Spatiotemporal Light Modulation for Optical Sectioning, Diffusion Measurement and Parallelized Experiments with a Dual-DMD Microscope

Spatiotemporal modulation of excitation adds a programmable control layer to fluorescence imaging, enhancing probe functionality and enabling contrast mechanisms and quantitative readouts beyond intensity- or time-resolved approaches. We present two approaches exploiting this control. Modulated Illumination in Space and Time for Optical Sectioning (MIST-OS) suppresses out-of-focus fluorescence using oscillating stripes and temporally resolved detection, while Fourier-Transform Fluorescence Activation and Diffusion Exchange (FT-FADE) applies Fourier-domain analysis to patterned photoactivation for local one-dimensional diffusion measurement. These developments, along with ongoing efforts to establish similar protocols and perform parallelized temporal experiments (also demonstrated here on bacteria), motivated the creation of a custom dual–DMD microscope, fully documented to enable adoption by other laboratories.