at 850 nm, 986±456; and at 900 nm, 337±232.


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

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

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

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

f2: 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.
Mentions: AF intensity varied between tissues from different individuals, as shown in Figure 2A-D (four donors). Pixel fluorescence intensity in autofluorescent beams decreased with 2P excitation wavelength; at 750 nm excitation, mean intensity was 2495±1331 (mean ± standard deviation (SD); maximum range: 1–65355); at 800 nm, mean intensity was 2448±1200; at 850 nm, 986±456; and at 900 nm, 337±232.

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.