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Impact of wavefront distortion and scattering on 2-photon microscopy in mammalian brain tissue.

Chaigneau E, Wright AJ, Poland SP, Girkin JM, Silver RA - Opt Express (2011)

Bottom Line: We have investigated the effect of brain tissue on the 2P point spread function (PSF₂p) by imaging fluorescent beads through living cortical slices.Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation.These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology & Pharmacology, University College London, London, WC1E 6BT,UK.

ABSTRACT
Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF₂p) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF₂p at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF₂p, thereby providing a basis for improved 2P microscopy.

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Effect of scattering on the PSF2P. (a) Conventions used when modeling the PSF2P. Ballistic photons form a cone that can be decomposed into beamlets of coordinates (r = sinθ / sinθΝΑ, ω). (b) Comparison of the xe and ye profiles of the measured cortical PSF2P with the theoretical, microscope and modeled PSF2P that accounts for the effect of Lse in the focal plane (Top panel) and along the optical axis (Bottom panel).
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g004: Effect of scattering on the PSF2P. (a) Conventions used when modeling the PSF2P. Ballistic photons form a cone that can be decomposed into beamlets of coordinates (r = sinθ / sinθΝΑ, ω). (b) Comparison of the xe and ye profiles of the measured cortical PSF2P with the theoretical, microscope and modeled PSF2P that accounts for the effect of Lse in the focal plane (Top panel) and along the optical axis (Bottom panel).

Mentions: In a purely scattering sample, attenuation of ballistic excitation light due to scattering leads to reduction of the effective NA and enlargement of the PSF2P [12]. To examine the contribution of statistically homogeneous scattering (static) in cortical tissue we compared the dimensions of the measured PSF2P with that predicted using a vector-based model of PSF2P that incorporated scattering. We determined the PSF2P by calculating the fluorescence at each image point (xp, yp, zp) using Richards and Wolf’s normalized diffraction integral [30] expressed in cylindrical coordinates (rp, ωp, zp). We introduced space dependent amplitude Α (θ,ω) and phase factors Φ (θ,ω) of the field in the back aperture of the objective, givingex=−ifλ∫0θNA∫02πcos0.5θsinθ(cosθ+(1−cosθ)sin2ω)A(θ,ω)eik(Φ(θ,ω)+rpsinθcos(ω−ωp)+zpcosθ)dθdω(7)ey=ifλ∫0θNA∫02πcos0.5θsinθ(1−cosθ)cosωsinωA(θ,ω)eik(Φ(θ,ω)+rpsinθcos(ω−ωp)+zpcosθ)dθdω(8)ez=ifλ∫0θNA∫02πcos0.5θsin2θcosωA(θ,ω)eik(Φ(θ,ω)+rpsinθcos(ω−ωp)+zpcosθ)dθdω(9)where (r, θ, ω) are spherical polar coordinates for the reference sphere with polar axis θ = 0 in the z direction and θΝΑ is the maximal acceptance angle of the objective (Fig. 4 (a)Fig. 4


Impact of wavefront distortion and scattering on 2-photon microscopy in mammalian brain tissue.

Chaigneau E, Wright AJ, Poland SP, Girkin JM, Silver RA - Opt Express (2011)

Effect of scattering on the PSF2P. (a) Conventions used when modeling the PSF2P. Ballistic photons form a cone that can be decomposed into beamlets of coordinates (r = sinθ / sinθΝΑ, ω). (b) Comparison of the xe and ye profiles of the measured cortical PSF2P with the theoretical, microscope and modeled PSF2P that accounts for the effect of Lse in the focal plane (Top panel) and along the optical axis (Bottom panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369558&req=5

g004: Effect of scattering on the PSF2P. (a) Conventions used when modeling the PSF2P. Ballistic photons form a cone that can be decomposed into beamlets of coordinates (r = sinθ / sinθΝΑ, ω). (b) Comparison of the xe and ye profiles of the measured cortical PSF2P with the theoretical, microscope and modeled PSF2P that accounts for the effect of Lse in the focal plane (Top panel) and along the optical axis (Bottom panel).
Mentions: In a purely scattering sample, attenuation of ballistic excitation light due to scattering leads to reduction of the effective NA and enlargement of the PSF2P [12]. To examine the contribution of statistically homogeneous scattering (static) in cortical tissue we compared the dimensions of the measured PSF2P with that predicted using a vector-based model of PSF2P that incorporated scattering. We determined the PSF2P by calculating the fluorescence at each image point (xp, yp, zp) using Richards and Wolf’s normalized diffraction integral [30] expressed in cylindrical coordinates (rp, ωp, zp). We introduced space dependent amplitude Α (θ,ω) and phase factors Φ (θ,ω) of the field in the back aperture of the objective, givingex=−ifλ∫0θNA∫02πcos0.5θsinθ(cosθ+(1−cosθ)sin2ω)A(θ,ω)eik(Φ(θ,ω)+rpsinθcos(ω−ωp)+zpcosθ)dθdω(7)ey=ifλ∫0θNA∫02πcos0.5θsinθ(1−cosθ)cosωsinωA(θ,ω)eik(Φ(θ,ω)+rpsinθcos(ω−ωp)+zpcosθ)dθdω(8)ez=ifλ∫0θNA∫02πcos0.5θsin2θcosωA(θ,ω)eik(Φ(θ,ω)+rpsinθcos(ω−ωp)+zpcosθ)dθdω(9)where (r, θ, ω) are spherical polar coordinates for the reference sphere with polar axis θ = 0 in the z direction and θΝΑ is the maximal acceptance angle of the objective (Fig. 4 (a)Fig. 4

Bottom Line: We have investigated the effect of brain tissue on the 2P point spread function (PSF₂p) by imaging fluorescent beads through living cortical slices.Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation.These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology & Pharmacology, University College London, London, WC1E 6BT,UK.

ABSTRACT
Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF₂p) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF₂p at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF₂p, thereby providing a basis for improved 2P microscopy.

Show MeSH
Related in: MedlinePlus