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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

Fluorescence and SNR enhancement of the PSF2P in the cortex with a DMM. (a) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom panel) 3D sketch of the cortex showing in blue a thalamocortical brain slice. (b) Single images of a bead acquired using the DMM in control conditions (CC) in the focal plane (top panel) and in a plane comprising the optical axis (z) (bottom panel, y axis indicated by dashed line in top panel) at a depth of 150 μm. (c) As for B but for the optimized mirror shape in the cortex (OMSc). Same laser power as (b). (d) Intensity projection (sum) of the z-stack of images of the bead shows that, in this example where there was no visible surrounding lobes, DMM optimization resulted in a decrease of the volume of the main lobe of the PSF2P and decrease in the background.
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g006: Fluorescence and SNR enhancement of the PSF2P in the cortex with a DMM. (a) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom panel) 3D sketch of the cortex showing in blue a thalamocortical brain slice. (b) Single images of a bead acquired using the DMM in control conditions (CC) in the focal plane (top panel) and in a plane comprising the optical axis (z) (bottom panel, y axis indicated by dashed line in top panel) at a depth of 150 μm. (c) As for B but for the optimized mirror shape in the cortex (OMSc). Same laser power as (b). (d) Intensity projection (sum) of the z-stack of images of the bead shows that, in this example where there was no visible surrounding lobes, DMM optimization resulted in a decrease of the volume of the main lobe of the PSF2P and decrease in the background.

Mentions: We then corrected for the wavefront distortions introduced by the cortex by optimizing the DMM shape using beads imaged through cortical slices (Fig. 6 (a)Fig. 6


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)

Fluorescence and SNR enhancement of the PSF2P in the cortex with a DMM. (a) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom panel) 3D sketch of the cortex showing in blue a thalamocortical brain slice. (b) Single images of a bead acquired using the DMM in control conditions (CC) in the focal plane (top panel) and in a plane comprising the optical axis (z) (bottom panel, y axis indicated by dashed line in top panel) at a depth of 150 μm. (c) As for B but for the optimized mirror shape in the cortex (OMSc). Same laser power as (b). (d) Intensity projection (sum) of the z-stack of images of the bead shows that, in this example where there was no visible surrounding lobes, DMM optimization resulted in a decrease of the volume of the main lobe of the PSF2P and decrease in the background.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g006: Fluorescence and SNR enhancement of the PSF2P in the cortex with a DMM. (a) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom panel) 3D sketch of the cortex showing in blue a thalamocortical brain slice. (b) Single images of a bead acquired using the DMM in control conditions (CC) in the focal plane (top panel) and in a plane comprising the optical axis (z) (bottom panel, y axis indicated by dashed line in top panel) at a depth of 150 μm. (c) As for B but for the optimized mirror shape in the cortex (OMSc). Same laser power as (b). (d) Intensity projection (sum) of the z-stack of images of the bead shows that, in this example where there was no visible surrounding lobes, DMM optimization resulted in a decrease of the volume of the main lobe of the PSF2P and decrease in the background.
Mentions: We then corrected for the wavefront distortions introduced by the cortex by optimizing the DMM shape using beads imaged through cortical slices (Fig. 6 (a)Fig. 6

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