Light sheet microscopy for particle tracking analysis and determination of diffusion type

Abstract

Extracellular vesicles (EVs) are membrane-bound vesicles including microvesicles and exosomes. EVs have attracted academic and clinical interest due to their emerging roles in intercellular communication and novel therapeutic targeting in various diseases. So, there is a necessity to develop a suitable method for detection of EVs. Herein, we developed a light sheet microscope-based system for the analysis of microsphere beads that mimic the characteristics of the EVs. Because light-sheet helps to minimize background fluorescence and photobleaching in single-molecule experiments, the proposed system demonstrated its potential for two-dimensional particle tracking analysis for determination of size and the type of diffusion by tracking their movement inside a liquid phase using Stokes-Einstein relation. Two types of motion (such as sedimentation and Brownian motion) were observed with different design of sample chambers. We found that sedimentation motion is critical in the determination of particle size. Further, we have successfully tracked the Brownian motion of particles and plotted particle size distribution with diffusion type for the polystyrene microsphere beads ranging from 1 μm to 0.2 μm. In addition, the particles mixed with different sizes were successfully differentiated. In conclusion, the proposed technique is still in adolescence phase; however, we believe that this approach will emerge as an important tool for the study of dynamic processes in complex biological specimens.