Rapid adaptive optical recovery of optimal resolution over large volumes

Abstract

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

Figure 1

Adaptive optics (AO) over a large volume in the living zebrafish brain. (a) 3D rendering after AO correction of a membrane-labeled subset of neurons imaged by TPE fluorescence microscopy. (b) Top, x–y maximum intensity projection (MIP) and bottom, an x–z orthoslice through the plane defined by the red line, of the neurons in the green box in a, before AO correction. (c) Same, except after AO correction. (d) Same, except after AO correction and subsequent deconvolution. Scale bars, 10 μm. Scale is the same in b, c and d. (e) x–y and x–z frequency space representation of the volume in b, showing substantial loss of resolution without AO. (f) Same for the volume in d, showing recovery of spatial frequencies with AO out to the diffraction limit in all three dimensions.

Figure 2

Spatial variability of aberrations across the living zebrafish brain. (a) x–y MIP (left) after AO correction of a ubiquitously expressed cell membrane marker as imaged by TPE fluorescence microscopy at a depth of 150 μm. Three numbered regions are shown at higher magnification before (left column) and after (center column) AO correction, along with the wavefront correction (right column) for each. Scale bars, 10 μm. (b) x–y MIP of oligodendrocytes close to the midline of the hindbrain in a different cell line, before AO correction. (c, d) Same region, after AO correction, using the wavefront corrections (insets) measured over only the indicated sub-volumes (white boxes). Scale bar, 10 μm. Scale is the same in b, c and d. Note the recovery of near-optimal resolution in the boxed regions in c and d. However, the corrective wavefronts from these regions provide only partial correction of aberrations elsewhere in their respective imaging areas.

Figure 3

Two color confocal imaging with AO provided by a de-scanned two-photon guide star, deep in the living zebrafish brain. 3D volume rendering (left) of oligodendrocytes (magenta) and neuronal nuclei (green) from the optic tectum through the midbrain. MIPs before (center column) and after (right column) AO correction across four sub-volumes spanning depths as shown (yellow rectangles, left) demonstrate the recovery of diffraction-limited resolution throughout the 200 μm deep imaging volume.