Limits...
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 confocal imaging in the cortex of a living mouse by direct wavefront sensing of a GS generated by TPE excitation of neurons expressing YFP.(a) Single-plane confocal images of membrane-labelled neuronal processes ∼10 μm below pia of a mouse (Thy1-ChR2-EYFP) measured with objective correction ring only (left) and correction ring adjustment plus AO (right). The images without AO have been digitally enhanced 2 × in brightness to improve visibility. Scale bars, 5 μm (first column); 2 μm (third column). (b) Confocal images of neuronal processes 3–17 μm below pia having cytosolic expression of YFP in a Thy1-YFPH mouse measured with correction ring adjustment, but either no AO (left) or AO plus deconvolution (right). Line cuts at far right compare the signal strength and resolution in the two cases when imaging two voids (coloured lines at left) likely caused by displacement of cytosolic YFP by the mitochondria within the dendrites. Representative images from >100 imaging sections in two mice. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490402&req=5

f4: AO confocal imaging in the cortex of a living mouse by direct wavefront sensing of a GS generated by TPE excitation of neurons expressing YFP.(a) Single-plane confocal images of membrane-labelled neuronal processes ∼10 μm below pia of a mouse (Thy1-ChR2-EYFP) measured with objective correction ring only (left) and correction ring adjustment plus AO (right). The images without AO have been digitally enhanced 2 × in brightness to improve visibility. Scale bars, 5 μm (first column); 2 μm (third column). (b) Confocal images of neuronal processes 3–17 μm below pia having cytosolic expression of YFP in a Thy1-YFPH mouse measured with correction ring adjustment, but either no AO (left) or AO plus deconvolution (right). Line cuts at far right compare the signal strength and resolution in the two cases when imaging two voids (coloured lines at left) likely caused by displacement of cytosolic YFP by the mitochondria within the dendrites. Representative images from >100 imaging sections in two mice. Scale bar, 5 μm.

Mentions: Although two-photon imaging is necessary to image fluorescence multiple scattering lengths deep within tissue, single-photon modalities such as confocal microscopy have intrinsically higher spatial resolution and can be applied in the superficial layers of the brain, where scattering is still low. Even here, however, AO can play an important role, as the multiple protective layers over the brain (for example, dura mater, arachnoid and pia mater) as well as slight tilt of the cranial window can impose substantial aberrations on both the excitation and emission light. Instead of painstakingly and invasively removing some of these protective layers18, our method can provide a quick AO correction using fluorescence from the same structures that are the target of the imaging. For example, we were able to use TPE to generate a descanned GS from membrane-specific EYFP expressed in apical dendrites in layer I of the mouse cortex (Thy1-ChR2-EYFP-transgenic mice). After AO correction, the dendritic branches and spines were clearly resolved as hollow, thanks to the membrane marker (Fig. 4a). With a different transgenic line (Thy1-YFPH) expressing cytosolic YFP in a subset of neurons, dark voids were occasionally observed in apical dendrites, possibly caused by mitochondria displacing the cytosolic YFP (Fig. 4b; Supplementary Movie 7). Thus, even in these superficial layers, AO can substantially improve imaging quality in vivo.


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 confocal imaging in the cortex of a living mouse by direct wavefront sensing of a GS generated by TPE excitation of neurons expressing YFP.(a) Single-plane confocal images of membrane-labelled neuronal processes ∼10 μm below pia of a mouse (Thy1-ChR2-EYFP) measured with objective correction ring only (left) and correction ring adjustment plus AO (right). The images without AO have been digitally enhanced 2 × in brightness to improve visibility. Scale bars, 5 μm (first column); 2 μm (third column). (b) Confocal images of neuronal processes 3–17 μm below pia having cytosolic expression of YFP in a Thy1-YFPH mouse measured with correction ring adjustment, but either no AO (left) or AO plus deconvolution (right). Line cuts at far right compare the signal strength and resolution in the two cases when imaging two voids (coloured lines at left) likely caused by displacement of cytosolic YFP by the mitochondria within the dendrites. Representative images from >100 imaging sections in two mice. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: AO confocal imaging in the cortex of a living mouse by direct wavefront sensing of a GS generated by TPE excitation of neurons expressing YFP.(a) Single-plane confocal images of membrane-labelled neuronal processes ∼10 μm below pia of a mouse (Thy1-ChR2-EYFP) measured with objective correction ring only (left) and correction ring adjustment plus AO (right). The images without AO have been digitally enhanced 2 × in brightness to improve visibility. Scale bars, 5 μm (first column); 2 μm (third column). (b) Confocal images of neuronal processes 3–17 μm below pia having cytosolic expression of YFP in a Thy1-YFPH mouse measured with correction ring adjustment, but either no AO (left) or AO plus deconvolution (right). Line cuts at far right compare the signal strength and resolution in the two cases when imaging two voids (coloured lines at left) likely caused by displacement of cytosolic YFP by the mitochondria within the dendrites. Representative images from >100 imaging sections in two mice. Scale bar, 5 μm.
Mentions: Although two-photon imaging is necessary to image fluorescence multiple scattering lengths deep within tissue, single-photon modalities such as confocal microscopy have intrinsically higher spatial resolution and can be applied in the superficial layers of the brain, where scattering is still low. Even here, however, AO can play an important role, as the multiple protective layers over the brain (for example, dura mater, arachnoid and pia mater) as well as slight tilt of the cranial window can impose substantial aberrations on both the excitation and emission light. Instead of painstakingly and invasively removing some of these protective layers18, our method can provide a quick AO correction using fluorescence from the same structures that are the target of the imaging. For example, we were able to use TPE to generate a descanned GS from membrane-specific EYFP expressed in apical dendrites in layer I of the mouse cortex (Thy1-ChR2-EYFP-transgenic mice). After AO correction, the dendritic branches and spines were clearly resolved as hollow, thanks to the membrane marker (Fig. 4a). With a different transgenic line (Thy1-YFPH) expressing cytosolic YFP in a subset of neurons, dark voids were occasionally observed in apical dendrites, possibly caused by mitochondria displacing the cytosolic YFP (Fig. 4b; Supplementary Movie 7). Thus, even in these superficial layers, AO can substantially improve imaging quality in vivo.

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