Label-free 3D tracking of biological micro-objects using RGB pinholes

Abstract

Defocusing digital particle image velocimetry is a method developed within the field of fluidics to measure the velocities of particles within a volume through image analysis. For three-dimensional (3D) tracking, approaches utilizing defocusing techniques have been proposed to enable depthwise positional measurements. Recently, there have been suggestions to simplify this approach by employing a triple pinhole mask and to further enhance it with color filters that allow distinguish particles, even under dense conditions. This paper presents an attempt to apply the defocusing method using a triple pinhole mask with red, green, and blue (RGB) color-coding filters, demonstrating the successful label-free 3D tracking of biological micro-objects at multiple positions within a volume. The application of this method in fluidics and its potential for biological analysis are highlighted through successful examples of 3D tracking involving micro-particles influenced by motor proteins, dividing cells, and swimming microorganisms.