Three-dimensional random-access confocal microscopy with 3D remote focusing system

Understanding biological activities in cells or deep tissues requires high-speed three-dimensional (3D) imaging. Substantial progress has been made with the emergence of 3D random-access microscopy. However, current solutions for fast 3D random-access imaging remain complex and costly. Herein we propose a simple, cost-effective, and fast 3D random-access confocal microscopy with remote focusing system. Our system shows isotropic response times across the x, y, and z axes, with a 34-fold improvement in axial response time over traditional piezo stages. We demonstrate its volumetric imaging performance with fluorescent particles and live cells. Furthermore, we validate the 3D random-access imaging capability of this system by continuously monitoring the signals in three different planes, showing a refresh rate of 500 Hz on two different positions in 3D. The simplicity, versatility, and affordability of our system promise widespread applications in research and industry. Haoyang Li, Quan Lu, Zhong Wang, and colleagues present a cost-effective and fast 3D random-access confocal microscopy. They demonstrate its performance of fluorescent particles and live Hela cells.