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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

Conventional implementation of deformable membrane mirror (DMM). (a) Schematic diagram of conventional DMM implementation. (b) PSF2P of the microscope including the DMM in control conditions in the focal plane (Top) and in a plane comprising the optical axis (Bottom) indicated by the dashed line on the top panel.
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g005: Conventional implementation of deformable membrane mirror (DMM). (a) Schematic diagram of conventional DMM implementation. (b) PSF2P of the microscope including the DMM in control conditions in the focal plane (Top) and in a plane comprising the optical axis (Bottom) indicated by the dashed line on the top panel.

Mentions: The DMM was inserted in the excitation path in a conventional 4f configuration, ensuring conjugation of the DMM and the galvanometers mirrors, which were reimaged onto the objective backaperture [19,21] (Fig. 5 (a)Fig. 5


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)

Conventional implementation of deformable membrane mirror (DMM). (a) Schematic diagram of conventional DMM implementation. (b) PSF2P of the microscope including the DMM in control conditions in the focal plane (Top) and in a plane comprising the optical axis (Bottom) indicated by the dashed line on the top panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g005: Conventional implementation of deformable membrane mirror (DMM). (a) Schematic diagram of conventional DMM implementation. (b) PSF2P of the microscope including the DMM in control conditions in the focal plane (Top) and in a plane comprising the optical axis (Bottom) indicated by the dashed line on the top panel.
Mentions: The DMM was inserted in the excitation path in a conventional 4f configuration, ensuring conjugation of the DMM and the galvanometers mirrors, which were reimaged onto the objective backaperture [19,21] (Fig. 5 (a)Fig. 5

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