Synchronization-based image reconstruction for three-dimensional wide-field confocal imaging of periodically moving objects beyond the frame rate

Abstract

We extend our previously proposed image reconstruction method, which allows confocal microscopes to capture periodically moving objects at frequencies beyond their frame rates, to three-dimensional and two-dimensional wide-field imaging. This extension is achieved by implementing a synchronization scheme between a confocal laser scanning microscope and a function generator to ensure consistent initial phase alignment across image sequences acquired at different focal depths or fields of view. The method was demonstrated by visualizing 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 single-particle analysis confirmed that the reconstructed images accurately captured the underlying particle dynamics. The extended approach requires no additional specialized hardware and can be readily integrated with conventional confocal microscopes. Thus, it extends the applicability of confocal imaging to the fast dynamics of periodic processes in biological and soft-matter systems.

Figures (4)

• Figure 1: (a) Block diagram of a confocal microscopy analysis system. The confocal microscope and function generator are synchronized to ensure a consistent absolute initial phase for each image sequence. (b) Schematic illustration of the image reconstruction process for the three-dimensional imaging of objects moving at frequencies exceeding the frame rate. Pixel size is not to scale. Representative raw and reconstructed confocal images of particles adhered to an aluminum bar oscillating at 100 Hz, acquired at two depths, are also shown.
• Figure 2: (a) x--z cross-sectional images at phases 0.4$\pi$ and 1.4$\pi$, corresponding to the phases at which the particle displacement along the x-axis is maximum. (b) Three-dimensional trajectories of 76 particles over one oscillation period. The displacements of a representative particle along the x-, y-, and z-axes are also shown. Solid lines indicate discrete Fourier transform fits. Multimedia available online.
• Figure 3: Displacement of individual particles along the $x$-axis over one pulse-wave generation period at different x-positions. Dotted lines represent the average displacement at each phase. Corresponding snapshots at the initial phase are shown above each graph. Multimedia available online.
• Figure 4: Maximum average displacement of colloidal particles as a function of their $x$-position during pulse-wave propagation.