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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.


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.
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f4: 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.

Mentions: Hoechst fluorescence intensity in the green channel (500–550 nm) was highest with 2P excitation at 750 nm, but emission intensity diminished with longer excitation wavelengths (compare Figure 1E,H; shown for 850 nm excitation). Relative intensity of Hoechst 33342 fluorescence diminished with increasing excitation wavelength (compare Figure 3A,C,B,D, blue ROIs; Figure 4, white bars), becoming virtually imperceptible with 900 nm excitation (see Figure 3E,F, blue ROIs).


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

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

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

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

f4: 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.
Mentions: Hoechst fluorescence intensity in the green channel (500–550 nm) was highest with 2P excitation at 750 nm, but emission intensity diminished with longer excitation wavelengths (compare Figure 1E,H; shown for 850 nm excitation). Relative intensity of Hoechst 33342 fluorescence diminished with increasing excitation wavelength (compare Figure 3A,C,B,D, blue ROIs; Figure 4, white bars), becoming virtually imperceptible with 900 nm excitation (see Figure 3E,F, blue ROIs).

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.