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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 morphological imaging deep inside the cortex of a living mouse.(a) 120 × 120 μm field-of-view single-plane TPE fluorescence images of neurons in a Thy1-YFPH mouse at 600–620 μm below pia after AO correction. Scale bar, 20 μm. (b) TPE fluorescence images of dendrites at 606, 606.5 and 608.5-μm depth taken with objective correction ring adjustment only (left) and correction ring adjustment plus AO (right). Scale bar, 5 μm. (c) SH sensor image (left) for an NIR GS produced by TPE excitation of directly injected ICG, and the corresponding corrective wavefront (right). Representative images from >500 image sections in five mice.
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f1: AO correction via direct wavefront sensing improves morphological imaging deep inside the cortex of a living mouse.(a) 120 × 120 μm field-of-view single-plane TPE fluorescence images of neurons in a Thy1-YFPH mouse at 600–620 μm below pia after AO correction. Scale bar, 20 μm. (b) TPE fluorescence images of dendrites at 606, 606.5 and 608.5-μm depth taken with objective correction ring adjustment only (left) and correction ring adjustment plus AO (right). Scale bar, 5 μm. (c) SH sensor image (left) for an NIR GS produced by TPE excitation of directly injected ICG, and the corresponding corrective wavefront (right). Representative images from >500 image sections in five mice.

Mentions: Reduced scattering at longer wavelength allows direct wavefront sensing at increasingly greater depths in the scattering mouse brain in vivo, as demonstrated by GSs with emission peaks of 530 (YFP), 580 (tdTomato) and 810 nm (indocyanine green, ICG; Supplementary Figs 1 and 2; Methods). In one example, we injected ICG in the mouse cortex and used TPE to generate a NIR GS for aberration-corrected in vivo morphological imaging of layer 5 pyramidal neurons sparsely labelled with YFP (Methods). For in vivo mouse brain imaging, cranial window introduces aberrations that can be corrected either by AO (Supplementary Fig. 3) or by adjusting the correction ring on the objective (Supplementary Fig. 4). We used the correction ring of a 1.1 numerical aperture (NA) water-immersion objective to pre-correct cranial-window-induced aberrations (Supplementary Fig. 4), which ensured that the subsequent AO correction predominantly reflected the aberration of the brain tissue itself. With a single wavefront measurement 600 μm below pia, dendritic spines could be clearly resolved over a large field of view (120 × 120 μm; Fig. 1a). Without the additional signal (up to 6 × ) afforded by AO, most of these spines were invisible (Fig. 1b). Resolution enhancement by AO was also obvious: the full width at half maximum of a dendritic spine neck 550 μm below pia was reduced from ∼660 nm before AO to ∼430 nm after AO (Supplementary Fig. 5). The corrective wavefront contained of large elements of coma, astigmatism and spherical aberration caused by the refractive index mismatch between brain tissue and water, as well as high-order modes resulting from the heterogeneity of the brain (Fig. 1c; Supplementary Fig. 6). This heterogeneity is also evident in the SH image, where individual GS images have complex intensity and shape variations (Fig. 1c). Our direct-wavefront-sensing approach can be applied to improve structural imaging at 700 μm (Supplementary Movie 1) or even 760 μm (Supplementary Movie 2) below pia, despite the presence of substantial brain motion at these depths. Even deeper corrections are limited by the cranial window size (Supplementary Fig. 7).


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 morphological imaging deep inside the cortex of a living mouse.(a) 120 × 120 μm field-of-view single-plane TPE fluorescence images of neurons in a Thy1-YFPH mouse at 600–620 μm below pia after AO correction. Scale bar, 20 μm. (b) TPE fluorescence images of dendrites at 606, 606.5 and 608.5-μm depth taken with objective correction ring adjustment only (left) and correction ring adjustment plus AO (right). Scale bar, 5 μm. (c) SH sensor image (left) for an NIR GS produced by TPE excitation of directly injected ICG, and the corresponding corrective wavefront (right). Representative images from >500 image sections in five mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490402&req=5

f1: AO correction via direct wavefront sensing improves morphological imaging deep inside the cortex of a living mouse.(a) 120 × 120 μm field-of-view single-plane TPE fluorescence images of neurons in a Thy1-YFPH mouse at 600–620 μm below pia after AO correction. Scale bar, 20 μm. (b) TPE fluorescence images of dendrites at 606, 606.5 and 608.5-μm depth taken with objective correction ring adjustment only (left) and correction ring adjustment plus AO (right). Scale bar, 5 μm. (c) SH sensor image (left) for an NIR GS produced by TPE excitation of directly injected ICG, and the corresponding corrective wavefront (right). Representative images from >500 image sections in five mice.
Mentions: Reduced scattering at longer wavelength allows direct wavefront sensing at increasingly greater depths in the scattering mouse brain in vivo, as demonstrated by GSs with emission peaks of 530 (YFP), 580 (tdTomato) and 810 nm (indocyanine green, ICG; Supplementary Figs 1 and 2; Methods). In one example, we injected ICG in the mouse cortex and used TPE to generate a NIR GS for aberration-corrected in vivo morphological imaging of layer 5 pyramidal neurons sparsely labelled with YFP (Methods). For in vivo mouse brain imaging, cranial window introduces aberrations that can be corrected either by AO (Supplementary Fig. 3) or by adjusting the correction ring on the objective (Supplementary Fig. 4). We used the correction ring of a 1.1 numerical aperture (NA) water-immersion objective to pre-correct cranial-window-induced aberrations (Supplementary Fig. 4), which ensured that the subsequent AO correction predominantly reflected the aberration of the brain tissue itself. With a single wavefront measurement 600 μm below pia, dendritic spines could be clearly resolved over a large field of view (120 × 120 μm; Fig. 1a). Without the additional signal (up to 6 × ) afforded by AO, most of these spines were invisible (Fig. 1b). Resolution enhancement by AO was also obvious: the full width at half maximum of a dendritic spine neck 550 μm below pia was reduced from ∼660 nm before AO to ∼430 nm after AO (Supplementary Fig. 5). The corrective wavefront contained of large elements of coma, astigmatism and spherical aberration caused by the refractive index mismatch between brain tissue and water, as well as high-order modes resulting from the heterogeneity of the brain (Fig. 1c; Supplementary Fig. 6). This heterogeneity is also evident in the SH image, where individual GS images have complex intensity and shape variations (Fig. 1c). Our direct-wavefront-sensing approach can be applied to improve structural imaging at 700 μm (Supplementary Movie 1) or even 760 μm (Supplementary Movie 2) below pia, despite the presence of substantial brain motion at these depths. Even deeper corrections are limited by the cranial window size (Supplementary Fig. 7).

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