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

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.

Figure 1. 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.

Figure 2. Exemplary spatial variation of sample-induced aberrations in the cortex of a living mouse.

(a) SH images and (b) their corresponding corrective wavefronts obtained at pia surface and three different regions (i, ii and iii) 400 μm below pia of a Thy1-YFPH mouse. (c) Images of regions i, ii and iii obtained with the corrective wavefronts in b, respectively. Scale bar, 20 μm. The inset (‘map of imaging locations') shows the relative positions of regions i, ii and iii. Scale bar, 100 μm (inset).

Figure 3. 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.

Figure 4. 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.