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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 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.
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f5: 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.

Mentions: Bleed-through of Hoechst 33342 fluorescence into the near red channel (565–605 nm) was analyzed at excitation wavelengths of 750, 800, 850, and 900 nm in the fixed tissues, as shown in Figure 5. ECM AF bleed-through intensity sharply decreased with longer wavelengths. Hoechst 33342 colabeling did not significantly alter the ECM AF bleed-through. Hoechst 33342–labeled nuclear bleed-through was an order of magnitude higher than ECM AF bleed-through across all excitation wavelengths. The Hoechst 33342 bleed-through intensity decreased with 2P excitation wavelength; 2354±1577 (mean ± SD) at 750 nm; 594±520 at 800 nm; 26±37 at 850 nm; and 9±13 at 900 nm excitation.


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

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

f5: 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.
Mentions: Bleed-through of Hoechst 33342 fluorescence into the near red channel (565–605 nm) was analyzed at excitation wavelengths of 750, 800, 850, and 900 nm in the fixed tissues, as shown in Figure 5. ECM AF bleed-through intensity sharply decreased with longer wavelengths. Hoechst 33342 colabeling did not significantly alter the ECM AF bleed-through. Hoechst 33342–labeled nuclear bleed-through was an order of magnitude higher than ECM AF bleed-through across all excitation wavelengths. The Hoechst 33342 bleed-through intensity decreased with 2P excitation wavelength; 2354±1577 (mean ± SD) at 750 nm; 594±520 at 800 nm; 26±37 at 850 nm; and 9±13 at 900 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.