In vivo volumetric imaging of biological dynamics in deep tissue via wavefront engineering

Abstract

Biological systems undergo dynamical changes continuously which span multiple spatial and temporal scales. To study these complex biological dynamics in vivo, high-speed volumetric imaging that can work at large imaging depth is highly desired. However, deep tissue imaging suffers from wavefront distortion, resulting in reduced Strehl ratio and image quality. Here we combine the two wavefront engineering methods developed in our lab, namely the optical phase-locked ultrasound lens based volumetric imaging and the iterative multiphoton adaptive compensation technique, and demonstrate in vivo volumetric imaging of microglial and mitochondrial dynamics at large depth in mouse brain cortex and lymph node, respectively.

Fig. 1

Diagram of the experimental setup. DBS: dichroic beam splitter, PBS: polarization beam splitter, QWP: quarter wave plate, UL: ultrasound lens, RL: relay lens, M: mirror, PLL: phase-lock loop, PD: photodetector, DM: deformable mirror, L: lens, PMT: photomultiplier tube. The inset: phase pattern for correcting the system aberration.

Fig. 2

Volumetric imaging of transient morphology of microglia in mouse brain cortex. (a) The phase pattern for full correction of both system and tissue induced wavefront distortions. (b, c) The maximum intensity projections of the microglia at depth 405-413 µm, with full correction and system correction respectively (Visualization 1). Scale bar: 10 µm. Power: 36 mW at 935 nm. (d) The signal intensity along the dashed line labeled in (c). (e-g) The transient morphologies of the microglia (Visualization 2) at depth 380-420 µm under the dura. Volume size: 98 × 49 × 40 µm³. The dashed circle in (f) labels a GFP-expressing cell patrolling around the brain cortex through a blood vessel. Laser power: 108 mW at 935 nm.

Fig. 3

Volumetric imaging of the dynamics of mitochondrial network in lymphocytes of mouse lymph node. (a) The phase pattern for full correction of both system and tissue induced wavefront distortions. (b, c) The volume views of mitochondria in lymphocytes, with full correction and system correction respectively. The volume is 17.6 × 17.6 × 18 µm³, at the depth of 340-364 µm under the surface. Laser power: 55 mW at 935 nm. (d, e) The images acquired at 348 µm depth, with full correction and system correction respectively. Scale bar: 5 µm. (f) The signal intensity along the dashed line labeled in (e). (g-i) The transient morphologies of the mitochondria in lymphocytes (Visualization 3). Volume size: 12 × 9 × 18 µm³. Laser power: 90 mW at 935 nm.