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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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Two-photon point spread function (PSF2P) characteristics in the cortex. (a) (Top left panel) experimental setup consisting of water immersion objective and beads used to measure the optical system PSF2P. Single images of beads acquired using the optical system in the focal plane (x-y) (Bottom left) and in a plane comprising the optical axis (z) (Bottom right). y axis indicated by a dashed line in the bottom left panel. Excitation wavelength (λ) = 725 nm. (b) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom left panel) 3D sketch of the cortex showing in blue a tangential slice in cortical layer II / III. (Top middle and right panels) x-y and y-z images of beads acquired by focusing through tangential slices at a depth of 150 μm. y axis indicated by a dashed line in the top middle panel. Same look up table as (a). (c) Gaussian fits of average PSF2P. (Top panel) x-y plane (Bottom panel) z axis. Error bars show the standard deviation.
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g002: Two-photon point spread function (PSF2P) characteristics in the cortex. (a) (Top left panel) experimental setup consisting of water immersion objective and beads used to measure the optical system PSF2P. Single images of beads acquired using the optical system in the focal plane (x-y) (Bottom left) and in a plane comprising the optical axis (z) (Bottom right). y axis indicated by a dashed line in the bottom left panel. Excitation wavelength (λ) = 725 nm. (b) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom left panel) 3D sketch of the cortex showing in blue a tangential slice in cortical layer II / III. (Top middle and right panels) x-y and y-z images of beads acquired by focusing through tangential slices at a depth of 150 μm. y axis indicated by a dashed line in the top middle panel. Same look up table as (a). (c) Gaussian fits of average PSF2P. (Top panel) x-y plane (Bottom panel) z axis. Error bars show the standard deviation.

Mentions: To investigate the effects of brain tissue on the PSF2P we first quantified the properties of our 2P microscope, which consisted of a femtosecond tuneable Laser (Tsunami, Newport-Spectra Physics), scanhead (Ultima, Prairie Technologies), upright microscope (BX51, Olympus), and IR antireflection coated water-immersion objective (Olympus LumPLanFL/IR 60x/0.90W). Green fluorescence light was collected selectively using an emission filter (HQ 525/70m-2P Chroma Technology) and detected using GaAsP photomultipliers (Hamamatsu H7422). Images were acquired with PrairieView acquisition software. The laser intensity was controlled using a Pockels-cell (Conoptics Model 302CE) and neutral density filter (NDC-50S-3M, Thorlabs) when necessary. A single layer of 200 nm diameter green fluorescent beads (FluoSpheres, Invitrogen) was fixed to the bottom of the recording chamber (Fig. 2 (a)Fig. 2


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)

Two-photon point spread function (PSF2P) characteristics in the cortex. (a) (Top left panel) experimental setup consisting of water immersion objective and beads used to measure the optical system PSF2P. Single images of beads acquired using the optical system in the focal plane (x-y) (Bottom left) and in a plane comprising the optical axis (z) (Bottom right). y axis indicated by a dashed line in the bottom left panel. Excitation wavelength (λ) = 725 nm. (b) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom left panel) 3D sketch of the cortex showing in blue a tangential slice in cortical layer II / III. (Top middle and right panels) x-y and y-z images of beads acquired by focusing through tangential slices at a depth of 150 μm. y axis indicated by a dashed line in the top middle panel. Same look up table as (a). (c) Gaussian fits of average PSF2P. (Top panel) x-y plane (Bottom panel) z axis. Error bars show the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g002: Two-photon point spread function (PSF2P) characteristics in the cortex. (a) (Top left panel) experimental setup consisting of water immersion objective and beads used to measure the optical system PSF2P. Single images of beads acquired using the optical system in the focal plane (x-y) (Bottom left) and in a plane comprising the optical axis (z) (Bottom right). y axis indicated by a dashed line in the bottom left panel. Excitation wavelength (λ) = 725 nm. (b) (Top left panel) Experimental setup used to measure the PSF2P in acute slices of cortex. (Bottom left panel) 3D sketch of the cortex showing in blue a tangential slice in cortical layer II / III. (Top middle and right panels) x-y and y-z images of beads acquired by focusing through tangential slices at a depth of 150 μm. y axis indicated by a dashed line in the top middle panel. Same look up table as (a). (c) Gaussian fits of average PSF2P. (Top panel) x-y plane (Bottom panel) z axis. Error bars show the standard deviation.
Mentions: To investigate the effects of brain tissue on the PSF2P we first quantified the properties of our 2P microscope, which consisted of a femtosecond tuneable Laser (Tsunami, Newport-Spectra Physics), scanhead (Ultima, Prairie Technologies), upright microscope (BX51, Olympus), and IR antireflection coated water-immersion objective (Olympus LumPLanFL/IR 60x/0.90W). Green fluorescence light was collected selectively using an emission filter (HQ 525/70m-2P Chroma Technology) and detected using GaAsP photomultipliers (Hamamatsu H7422). Images were acquired with PrairieView acquisition software. The laser intensity was controlled using a Pockels-cell (Conoptics Model 302CE) and neutral density filter (NDC-50S-3M, Thorlabs) when necessary. A single layer of 200 nm diameter green fluorescent beads (FluoSpheres, Invitrogen) was fixed to the bottom of the recording chamber (Fig. 2 (a)Fig. 2

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