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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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Spatial dependence of wavefront distortions in the cortex. (a) (Left) A group of beads imaged through a 150 μm thalamocortical slice for the DMM set to control conditions (CC). The DMM shape was optimized on the central bead giving the optimized mirror shape in the cortex (OMSc). (Right) Using OMSc improved bead definition and fluorescence intensity in the lower half of the image but not in the upper half of the image, illustrating that wavefront distortions vary across a cortical slice. (b) Protocol used to examine the variability of wavefront distortions in the cortex: a first cortical column (C1) was positioned at the center of the field of view (indicated by the square box) and a first DMM shape optimization was performed there, giving OMSc (1). Then a neighboring cortical column (C2) was positioned at the center of the field of view, a second DMM shape optimization was performed, giving OMSc (2). Last a bead at the center of C2 was imaged using OMSc (1), OMSc (2) and CC. (c) Bead fluorescence using OMSc (1) was significantly smaller than using OMSc (2) (*) (p < 0.011, n = 8, paired t-test). Grey symbols: individual experiments, colored symbols: mean. The sem is smaller than the colored symbols.
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g008: Spatial dependence of wavefront distortions in the cortex. (a) (Left) A group of beads imaged through a 150 μm thalamocortical slice for the DMM set to control conditions (CC). The DMM shape was optimized on the central bead giving the optimized mirror shape in the cortex (OMSc). (Right) Using OMSc improved bead definition and fluorescence intensity in the lower half of the image but not in the upper half of the image, illustrating that wavefront distortions vary across a cortical slice. (b) Protocol used to examine the variability of wavefront distortions in the cortex: a first cortical column (C1) was positioned at the center of the field of view (indicated by the square box) and a first DMM shape optimization was performed there, giving OMSc (1). Then a neighboring cortical column (C2) was positioned at the center of the field of view, a second DMM shape optimization was performed, giving OMSc (2). Last a bead at the center of C2 was imaged using OMSc (1), OMSc (2) and CC. (c) Bead fluorescence using OMSc (1) was significantly smaller than using OMSc (2) (*) (p < 0.011, n = 8, paired t-test). Grey symbols: individual experiments, colored symbols: mean. The sem is smaller than the colored symbols.

Mentions: We then tested whether wavefront correction at a particular cortical location was effective in correcting for wavefront distortions introduced by the surrounding regions. Figure 8 (a)Fig. 8


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)

Spatial dependence of wavefront distortions in the cortex. (a) (Left) A group of beads imaged through a 150 μm thalamocortical slice for the DMM set to control conditions (CC). The DMM shape was optimized on the central bead giving the optimized mirror shape in the cortex (OMSc). (Right) Using OMSc improved bead definition and fluorescence intensity in the lower half of the image but not in the upper half of the image, illustrating that wavefront distortions vary across a cortical slice. (b) Protocol used to examine the variability of wavefront distortions in the cortex: a first cortical column (C1) was positioned at the center of the field of view (indicated by the square box) and a first DMM shape optimization was performed there, giving OMSc (1). Then a neighboring cortical column (C2) was positioned at the center of the field of view, a second DMM shape optimization was performed, giving OMSc (2). Last a bead at the center of C2 was imaged using OMSc (1), OMSc (2) and CC. (c) Bead fluorescence using OMSc (1) was significantly smaller than using OMSc (2) (*) (p < 0.011, n = 8, paired t-test). Grey symbols: individual experiments, colored symbols: mean. The sem is smaller than the colored symbols.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g008: Spatial dependence of wavefront distortions in the cortex. (a) (Left) A group of beads imaged through a 150 μm thalamocortical slice for the DMM set to control conditions (CC). The DMM shape was optimized on the central bead giving the optimized mirror shape in the cortex (OMSc). (Right) Using OMSc improved bead definition and fluorescence intensity in the lower half of the image but not in the upper half of the image, illustrating that wavefront distortions vary across a cortical slice. (b) Protocol used to examine the variability of wavefront distortions in the cortex: a first cortical column (C1) was positioned at the center of the field of view (indicated by the square box) and a first DMM shape optimization was performed there, giving OMSc (1). Then a neighboring cortical column (C2) was positioned at the center of the field of view, a second DMM shape optimization was performed, giving OMSc (2). Last a bead at the center of C2 was imaged using OMSc (1), OMSc (2) and CC. (c) Bead fluorescence using OMSc (1) was significantly smaller than using OMSc (2) (*) (p < 0.011, n = 8, paired t-test). Grey symbols: individual experiments, colored symbols: mean. The sem is smaller than the colored symbols.
Mentions: We then tested whether wavefront correction at a particular cortical location was effective in correcting for wavefront distortions introduced by the surrounding regions. Figure 8 (a)Fig. 8

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