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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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Contributions of wavefront distortion and scattering to the cortical PSF2P and their effects on 2P microscopy. (a) Image of modeled microscope PSF2P assuming NA 0.7 in x-y plane (top) and y-z plane (bottom panel), y axis indicated by dashed line in top panel. (b) Example of a modeled PSF2P taking into account the optical aberrations corrected using the DMM in the cortex assuming NA 0.7. 2P excitation was normalized by its maximal value in (a) and (b). (c) Quantification of the average 2P excitation (integral of the distribution of the squared intensity of excitation light in the PSF2P) in the central 3D Gaussian lobe and in the surrounding lobes, for the modeled ideal PSF2P, the measured microscope PSF2P, the modeled PSF2P including measured cortical distortions and the experimentally measured cortical PSF2P, all at NA = 0.7. (*) p < 0.003, t-test, (**) p < 0.001, t-test. (d) Modeling the effects of wavefront distortions and scattering on fluorescence. (Top panel) The predicted fluorescence emitted by homogeneously labeled spherical objects using an ideal microscope, in the presence of scattering (Lse = 77 μm) and in the presence of optical aberrations, plotted versus the size of the object. Furthermore, to determine effects of the surrounding lobes, the fluorescence emitted by the main Gaussian core of the PSF2P was calculated in the presence of optical aberrations (dotted red line). (Bottom panel) Plots from top panel were normalized to the maximum value.
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g012: Contributions of wavefront distortion and scattering to the cortical PSF2P and their effects on 2P microscopy. (a) Image of modeled microscope PSF2P assuming NA 0.7 in x-y plane (top) and y-z plane (bottom panel), y axis indicated by dashed line in top panel. (b) Example of a modeled PSF2P taking into account the optical aberrations corrected using the DMM in the cortex assuming NA 0.7. 2P excitation was normalized by its maximal value in (a) and (b). (c) Quantification of the average 2P excitation (integral of the distribution of the squared intensity of excitation light in the PSF2P) in the central 3D Gaussian lobe and in the surrounding lobes, for the modeled ideal PSF2P, the measured microscope PSF2P, the modeled PSF2P including measured cortical distortions and the experimentally measured cortical PSF2P, all at NA = 0.7. (*) p < 0.003, t-test, (**) p < 0.001, t-test. (d) Modeling the effects of wavefront distortions and scattering on fluorescence. (Top panel) The predicted fluorescence emitted by homogeneously labeled spherical objects using an ideal microscope, in the presence of scattering (Lse = 77 μm) and in the presence of optical aberrations, plotted versus the size of the object. Furthermore, to determine effects of the surrounding lobes, the fluorescence emitted by the main Gaussian core of the PSF2P was calculated in the presence of optical aberrations (dotted red line). (Bottom panel) Plots from top panel were normalized to the maximum value.

Mentions: Figures 12 (a) and (b)Fig. 12


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)

Contributions of wavefront distortion and scattering to the cortical PSF2P and their effects on 2P microscopy. (a) Image of modeled microscope PSF2P assuming NA 0.7 in x-y plane (top) and y-z plane (bottom panel), y axis indicated by dashed line in top panel. (b) Example of a modeled PSF2P taking into account the optical aberrations corrected using the DMM in the cortex assuming NA 0.7. 2P excitation was normalized by its maximal value in (a) and (b). (c) Quantification of the average 2P excitation (integral of the distribution of the squared intensity of excitation light in the PSF2P) in the central 3D Gaussian lobe and in the surrounding lobes, for the modeled ideal PSF2P, the measured microscope PSF2P, the modeled PSF2P including measured cortical distortions and the experimentally measured cortical PSF2P, all at NA = 0.7. (*) p < 0.003, t-test, (**) p < 0.001, t-test. (d) Modeling the effects of wavefront distortions and scattering on fluorescence. (Top panel) The predicted fluorescence emitted by homogeneously labeled spherical objects using an ideal microscope, in the presence of scattering (Lse = 77 μm) and in the presence of optical aberrations, plotted versus the size of the object. Furthermore, to determine effects of the surrounding lobes, the fluorescence emitted by the main Gaussian core of the PSF2P was calculated in the presence of optical aberrations (dotted red line). (Bottom panel) Plots from top panel were normalized to the maximum value.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g012: Contributions of wavefront distortion and scattering to the cortical PSF2P and their effects on 2P microscopy. (a) Image of modeled microscope PSF2P assuming NA 0.7 in x-y plane (top) and y-z plane (bottom panel), y axis indicated by dashed line in top panel. (b) Example of a modeled PSF2P taking into account the optical aberrations corrected using the DMM in the cortex assuming NA 0.7. 2P excitation was normalized by its maximal value in (a) and (b). (c) Quantification of the average 2P excitation (integral of the distribution of the squared intensity of excitation light in the PSF2P) in the central 3D Gaussian lobe and in the surrounding lobes, for the modeled ideal PSF2P, the measured microscope PSF2P, the modeled PSF2P including measured cortical distortions and the experimentally measured cortical PSF2P, all at NA = 0.7. (*) p < 0.003, t-test, (**) p < 0.001, t-test. (d) Modeling the effects of wavefront distortions and scattering on fluorescence. (Top panel) The predicted fluorescence emitted by homogeneously labeled spherical objects using an ideal microscope, in the presence of scattering (Lse = 77 μm) and in the presence of optical aberrations, plotted versus the size of the object. Furthermore, to determine effects of the surrounding lobes, the fluorescence emitted by the main Gaussian core of the PSF2P was calculated in the presence of optical aberrations (dotted red line). (Bottom panel) Plots from top panel were normalized to the maximum value.
Mentions: Figures 12 (a) and (b)Fig. 12

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