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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

Multiphoton microscopy setup for simultaneous third harmonic generation (THG) and two-photon autofluorescence (TPAF) imaging of retina. A: A femtosecond fiber laser oscillator generates a 1044-nm laser beam to target the cross sections of full-thickness mouse retina. The laser beam is scanned and focused across the sample with a 60X microscope objective (OBJ). A dichroic mirror (DM1) separates the excitation laser beam (1044 nm) from the TPAF signal (522 to 600 nm) traveling in the backward (epi) direction and detected with a photomultiplier (PMT1). A high numerical aperture condenser lens (CL) collects the forward propagating THG signal. A second dichroic mirror and a narrow band filter sets separate the forward propagating TPAF and excitation beams from the THG signal (348 nm), which is detected with a photomultiplier (PMT2), DM1: dichroic mirror, SL: scanning lens, TL: tube lens, OBJ: objective, S: sample, CL: condenser lens, DM2: dichroic and narrow band filter, PMT: photomultiplier. B: A Jablonski diagram showing the interaction of multiple infrared photons between the electronic ground state and the electronic virtual state. In THG, three infrared excitation photons are instantaneously up-converted into a single photon of three times the energy. C: Under multiphoton microscopic conditions, no THG signal is generated inside a homogenous medium, i.e., the aqueous media of the cytoplasm. D: At the interface of two media with different nonlinear susceptibilities, i.e., the aqueous media and the lipidic organelle, significant THG is generated at the focus of the excitation beam. E: The THG signal from the lipid-rich nuclear membrane (+), cell membrane (*), and lipid droplets (x) of an opossum kidney (OK) cell create a biomarker for lipid-specific functional microscopy. F: THG signal from the pigment granules (x) of a cultured human fetal RPE (hfRPE) cell. The region with no THG signal (+) represents the nuclei.
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f1: Multiphoton microscopy setup for simultaneous third harmonic generation (THG) and two-photon autofluorescence (TPAF) imaging of retina. A: A femtosecond fiber laser oscillator generates a 1044-nm laser beam to target the cross sections of full-thickness mouse retina. The laser beam is scanned and focused across the sample with a 60X microscope objective (OBJ). A dichroic mirror (DM1) separates the excitation laser beam (1044 nm) from the TPAF signal (522 to 600 nm) traveling in the backward (epi) direction and detected with a photomultiplier (PMT1). A high numerical aperture condenser lens (CL) collects the forward propagating THG signal. A second dichroic mirror and a narrow band filter sets separate the forward propagating TPAF and excitation beams from the THG signal (348 nm), which is detected with a photomultiplier (PMT2), DM1: dichroic mirror, SL: scanning lens, TL: tube lens, OBJ: objective, S: sample, CL: condenser lens, DM2: dichroic and narrow band filter, PMT: photomultiplier. B: A Jablonski diagram showing the interaction of multiple infrared photons between the electronic ground state and the electronic virtual state. In THG, three infrared excitation photons are instantaneously up-converted into a single photon of three times the energy. C: Under multiphoton microscopic conditions, no THG signal is generated inside a homogenous medium, i.e., the aqueous media of the cytoplasm. D: At the interface of two media with different nonlinear susceptibilities, i.e., the aqueous media and the lipidic organelle, significant THG is generated at the focus of the excitation beam. E: The THG signal from the lipid-rich nuclear membrane (+), cell membrane (*), and lipid droplets (x) of an opossum kidney (OK) cell create a biomarker for lipid-specific functional microscopy. F: THG signal from the pigment granules (x) of a cultured human fetal RPE (hfRPE) cell. The region with no THG signal (+) represents the nuclei.

Mentions: The retina images were acquired with a custom-built multiphoton microscope platform optimized for simultaneous THG and TPAF imaging (Figure 1A). The laser system was a custom-built all-normal-dispersion femtosecond fiber laser [16] generating 600 mW of 1044 nm femtosecond pulses with a repetition rate of 63 MHz. The laser pulses were pre-compressed to about 200 fs at the sample. Raster scanning of the laser focus across the sample was achieved with two non-resonant galvanometric mirrors (6220HM60B; Cambridge Technology, Watertown, MA). The pulse train with an average power of 8 mW (incident at the sample) was focused with an Olympus UPlanSApo 60X/1.2 NA water objective (Olympus). Due to the small scanning mirrors (5-mm clear aperture), the focusing objective was well underfilled resulting in a decrease in the effective numerical aperture from 1.2 to 0.7. Imaging was performed on a custom-built upright microscope equipped with two single-photon counting photomultiplier tubes (PMTs) detectors; one in the epidetection (backward direction) collected TPAF signal through the focusing objective lens, and the other in the forward direction collected the THG signal with a custom-built compound collection lens with an NA of 0.9. A dichroic mirror (FF705-Di01, Semrock, Rochester, NY) and a bandpass filter (HQ550/100 M-2p, Chroma Technology Corp, Bellows Falls, VT; transmission from 500 to 600 nm signal) were used in the epidetection to separate the TPAF from the incident excitation beam. Note that the large transmission window of the TPAF bandpass filter would allow for detection of any second harmonic (SH) signal generated by the sample. However, due to the lack of collagen in the retina, we did not expect any SH signal [17]. The forward THG signal was filtered with a second dichroic mirror (FF409-Di03, Semrock, Rochester, NY) and a bandpass filter (FF01–390/SP-25, Semrock).


Third harmonic generation microscopy of a mouse retina.

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

Multiphoton microscopy setup for simultaneous third harmonic generation (THG) and two-photon autofluorescence (TPAF) imaging of retina. A: A femtosecond fiber laser oscillator generates a 1044-nm laser beam to target the cross sections of full-thickness mouse retina. The laser beam is scanned and focused across the sample with a 60X microscope objective (OBJ). A dichroic mirror (DM1) separates the excitation laser beam (1044 nm) from the TPAF signal (522 to 600 nm) traveling in the backward (epi) direction and detected with a photomultiplier (PMT1). A high numerical aperture condenser lens (CL) collects the forward propagating THG signal. A second dichroic mirror and a narrow band filter sets separate the forward propagating TPAF and excitation beams from the THG signal (348 nm), which is detected with a photomultiplier (PMT2), DM1: dichroic mirror, SL: scanning lens, TL: tube lens, OBJ: objective, S: sample, CL: condenser lens, DM2: dichroic and narrow band filter, PMT: photomultiplier. B: A Jablonski diagram showing the interaction of multiple infrared photons between the electronic ground state and the electronic virtual state. In THG, three infrared excitation photons are instantaneously up-converted into a single photon of three times the energy. C: Under multiphoton microscopic conditions, no THG signal is generated inside a homogenous medium, i.e., the aqueous media of the cytoplasm. D: At the interface of two media with different nonlinear susceptibilities, i.e., the aqueous media and the lipidic organelle, significant THG is generated at the focus of the excitation beam. E: The THG signal from the lipid-rich nuclear membrane (+), cell membrane (*), and lipid droplets (x) of an opossum kidney (OK) cell create a biomarker for lipid-specific functional microscopy. F: THG signal from the pigment granules (x) of a cultured human fetal RPE (hfRPE) cell. The region with no THG signal (+) represents the nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Multiphoton microscopy setup for simultaneous third harmonic generation (THG) and two-photon autofluorescence (TPAF) imaging of retina. A: A femtosecond fiber laser oscillator generates a 1044-nm laser beam to target the cross sections of full-thickness mouse retina. The laser beam is scanned and focused across the sample with a 60X microscope objective (OBJ). A dichroic mirror (DM1) separates the excitation laser beam (1044 nm) from the TPAF signal (522 to 600 nm) traveling in the backward (epi) direction and detected with a photomultiplier (PMT1). A high numerical aperture condenser lens (CL) collects the forward propagating THG signal. A second dichroic mirror and a narrow band filter sets separate the forward propagating TPAF and excitation beams from the THG signal (348 nm), which is detected with a photomultiplier (PMT2), DM1: dichroic mirror, SL: scanning lens, TL: tube lens, OBJ: objective, S: sample, CL: condenser lens, DM2: dichroic and narrow band filter, PMT: photomultiplier. B: A Jablonski diagram showing the interaction of multiple infrared photons between the electronic ground state and the electronic virtual state. In THG, three infrared excitation photons are instantaneously up-converted into a single photon of three times the energy. C: Under multiphoton microscopic conditions, no THG signal is generated inside a homogenous medium, i.e., the aqueous media of the cytoplasm. D: At the interface of two media with different nonlinear susceptibilities, i.e., the aqueous media and the lipidic organelle, significant THG is generated at the focus of the excitation beam. E: The THG signal from the lipid-rich nuclear membrane (+), cell membrane (*), and lipid droplets (x) of an opossum kidney (OK) cell create a biomarker for lipid-specific functional microscopy. F: THG signal from the pigment granules (x) of a cultured human fetal RPE (hfRPE) cell. The region with no THG signal (+) represents the nuclei.
Mentions: The retina images were acquired with a custom-built multiphoton microscope platform optimized for simultaneous THG and TPAF imaging (Figure 1A). The laser system was a custom-built all-normal-dispersion femtosecond fiber laser [16] generating 600 mW of 1044 nm femtosecond pulses with a repetition rate of 63 MHz. The laser pulses were pre-compressed to about 200 fs at the sample. Raster scanning of the laser focus across the sample was achieved with two non-resonant galvanometric mirrors (6220HM60B; Cambridge Technology, Watertown, MA). The pulse train with an average power of 8 mW (incident at the sample) was focused with an Olympus UPlanSApo 60X/1.2 NA water objective (Olympus). Due to the small scanning mirrors (5-mm clear aperture), the focusing objective was well underfilled resulting in a decrease in the effective numerical aperture from 1.2 to 0.7. Imaging was performed on a custom-built upright microscope equipped with two single-photon counting photomultiplier tubes (PMTs) detectors; one in the epidetection (backward direction) collected TPAF signal through the focusing objective lens, and the other in the forward direction collected the THG signal with a custom-built compound collection lens with an NA of 0.9. A dichroic mirror (FF705-Di01, Semrock, Rochester, NY) and a bandpass filter (HQ550/100 M-2p, Chroma Technology Corp, Bellows Falls, VT; transmission from 500 to 600 nm signal) were used in the epidetection to separate the TPAF from the incident excitation beam. Note that the large transmission window of the TPAF bandpass filter would allow for detection of any second harmonic (SH) signal generated by the sample. However, due to the lack of collagen in the retina, we did not expect any SH signal [17]. The forward THG signal was filtered with a second dichroic mirror (FF409-Di03, Semrock, Rochester, NY) and a bandpass filter (FF01–390/SP-25, Semrock).

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