Microscopy total internal reflection

Total internal reflection microscopy (TIRM) was introduced in 1987 by Prieve et al. [343]. TIRM allows to probe the interaction of a single microsphere with a transparent flat plate. In a TIRM experiment, a microsphere is allowed to sediment toward the plate. The technique relies on repulsive forces between sphere and plate. This repulsion will typically result from electric double layer or steric forces. They keep the sphere from getting into contact with the plate. Thermal fluctuations will constantly change the precise distance. The distance between sphere and plate is monitored by the light intensity scattered from the particle when illuminated by an evanescent wave and can be determined with a resolution of w 1 nm. By recording the fluctuations in vertical position of the sphere due to Brownian motion, the potential energy of interaction and the diffusion coefficient of the sphere can be deduced. For overviews of the technique, see Refs [344, 345]. 

X is the laser wavelength. The actual angle of incidence 0 of the laser beam has to be above the critical angle. Equation (3.27) shows that we can adjust the penetration depth Xev by changing the angle of incidence. 

At a wavelength of633 run zinc crown glass has a refractive index of tti = 1.52. The critical angle with water (n2 = 1.33) is 0c = 61°. For an angle of incidence of 0 = 65°, the decay length of the evanescent field is Xev = 140 nm. For 0c = 85°, it decreases to = 70nm. 

For the dependence of the scattered intensity I(z) on distance z, one assumes that I (z) decays with distance z of the particle from the planar surface in the same way as the intensity of the evanescent wave itself [346]  

h is the intensity at the surface. Together with the exponential dependence, this leads to a distance resolution of about 1 nm [344]. 

The interaction potentials between a single particle and a wall can be obtained using evaneseent field scattering in total internal reflection microscopy (TIRM) [69, 70]. The fluctuations of the separation distance resulting from thermal motion can be direetly detected from the scattered intensity. In a typical TIRM set-up a laser beam is directed via a prism to the glass/solution interface as sketched in Fig. 2.36, with an incident angle that is chosen such that total reflection occurs. The eleetric field of the laser beam penetrates the interface causing an 

Why does the scattered intensity due to a colloid decrease with increasing distance from the surface  

An optical trap can be set up to prevent the colloidal particle from moving out of the microscope s observation area. For this purpose a second laser beam has to 

In Hirschfeld s data, the background signal was about 100 photocounts, while the photon burst amplitude was about 400 photocoimts larger. That is, ii = 100 and = 400. Also, the probability that a molecule was present in the laser beam looks to be about Pq = 0.2 by definition, the probability that a molecule was not present is Pq = 0.8. Using the equations from above, the optimum threshold, in a Bayes sense, is given by 

---

D.C. Prieve and N.A. Frej Total Internal Reflection Microscopy A Quantitative Tool for the Measurement of Colloidal Forces. Langmuir 6, 396 (1990). 

Until fairly recently, the theories described in Secs. II and III for particle-surface interactions could not be verified by direct measurement, although plate-plate interactions could be studied by using the surface forces apparatus (SFA) [61,62]. However, in the past decade two techniques have been developed that specifically allow one to examine particles near surfaces, those being total internal reflection microscopy (TIRM) and an adapted version of atomic force microscopy (AFM). These two methods are, in a sense, complementary. In TIRM, one measures the position of a force-and torque-free, colloidal particle approximately 7-15 fim in dimension as it interacts with a nearby surface. In the AFM method, a small (3.5-10 jam) sphere is attached to the cantilever tip of an atomic force microscope, and when the tip is placed near a surface, the force measured is exactly the particle-surface interaction force. Hence, in TIRM one measures the position of a force-free particle, while in AFM one measures the force on a particle held at a fixed position. 

Evanescent wave microscopy or total internal reflection microscopy (TIRM) has been employed in the fields of biology and chemistry since the 1970s. The TIRM technique has long been used in cell biology studies and more recently cell-substrate contacts, vesicle fusion, and single-molecule observation. Here, cells on a microscope cover glass are illuminated by an... 

Paige, MF, Bjerneld, EJ, and Moerner, WE, A comparison of through-the-objective total internal reflection microscopy and epifluorescence microscopy for single-molecule fluorescence Imaging. Single Molecules (2001) 191-201. 

In this section we summarize experimental methods that enable measuring (depletion) interaction potentials between particles [64]. We distinguish pair interactions (Sects. 2.6.1-2.6.3) and many-body interactions (Sect. 2.6.4). The latter can be measured indirectly using scattering techniques or microscopy, whereas for pair interactions direct methods are available. Common instruments for investigating such pair interactions are the surface force apparams (SFA) [65], optical tweezers [66, 67], atomic force microscopy (AFM) [68], and total internal reflection microscopy (TIRM) [69, 70]. 

In total internal reflection microscopy, the light is confined only to the z-direction, resulting in the diffraction-limited resolution in the xy-plane. In the last two decades a novel optical microscopy has been developed aiming at high spatial resolution by the three-dimensionally localized optical field, optical near-field . Details on the structural analysis using the near-field microscopy is described in the next section. 

There are a number of force measuring techniques from which the interactions between particles can be measured Surface Force Apparatus (SFA), Atomic Force Microscopy (AFM), Total Internal Reflection Microscopy (TIRM), Optical Tweezers (OT), Micropipette Aspiration (MPA). The SFA and AFM are currently the most versatile in that the surface forces and separations can be accurately controlled and measured over a large range. 

---