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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

Optimization of the DMM shape on a cellular element. (a) Fine dendrite imaged with DMM in control conditions (CC). (b) Fluorescence change during the DMM shape optimization on a dendrite (arrow in panel A). (c) Same cellular element imaged using the optimized mirror shape (OMSc) and the same laser power as in CC.
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g011: Optimization of the DMM shape on a cellular element. (a) Fine dendrite imaged with DMM in control conditions (CC). (b) Fluorescence change during the DMM shape optimization on a dendrite (arrow in panel A). (c) Same cellular element imaged using the optimized mirror shape (OMSc) and the same laser power as in CC.

Mentions: Since it is difficult to distribute beads in living brain we examined the feasibility of optimizing the DMM shape on fine neuronal processes. DMM shape optimizations could be successfully achieved on small dendrites or spines from fluorescent-labeled neurons located at an average depth of 85 ± 24 μm (n = 9) below the surface of the brain slice, provided that the laser intensity used during the optimization was kept low (Fig. 11Fig. 11


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)

Optimization of the DMM shape on a cellular element. (a) Fine dendrite imaged with DMM in control conditions (CC). (b) Fluorescence change during the DMM shape optimization on a dendrite (arrow in panel A). (c) Same cellular element imaged using the optimized mirror shape (OMSc) and the same laser power as in CC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g011: Optimization of the DMM shape on a cellular element. (a) Fine dendrite imaged with DMM in control conditions (CC). (b) Fluorescence change during the DMM shape optimization on a dendrite (arrow in panel A). (c) Same cellular element imaged using the optimized mirror shape (OMSc) and the same laser power as in CC.
Mentions: Since it is difficult to distribute beads in living brain we examined the feasibility of optimizing the DMM shape on fine neuronal processes. DMM shape optimizations could be successfully achieved on small dendrites or spines from fluorescent-labeled neurons located at an average depth of 85 ± 24 μm (n = 9) below the surface of the brain slice, provided that the laser intensity used during the optimization was kept low (Fig. 11Fig. 11

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