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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 correction for the light efficient DMM configuration. (a) Experimental protocol: an optimization was performed at the center of the field of view (position 1), at a particular cortical location. The cortical location was moved across the field of view at distances of 50 μm (position 2) or 100 μm (position 3) away from the optical axis, and the fluorescence and SNR obtained using the optimized mirror shape (OMSc) and in control conditions (CC) were measured. (b - c) The change in fluorescence (b) and SNR (c) across the field of view using the OMS performed at position 1. There was no significant change in these parameters with distance to the optical axis (p > 0.17, paired t-test). Grey symbols: individual experiments, colored symbols: mean, black bars: sem.
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g010: Spatial dependence of wavefront correction for the light efficient DMM configuration. (a) Experimental protocol: an optimization was performed at the center of the field of view (position 1), at a particular cortical location. The cortical location was moved across the field of view at distances of 50 μm (position 2) or 100 μm (position 3) away from the optical axis, and the fluorescence and SNR obtained using the optimized mirror shape (OMSc) and in control conditions (CC) were measured. (b - c) The change in fluorescence (b) and SNR (c) across the field of view using the OMS performed at position 1. There was no significant change in these parameters with distance to the optical axis (p > 0.17, paired t-test). Grey symbols: individual experiments, colored symbols: mean, black bars: sem.

Mentions: To quantify the spatial dependence of cortical wavefront corrections with this configuration, we investigated the field of view over which a DMM OMSc provided fluorescence and SNR enhancement (as described in 4.1.1). There was no significant change in the fluorescence and SNR enhancements with distance from the optical axis (Fig. 10 (a-c)Fig. 10


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 correction for the light efficient DMM configuration. (a) Experimental protocol: an optimization was performed at the center of the field of view (position 1), at a particular cortical location. The cortical location was moved across the field of view at distances of 50 μm (position 2) or 100 μm (position 3) away from the optical axis, and the fluorescence and SNR obtained using the optimized mirror shape (OMSc) and in control conditions (CC) were measured. (b - c) The change in fluorescence (b) and SNR (c) across the field of view using the OMS performed at position 1. There was no significant change in these parameters with distance to the optical axis (p > 0.17, paired t-test). Grey symbols: individual experiments, colored symbols: mean, black bars: sem.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g010: Spatial dependence of wavefront correction for the light efficient DMM configuration. (a) Experimental protocol: an optimization was performed at the center of the field of view (position 1), at a particular cortical location. The cortical location was moved across the field of view at distances of 50 μm (position 2) or 100 μm (position 3) away from the optical axis, and the fluorescence and SNR obtained using the optimized mirror shape (OMSc) and in control conditions (CC) were measured. (b - c) The change in fluorescence (b) and SNR (c) across the field of view using the OMS performed at position 1. There was no significant change in these parameters with distance to the optical axis (p > 0.17, paired t-test). Grey symbols: individual experiments, colored symbols: mean, black bars: sem.
Mentions: To quantify the spatial dependence of cortical wavefront corrections with this configuration, we investigated the field of view over which a DMM OMSc provided fluorescence and SNR enhancement (as described in 4.1.1). There was no significant change in the fluorescence and SNR enhancements with distance from the optical axis (Fig. 10 (a-c)Fig. 10

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