Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and suffers a reduced acquisition rate, which is critical to document neuronal dynamics properly. In this study, we implemented an electro-tunable lens (ETL) in the line-scan confocal microscopy, enabling the volumetric acquisition at the rate of 20 frames per second with the maximum volume of interest of 315 × 315 × 80 μm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 μm and 90.4 ± 2.1 μm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth elongation by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of cleared mouse brain ex vivo samples and in vivo brains. The current study foregrounds the successful application of resonant ETL for constructing a basis for a high-performance 3D line-scan confocal microscopy system, which will enhance our understanding of various dynamic biological processes.