Tunable TIRF Microscopy: Simultaneous Visualization of Intracellular Dynamics at Multiple Depths

We propose and demonstrate tunable total internal reflection fluorescence (TIRF) microscopy, a new method to achieve dynamic customized depth‐selective fluorescence excitation across the field of view. By simply adding a digital micromirror device (DMD) and a spatial filter to a conventional fluorescence microscope, we show that different parts of the sample can be illuminated simultaneously with varying excitation light volumes, enabling a new versatile type of TIRF microscopy where the imaging depths can be customized to the user's needs. As the method does not use any moving parts, dynamic modulation of the optimized excitation field depth can be easily realized at speeds only limited by DMD refresh rates. Using tunable TIRF, we exploit the simultaneous orthogonal control of the illumination volume to visualize the vesicle transport near the bottom surface of the cell while simultaneously imaging the movement of membrane structures at elevated volumes in human umbilical vein endothelial cells (HUVECs) and HeLa cells, which is unfeasible using conventional TIRF microscopy.