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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

Compensating for optical aberrations with light-efficient DMM implementation. (a) Light efficient configuration with the DMM implemented at 45°. (b) PSF2P of the microscope with the light-efficient DMM in control conditions in the focal plane (Left) and in a plane comprising the optical axis (Right). Y axis indicated by the dashed line on the top panel. (c) Single images of a bead under a cortical slice at a depth of 150 μm acquired using the DMM in control conditions (CC) in the focal plane. (d) As for (c) but for the optimized mirror shape in the cortex (OMSc). Same laser power as (c). (e) Z stacks of images of the previous bead were acquired and normalised to the maximal fluorescence for the DMM in CC. Focal plane (top panel) and plane comprising the optical axis (z) (bottom panel). Arrow indicates a surrounding lobe that disappeared after the DMM optimization. x axis indicated by dashed line in top panel. (f) Same as (e) but using the OMSc. (g) Maximum intensity projection (MIP) of data from (e). (h) MIP of data from (f).
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g009: Compensating for optical aberrations with light-efficient DMM implementation. (a) Light efficient configuration with the DMM implemented at 45°. (b) PSF2P of the microscope with the light-efficient DMM in control conditions in the focal plane (Left) and in a plane comprising the optical axis (Right). Y axis indicated by the dashed line on the top panel. (c) Single images of a bead under a cortical slice at a depth of 150 μm acquired using the DMM in control conditions (CC) in the focal plane. (d) As for (c) but for the optimized mirror shape in the cortex (OMSc). Same laser power as (c). (e) Z stacks of images of the previous bead were acquired and normalised to the maximal fluorescence for the DMM in CC. Focal plane (top panel) and plane comprising the optical axis (z) (bottom panel). Arrow indicates a surrounding lobe that disappeared after the DMM optimization. x axis indicated by dashed line in top panel. (f) Same as (e) but using the OMSc. (g) Maximum intensity projection (MIP) of data from (e). (h) MIP of data from (f).

Mentions: The conventional DMM configuration used so far resulted in the loss of about 50% of excitation light, due to the incoming and outgoing beams passing through a polarization beam splitter and quarter-wave plate. Since both the power of the excitation light and the effective NA are limiting for deep tissue imaging and for performing photolysis, we explored the possibility of using an alternative DMM implementation (Fig. 9 (a)Fig. 9


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)

Compensating for optical aberrations with light-efficient DMM implementation. (a) Light efficient configuration with the DMM implemented at 45°. (b) PSF2P of the microscope with the light-efficient DMM in control conditions in the focal plane (Left) and in a plane comprising the optical axis (Right). Y axis indicated by the dashed line on the top panel. (c) Single images of a bead under a cortical slice at a depth of 150 μm acquired using the DMM in control conditions (CC) in the focal plane. (d) As for (c) but for the optimized mirror shape in the cortex (OMSc). Same laser power as (c). (e) Z stacks of images of the previous bead were acquired and normalised to the maximal fluorescence for the DMM in CC. Focal plane (top panel) and plane comprising the optical axis (z) (bottom panel). Arrow indicates a surrounding lobe that disappeared after the DMM optimization. x axis indicated by dashed line in top panel. (f) Same as (e) but using the OMSc. (g) Maximum intensity projection (MIP) of data from (e). (h) MIP of data from (f).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g009: Compensating for optical aberrations with light-efficient DMM implementation. (a) Light efficient configuration with the DMM implemented at 45°. (b) PSF2P of the microscope with the light-efficient DMM in control conditions in the focal plane (Left) and in a plane comprising the optical axis (Right). Y axis indicated by the dashed line on the top panel. (c) Single images of a bead under a cortical slice at a depth of 150 μm acquired using the DMM in control conditions (CC) in the focal plane. (d) As for (c) but for the optimized mirror shape in the cortex (OMSc). Same laser power as (c). (e) Z stacks of images of the previous bead were acquired and normalised to the maximal fluorescence for the DMM in CC. Focal plane (top panel) and plane comprising the optical axis (z) (bottom panel). Arrow indicates a surrounding lobe that disappeared after the DMM optimization. x axis indicated by dashed line in top panel. (f) Same as (e) but using the OMSc. (g) Maximum intensity projection (MIP) of data from (e). (h) MIP of data from (f).
Mentions: The conventional DMM configuration used so far resulted in the loss of about 50% of excitation light, due to the incoming and outgoing beams passing through a polarization beam splitter and quarter-wave plate. Since both the power of the excitation light and the effective NA are limiting for deep tissue imaging and for performing photolysis, we explored the possibility of using an alternative DMM implementation (Fig. 9 (a)Fig. 9

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