3D Confocal Spinning Disc Imaging with an Electro-Optical Tunable Lens─Experiments and Theory

Three-dimensional high-resolution imaging of dynamic objects are still limited in applications because axial sample scans are slow or even impossible. Here we present a confocal spinning disk microscopy system in fluorescence and brightfield mode incorporating an electro-optical tunable lens (ETL) for rapid axial refocusing. We demonstrate high-speed volumetric imaging with a NA = 1.2 lens by synchronizing 50–200 Hz camera frame rates with ETL refocusing, without the need for sample movement. In a 170 × 130 × 40 μm3 volume we demonstrate efficient optical sectioning without significant optical aberrations. We provide a theoretical estimate for the number of photons transferred to the camera and estimate the spatial frequency transfer for a finite pinhole size, defining lateral and axial resolution. We image the 3D diffusion of 0.2 and 1 μm beads in water and discuss and model the time-dependent shape changes of the point spread and transfer functions. We demonstrate the benefits of fast refocusing by showing the filopodia-dynamics of actin labeled macrophage cells and the orientation of intercellular tunnelling nanotubes filled with microtubules.