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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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Related in: MedlinePlus

Comparing the effects of static, statistically homogeneous scattering and wavefront distortion on excitation photons in 2P microscopy. Brain tissue is made of particles of a wide range of size and refractive index. Particles that are smaller than the wavelength of light create a statistically homogeneous effect. This decreases the power of ballistic photons while leaving the wavefront undistorted. Particles whose size is larger than the wavelength of light induce wavefront distortion.
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g001: Comparing the effects of static, statistically homogeneous scattering and wavefront distortion on excitation photons in 2P microscopy. Brain tissue is made of particles of a wide range of size and refractive index. Particles that are smaller than the wavelength of light create a statistically homogeneous effect. This decreases the power of ballistic photons while leaving the wavefront undistorted. Particles whose size is larger than the wavelength of light induce wavefront distortion.

Mentions: The PSF2P can be adversely affected by several physical processes when imaging biological tissue, including absorption, statistically homogeneous scattering [8] and optical aberrations [9]. In brain tissue, absorption is usually negligible [10,11]. Statistically homogeneous scattering and optical aberrations due to wavefront distortions both arise from local variations of refractive index, which disperse and delay a fraction of the ballistic photons. However, the relative contributions of these effects on light propagation depend on the size of the structures within the sample (Fig. 1Fig. 1


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)

Comparing the effects of static, statistically homogeneous scattering and wavefront distortion on excitation photons in 2P microscopy. Brain tissue is made of particles of a wide range of size and refractive index. Particles that are smaller than the wavelength of light create a statistically homogeneous effect. This decreases the power of ballistic photons while leaving the wavefront undistorted. Particles whose size is larger than the wavelength of light induce wavefront distortion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g001: Comparing the effects of static, statistically homogeneous scattering and wavefront distortion on excitation photons in 2P microscopy. Brain tissue is made of particles of a wide range of size and refractive index. Particles that are smaller than the wavelength of light create a statistically homogeneous effect. This decreases the power of ballistic photons while leaving the wavefront undistorted. Particles whose size is larger than the wavelength of light induce wavefront distortion.
Mentions: The PSF2P can be adversely affected by several physical processes when imaging biological tissue, including absorption, statistically homogeneous scattering [8] and optical aberrations [9]. In brain tissue, absorption is usually negligible [10,11]. Statistically homogeneous scattering and optical aberrations due to wavefront distortions both arise from local variations of refractive index, which disperse and delay a fraction of the ballistic photons. However, the relative contributions of these effects on light propagation depend on the size of the structures within the sample (Fig. 1Fig. 1

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