Synchronization-based image reconstruction for three-dimensional wide-field confocal imaging of periodically moving...

Confocal microscopy enables high-resolution imaging deep within samples but is fundamentally limited by its low frame rate, making the visualization of fast dynamics challenging. We developed a synchronization-based image reconstruction method that extends conventional laser scanning confocal microscopes to three-dimensional and two-dimensional wide-field imaging of periodically moving objects at frequencies beyond the nominal frame rate. The method synchronizes a confocal microscope with a function generator to ensure consistent initial phase alignment across image sequences acquired at different focal depths or fields of view. Using this approach, we visualized the three-dimensional motion of silica particles attached to an aluminum bar oscillating at 100 Hz and the two-dimensional wide-field response of colloidal particles subjected to periodic pulsed excitation. Quantitative analysis of particle trajectories confirmed that the reconstructed images captured the underlying particle dynamics. Because the method requires no additional specialized imaging hardware and preserves the intrinsic imaging performance of confocal microscopy, it can be readily integrated with conventional confocal microscopes for investigating the fast dynamics of periodic processes in biological and soft-matter systems.