Study on the statistical intensity distribution (SID) of fluorescent nanoparticles in TIRFM measurement

Total internal reflection fluorescence microscopy (TIRFM) based on the evanescent wave provides a powerful imaging tool to measure nanoparticle dynamics near the interfaces in nanofluidic and biophysical systems. The most dramatic advantage of this technique is encoding the nanoparticle motion in the vertical dimension into intensity variation. However, large measurement uncertainty was found in previous works, which has been a major obstacle for applying TIRFM to nanofluidic studies. In this study, we investigate the statistical intensity distribution (SID) of the fluorescent nanoparticles in the evanescent field. We establish a theoretical description of the SID by considering the Boltzmann distribution of the nanoparticle concentration, the statistical particle size variation, and the focal plane thickness of the objective. The theoretical results show excellent agreement with the experimental SID histogram. We then develop a method to precisely determine the base intensity I-0 and decode the vertical positions of nanoparticles, by which the measurement uncertainty of TIRFM can be significantly reduced. This SID method is verified by the nanoparticle tracking velocimetry measurement near the wall using nanoparticles with diameters of 100nm or 250nm as tracers. Furthermore, our experiments show that the SID of nanoparticles is sensitive to the salt concentration of the solution and the nanoparticle properties, implying the possibility of using SID as a nano-probe to explore solution or colloidal properties in nanofluidics