RESEARCH ARTICLE C. H. Margraves Æ C. K. Choi Æ K. D. Kihm Measurements of the minimum elevation of nano-particles by 3D nanoscale tracking using ratiometric evanescent wave imaging Received: 20 December 2005 / Revised: 6 April 2006 / Accepted: 10 April 2006 / Published online: 10 May 2006 Ó Springer-Verlag 2006 Abstract Effect of saline concentration on the minimum elevation of nanoparticles has been examined under the electric double layer interactions with the substrate glass surface. The use of ratiometric total internal reflection fluorescence microscopy (R-TIRFM) allows three- dimensional tracking of nanoparticles in the near-wall region within less than 1 lm from the surface. The measurements of minimum elevation were made for polystyrene fluorescent nanospheres of 100, 250, and 500 nm in radii (SG = 1.05) for the salinity ranging from 0.1 to 10 mM. Special care was taken to insure cleaned surface conditions by elaborate sonication and rinsing of the glass substrate. The laser illumination intensity and duration also had to be carefully examined to minimize photobleaching of the fluorescence emission from particles. It is reported that the minimum elevation decreases with increasing saline concentration and with increasing particle sizes, for the first time experimentally and quantitatively to the authors’ knowledge. 1 Introduction A growing area of interest in the biomedical community is the examination of ligand–receptor, antigen–antibody, and drug–cell interactions. Of particular importance are the forces that cause or prevent these reactions from occurring. By gaining a better understanding of these forces it may be possible to promote certain desired interactions while reducing or eliminating others. Due to the fact that these interactions occur at very small length scales, popularly defined as the near-wall region 1 , total internal reflection fluorescence microscopy (TIRFM) provides an excellent tool for examination. TIRFM creates an evanescent wave field typically sev- eral hundred nanometers in penetration depth (Axelrod et al. 1992), which can be used to excite fluorescent particles as they fall into the illuminated zone. For years this technique has been used in several biological applications such as in vitro and in vivo single-molecule detections in cell biology (Sako et al. 2000; Ishijima and Yanagida 2001; Axelrod 2001). The use of TIRFM has been employed to observe a two-dimensional flow field in the near-wall region of a microchannel (Zettner and Yoda 2003). Their use of a prism-based TIRF system, however, generated excessive noise possibly occurring from the stray rays and the accuracy of the near-wall region sampling is question- able. The ratiometric analysis of TIRFM images, so called R-TIRFM, has allowed three-dimensional track- ing of the near-wall hindered Brownian motion of nanoparticles (Kihm et al. 2004), where a more accurate lens-based TIRF system was used to record images with substantially enhanced signal-to-noise ratios. Banerjee and Kihm (2005) presented the discrepancy between their measurements of hindered Brownian motion and predictions based on the hydrodynamic theories of near-wall hindered diffusion (Brenner 1961; Goldman et al. 1967). They have shown that the primary reason for the discrepancy, particularly for smaller nanoparticles, can be attributed to the electrophoretic hindering effects including the repulsive forces driven by the electric double layers (EDL) formed on both surfaces of particles and the substrate wall. An important force to be examined with TIRFM is the electrostatic force created by the EDL, of nanometer C. H. Margraves Æ C. K. Choi Æ K. D. Kihm (&) Micro/Nano-Scale Fluidics and Energy Transport (MINSFET) Laboratory, Mechanical, Aerospace, and Biomedical Engineering Department, University of Tennessee, Knoxville TN 37996-2210, USA Tel.: +1-865-9745292 Fax: +1-865-9745274 E-mail: kkihm@utk.edu URL: http://minsfet.utk.edu/ 1 The near-wall region is considered to be within one micron of the solid surface, in which both lateral and normal Brownian motions are substantially hindered due to the proximity of the no-slip wall (Brenner 1961; Goldman et al. 1967). Experiments in Fluids (2006) 41: 173–183 DOI 10.1007/s00348-006-0151-8