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Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue.

Wang K, Sun W, Richie CT, Harvey BK, Betzig E, Ji N - Nat Commun (2015)

Bottom Line: Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens.Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars.The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA.

ABSTRACT
Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars. The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

No MeSH data available.


Related in: MedlinePlus

AO correction via direct wavefront sensing improves functional calcium imaging deep inside the cortex of a living mouse.(a) Calcium responses evoked by drifting-grating stimulation 400 and 500 μm below pia in the primary visual cortex of a mouse (Thy1-GCaMP6s line GP4.3) before (left panel) and after (right panel) correction. Brightness of each pixel reflects its s.d. across 800 frames imaged during five repetitions of the drifting-grating stimulus set, and is correlated with the local calcium transient magnitude. Scale bar, 20 μm. (b) Calcium transients at regions of interest (ROI) i–vi, before and after AO correction. Solid colours label averaged transients; faded colours label transients during specific repetitions. Top panel indicates the orientations and drifting directions of the grating stimuli. Representative images from >20 imaging sections in three mice.
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f3: AO correction via direct wavefront sensing improves functional calcium imaging deep inside the cortex of a living mouse.(a) Calcium responses evoked by drifting-grating stimulation 400 and 500 μm below pia in the primary visual cortex of a mouse (Thy1-GCaMP6s line GP4.3) before (left panel) and after (right panel) correction. Brightness of each pixel reflects its s.d. across 800 frames imaged during five repetitions of the drifting-grating stimulus set, and is correlated with the local calcium transient magnitude. Scale bar, 20 μm. (b) Calcium transients at regions of interest (ROI) i–vi, before and after AO correction. Solid colours label averaged transients; faded colours label transients during specific repetitions. Top panel indicates the orientations and drifting directions of the grating stimuli. Representative images from >20 imaging sections in three mice.

Mentions: Widely used in medical diagnostics13, injection of ICG allows this direct wavefront-sensing approach to be applied to a wide variety of tissues in vivo. In the mouse brain, it can improve not only morphological imaging but also the sensitivity when measuring neural activity at 500-μm depth in cells expressing the calcium indicator GCaMP6s1415 (Thy1-GCaMP6s GP4.3 mouse; Supplementary Movie 3). One caveat, however, is that we observed heightened spontaneous activity of GCaMP6s in cortical regions injected with ICG. To eliminate this, we replaced ICG with the NIR protein iRFP NIR fluorescence protein iRFP71316 (peak emission at 713 nm) expressed via a viral vector in a subset of neurons in the primary visual cortex of the Thy1-GCaMP6s GP4.3 mice. With this marker, we were able to use a TPE-generated NIR GS to obtain corrective wavefronts down to 500 μm below pia, which then led to markedly improved sensitivity to evoked calcium responses in the visual cortex. Indeed, after AO correction, calcium transients could be detected in many more neurites at depths of 400 (Fig. 3; Supplementary Movie 4), 500 (Fig. 3; Supplementary Movie 5) and 600 μm (Supplementary Movie 6). Several factors contribute to these drastic improvements: calcium indicators usually have a heterogeneous labelling density in the brain. The more strongly labelled somata or neurites constitute the structures of interest, which are surrounded by the axons and dendrites of other more weakly labelled neurons. Calcium transients from these weakly labelled neuropil, or the ‘neuropil contaminations'17, represent the average response of many neurons and are not selective to the orientation of our visual stimuli. For a fine neurite, calcium transients within the laser focus are composed of signals from both the neurite and its surrounding neuropil. In a focus enlarged and dimmed by aberration, the calcium transient of the neurite may be overwhelmed by that of the neuropil. AO correction, however, both reduces the focal volume and increases the focal intensity, which enhances the neurite signal a lot more than the neuropil background. As a result, the orientation selectivity of many neurites is only detectable after AO correction raises the neurite calcium transients above that of the neuropil background5. This suggests that, to have an accurate characterization of neuronal responses at synaptic resolution, correction of brain-induced aberrations is essential.


Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue.

Wang K, Sun W, Richie CT, Harvey BK, Betzig E, Ji N - Nat Commun (2015)

AO correction via direct wavefront sensing improves functional calcium imaging deep inside the cortex of a living mouse.(a) Calcium responses evoked by drifting-grating stimulation 400 and 500 μm below pia in the primary visual cortex of a mouse (Thy1-GCaMP6s line GP4.3) before (left panel) and after (right panel) correction. Brightness of each pixel reflects its s.d. across 800 frames imaged during five repetitions of the drifting-grating stimulus set, and is correlated with the local calcium transient magnitude. Scale bar, 20 μm. (b) Calcium transients at regions of interest (ROI) i–vi, before and after AO correction. Solid colours label averaged transients; faded colours label transients during specific repetitions. Top panel indicates the orientations and drifting directions of the grating stimuli. Representative images from >20 imaging sections in three mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490402&req=5

f3: AO correction via direct wavefront sensing improves functional calcium imaging deep inside the cortex of a living mouse.(a) Calcium responses evoked by drifting-grating stimulation 400 and 500 μm below pia in the primary visual cortex of a mouse (Thy1-GCaMP6s line GP4.3) before (left panel) and after (right panel) correction. Brightness of each pixel reflects its s.d. across 800 frames imaged during five repetitions of the drifting-grating stimulus set, and is correlated with the local calcium transient magnitude. Scale bar, 20 μm. (b) Calcium transients at regions of interest (ROI) i–vi, before and after AO correction. Solid colours label averaged transients; faded colours label transients during specific repetitions. Top panel indicates the orientations and drifting directions of the grating stimuli. Representative images from >20 imaging sections in three mice.
Mentions: Widely used in medical diagnostics13, injection of ICG allows this direct wavefront-sensing approach to be applied to a wide variety of tissues in vivo. In the mouse brain, it can improve not only morphological imaging but also the sensitivity when measuring neural activity at 500-μm depth in cells expressing the calcium indicator GCaMP6s1415 (Thy1-GCaMP6s GP4.3 mouse; Supplementary Movie 3). One caveat, however, is that we observed heightened spontaneous activity of GCaMP6s in cortical regions injected with ICG. To eliminate this, we replaced ICG with the NIR protein iRFP NIR fluorescence protein iRFP71316 (peak emission at 713 nm) expressed via a viral vector in a subset of neurons in the primary visual cortex of the Thy1-GCaMP6s GP4.3 mice. With this marker, we were able to use a TPE-generated NIR GS to obtain corrective wavefronts down to 500 μm below pia, which then led to markedly improved sensitivity to evoked calcium responses in the visual cortex. Indeed, after AO correction, calcium transients could be detected in many more neurites at depths of 400 (Fig. 3; Supplementary Movie 4), 500 (Fig. 3; Supplementary Movie 5) and 600 μm (Supplementary Movie 6). Several factors contribute to these drastic improvements: calcium indicators usually have a heterogeneous labelling density in the brain. The more strongly labelled somata or neurites constitute the structures of interest, which are surrounded by the axons and dendrites of other more weakly labelled neurons. Calcium transients from these weakly labelled neuropil, or the ‘neuropil contaminations'17, represent the average response of many neurons and are not selective to the orientation of our visual stimuli. For a fine neurite, calcium transients within the laser focus are composed of signals from both the neurite and its surrounding neuropil. In a focus enlarged and dimmed by aberration, the calcium transient of the neurite may be overwhelmed by that of the neuropil. AO correction, however, both reduces the focal volume and increases the focal intensity, which enhances the neurite signal a lot more than the neuropil background. As a result, the orientation selectivity of many neurites is only detectable after AO correction raises the neurite calcium transients above that of the neuropil background5. This suggests that, to have an accurate characterization of neuronal responses at synaptic resolution, correction of brain-induced aberrations is essential.

Bottom Line: Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens.Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars.The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA.

ABSTRACT
Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars. The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

No MeSH data available.


Related in: MedlinePlus