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Third harmonic generation microscopy of a mouse retina.

Masihzadeh O, Lei TC, Domingue SR, Kahook MY, Bartels RA, Ammar DA - Mol. Vis. (2015)

Bottom Line: In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei.Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution.In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO.

ABSTRACT

Purpose: To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities.

Methods: A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei.

Results: Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules.

Conclusions: THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.

No MeSH data available.


Related in: MedlinePlus

Comparison of the inner regions of the retina. A: Without a fluorescent nuclear dye (ethidium bromide homodimer; EthD-1) counterstain. B: With nuclear fluorescent dye. Panels A and B show the ganglion cell layer imaged with third harmonic generation (THG) and two-photon autofluorescence (TPAF). The EthD-1 nuclear staining (Panel B, x), coincides with the THG signal from the ganglion cell nuclei (Panel A, *). Panel C shows an adjacent histological section stained with hematoxylin and eosin. The scale bar represents 20 µm.
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f5: Comparison of the inner regions of the retina. A: Without a fluorescent nuclear dye (ethidium bromide homodimer; EthD-1) counterstain. B: With nuclear fluorescent dye. Panels A and B show the ganglion cell layer imaged with third harmonic generation (THG) and two-photon autofluorescence (TPAF). The EthD-1 nuclear staining (Panel B, x), coincides with the THG signal from the ganglion cell nuclei (Panel A, *). Panel C shows an adjacent histological section stained with hematoxylin and eosin. The scale bar represents 20 µm.

Mentions: Figure 3, Figure 4, and Figure 5 show the regions of the retina imaged with THG and TPAF with and without a fluorescent nuclear dye (EthD-1) counterstain alongside an adjacent histological section stained with hematoxylin and eosin. When the outer retina of the albino mouse eye is examined (Figure 3), the ONL shows strong THG from the nuclear membrane with no THG within the photoreceptor nuclei (Figure 3A), which are apparent in an adjacent section of the retina containing nuclear dye (Figure 3B). The fluorescently labeled photoreceptor nuclei emit a bright fluorescent (green) signal that does not overlap the THG signal (Figure 3B). In Figure 3C, the corresponding histological section shows the characteristic mature rod nuclei, with tightly packed heterochromatin stained intensely blue by hematoxylin. In the inner and outer segment region, the difference in light/dark eosin staining (Figure 3C) shows that the outer segments contain a large degree of tightly-packed protein and lipid disks compared to the inner segments. This difference was detected in the increase in the TPAF signal in the outer segments (Figure 3A,B). The outer segment discs appear to generate a diffuse THG signal. The source of this signal was not completely elucidated but could be due to the lipids inside the disk membrane that were not resolved.


Third harmonic generation microscopy of a mouse retina.

Masihzadeh O, Lei TC, Domingue SR, Kahook MY, Bartels RA, Ammar DA - Mol. Vis. (2015)

Comparison of the inner regions of the retina. A: Without a fluorescent nuclear dye (ethidium bromide homodimer; EthD-1) counterstain. B: With nuclear fluorescent dye. Panels A and B show the ganglion cell layer imaged with third harmonic generation (THG) and two-photon autofluorescence (TPAF). The EthD-1 nuclear staining (Panel B, x), coincides with the THG signal from the ganglion cell nuclei (Panel A, *). Panel C shows an adjacent histological section stained with hematoxylin and eosin. The scale bar represents 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440497&req=5

f5: Comparison of the inner regions of the retina. A: Without a fluorescent nuclear dye (ethidium bromide homodimer; EthD-1) counterstain. B: With nuclear fluorescent dye. Panels A and B show the ganglion cell layer imaged with third harmonic generation (THG) and two-photon autofluorescence (TPAF). The EthD-1 nuclear staining (Panel B, x), coincides with the THG signal from the ganglion cell nuclei (Panel A, *). Panel C shows an adjacent histological section stained with hematoxylin and eosin. The scale bar represents 20 µm.
Mentions: Figure 3, Figure 4, and Figure 5 show the regions of the retina imaged with THG and TPAF with and without a fluorescent nuclear dye (EthD-1) counterstain alongside an adjacent histological section stained with hematoxylin and eosin. When the outer retina of the albino mouse eye is examined (Figure 3), the ONL shows strong THG from the nuclear membrane with no THG within the photoreceptor nuclei (Figure 3A), which are apparent in an adjacent section of the retina containing nuclear dye (Figure 3B). The fluorescently labeled photoreceptor nuclei emit a bright fluorescent (green) signal that does not overlap the THG signal (Figure 3B). In Figure 3C, the corresponding histological section shows the characteristic mature rod nuclei, with tightly packed heterochromatin stained intensely blue by hematoxylin. In the inner and outer segment region, the difference in light/dark eosin staining (Figure 3C) shows that the outer segments contain a large degree of tightly-packed protein and lipid disks compared to the inner segments. This difference was detected in the increase in the TPAF signal in the outer segments (Figure 3A,B). The outer segment discs appear to generate a diffuse THG signal. The source of this signal was not completely elucidated but could be due to the lipids inside the disk membrane that were not resolved.

Bottom Line: In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei.Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution.In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO.

ABSTRACT

Purpose: To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities.

Methods: A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei.

Results: Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules.

Conclusions: THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.

No MeSH data available.


Related in: MedlinePlus