Developing Scattering Morphology Resolved Total Internal Reflection Microscopy (SMR-TIRM) for Orientation Detection of Colloidal Ellipsoids

Abstract

Micrometer scale colloidal particles experiencing ∼kT scale interactions and suspended in a fluid are relevant to a broad spectrum of applications. Often, colloidal particles are anisotropic, either by design or by nature. Yet, there are few techniques by which ∼kT scale interactions of anisotropic particles can be measured. Herein, we present the initial development of scattering morphology resolved total internal reflection microscopy (SMR-TIRM). The hypothesis of this work is that the morphology of light scattered by an anisotropic particle from an evanescent wave is a sensitive function of particle orientation. This hypothesis was tested with experiments and simulations mapping the scattered light from colloidal ellipsoids at systemically varied orientations. Scattering morphologies were first fitted with a two-dimensional (2D) Gaussian surface. The fitted morphology was parameterized by the morphology’s orientation angle M ϕ and aspect ratio M AR. Data from both experiments and simulations show M ϕ to be a function of the particle azimuthal angle, while M AR was a sensitive function of the polar angle. This analysis shows that both azimuthal and polar angles of a colloidal ellipsoid could be resolved from scattering morphology as well or better than using bright-field microscopy. The integrated scattering intensity, which will be used for determining the separation distance, was also found to be a sensitive function of particle orientation. A procedure for interpreting these confounding effects was developed that in principle would uniquely determine the separation distance, the azimuthal angle, and the polar angle. Tracking these three quantities is necessary for calculating the potential energy landscape sampled by a colloidal ellipsoid.

Catch bonds in sickle cell disease: shear-enhanced adhesion of red blood cells to laminin

“…It should be noted that at t 3 , the sRBC was released from the boundary (LN surface) and the integrated TIR intensity during this phase dropped to approximately 0.5. This kind of intensity change is roughly consistent with the average distance between sRBC and the LN surface increasing on the order of ∼ O (10 nm) from the initial equilibrium distance, although a more precise measure of height change would require a deeper understanding of how scattering depends on the orientation and shape of the RBC (37,38). The sRBC displayed a dragging motion until t 4 and tumbled at t 5 .…”

“…Our team is developing a new class of TIRM, named Scattering Morphology Resolved TIRM (SMR-TIRM), suitable for probing interactions between anisotropic particles and a neighboring surface. We recently described the implementation of SMR-TIRM on anisotropic particles (37, 38). This improvement expands the applicability of TIRM to biological targets.…”

“…The video is then cropped and saved as .mp4 in Matlab using an automated routine we developed. (38) The detachment is initiated in a flow cytometry staining buffer (RnD Systems, Minneapolis, MN) by ramped flow as described in Fig. 4A at a ramping rate of 2.5 dyne/cm 2 /min.…”

Scattering Morphology Resolved Total Internal Reflection Microscopy (SMR-TIRM) Of Colloidal Spheres

“…One challenge with the application of conventional TIRM, however, is utilizing this technique with novel anisotropic particles [17]. Particles with a non-spherical shape or anisotropic optical properties will cause light scattered from the particle to depend upon not only the separation distance, but also its orientation with respect to the evanescent wave [18,19]. Decoding the signature of this scattered light for a specific type of anisotropic particle is a current challenge that Scattering Morphology Resolved Total Internal Reflection Microscopy (SMR-TIRM) intends to address.…”

“…SMR-TIRM uses the morphology of scattering in addition to the integrated intensity. Recent experimental and simulation results have shown that both the morphology and integrated intensity sensitively depend upon the orientation of an ellipsoidal colloid [18]. Primarily, this information could be used to estimate the orientation and position of the ellipsoid, thereby providing the necessary information to assemble the potential energy landscape for an anisotropic particle [20].…”