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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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Excitation mean free path in the cortex. (a) Maximum intensity projection along the z axis of a z-stack in layer II / III showing Alexa 488 filled pyramidal cell with dendrites spanning 200 μm in x-y and 84 μm in z. The scattering length or mean free path of excitation light (Lse) was estimated from the fluorescence of the dendrites at different depths. (b) Relationship between the fluorescence (F) divided by the square of the laser Power (P) and normalized by its value at the cortical slice surface (β) versus depth. Lse was calculated from an exponential fit (line) at a wavelength of 725 nm. (c) Dependence of Lse on wavelength (n = 4 −8 cells). Error bars give the standard error of the mean (sem), which is smaller than the symbols for λ ≤ 850 nm.
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g003: Excitation mean free path in the cortex. (a) Maximum intensity projection along the z axis of a z-stack in layer II / III showing Alexa 488 filled pyramidal cell with dendrites spanning 200 μm in x-y and 84 μm in z. The scattering length or mean free path of excitation light (Lse) was estimated from the fluorescence of the dendrites at different depths. (b) Relationship between the fluorescence (F) divided by the square of the laser Power (P) and normalized by its value at the cortical slice surface (β) versus depth. Lse was calculated from an exponential fit (line) at a wavelength of 725 nm. (c) Dependence of Lse on wavelength (n = 4 −8 cells). Error bars give the standard error of the mean (sem), which is smaller than the symbols for λ ≤ 850 nm.

Mentions: The fine processes (dendrites) of an individual neuron can span focal depths of hundreds of micrometers. We used this property to determine Lse, by imaging these small structures at various depths. To do this, single cortical neurons in layer II / III (thalamocortical slices) were whole-cell patch-clamped and filled with an internal solution containing (in millimoles of compound used to make a liter of solution): 130 KMeSO3, 10 Na2Phosphocreatine, 10 HEPES, 4 MgCl2, 0.1 EGTA, 0.3 NaGTP, 4 Na2ATP, and a green emitting fluorescent dye, 0.2 mΜ fluo-4 (Invitrogen) or Alexa 488 (Invitrogen). After the dye had reached diffusion equilibrium within the cell, a z-stack of images of the dendritic tree was acquired (using epi-fluorescence collection) at a range of excitation wavelengths (725 ≤ λ ≤ 950 nm in n = 8 cells), using 4 μs dwell time and averages of 2 images (Fig. 3 (a)Fig. 3


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)

Excitation mean free path in the cortex. (a) Maximum intensity projection along the z axis of a z-stack in layer II / III showing Alexa 488 filled pyramidal cell with dendrites spanning 200 μm in x-y and 84 μm in z. The scattering length or mean free path of excitation light (Lse) was estimated from the fluorescence of the dendrites at different depths. (b) Relationship between the fluorescence (F) divided by the square of the laser Power (P) and normalized by its value at the cortical slice surface (β) versus depth. Lse was calculated from an exponential fit (line) at a wavelength of 725 nm. (c) Dependence of Lse on wavelength (n = 4 −8 cells). Error bars give the standard error of the mean (sem), which is smaller than the symbols for λ ≤ 850 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g003: Excitation mean free path in the cortex. (a) Maximum intensity projection along the z axis of a z-stack in layer II / III showing Alexa 488 filled pyramidal cell with dendrites spanning 200 μm in x-y and 84 μm in z. The scattering length or mean free path of excitation light (Lse) was estimated from the fluorescence of the dendrites at different depths. (b) Relationship between the fluorescence (F) divided by the square of the laser Power (P) and normalized by its value at the cortical slice surface (β) versus depth. Lse was calculated from an exponential fit (line) at a wavelength of 725 nm. (c) Dependence of Lse on wavelength (n = 4 −8 cells). Error bars give the standard error of the mean (sem), which is smaller than the symbols for λ ≤ 850 nm.
Mentions: The fine processes (dendrites) of an individual neuron can span focal depths of hundreds of micrometers. We used this property to determine Lse, by imaging these small structures at various depths. To do this, single cortical neurons in layer II / III (thalamocortical slices) were whole-cell patch-clamped and filled with an internal solution containing (in millimoles of compound used to make a liter of solution): 130 KMeSO3, 10 Na2Phosphocreatine, 10 HEPES, 4 MgCl2, 0.1 EGTA, 0.3 NaGTP, 4 Na2ATP, and a green emitting fluorescent dye, 0.2 mΜ fluo-4 (Invitrogen) or Alexa 488 (Invitrogen). After the dye had reached diffusion equilibrium within the cell, a z-stack of images of the dendritic tree was acquired (using epi-fluorescence collection) at a range of excitation wavelengths (725 ≤ λ ≤ 950 nm in n = 8 cells), using 4 μs dwell time and averages of 2 images (Fig. 3 (a)Fig. 3

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