Total Internal Reflection Microscopy TIRM

Total Internal Reflection Microscopy serves, e.g., for obtaining the height distribution function between an - in most cases spherical - object and a surface from quantitative, time-resolved measurements of the intensity of light scattered within the evanescent wave. From the height distribution function one might find the interaction energy between object and surface. 

The instantaneous separation distance is determined by measuring the intensity of evanescent light scattered by the object. The intensity of the light flickers with time as the distance between the object and the lower microscope slide changes due to Brownian motion. Because of the nonuniform illumination of the liquid by the evanescent wave, the amount of light scattered by the object is a measure of its proximity to the interface. Let us measure the intensity change while the distance to the slide is varied. Then the intensity as 

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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... 

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]. 

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... 

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. 

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]. 

In this chapter we review some data on the interactions between two soUd-liquid or two air-liquid interfaces obtained with a range of surface force techniques. It is beyond the purpose of this chapter to describe the merits and drawbacks of the various methods and the interested reader is referred to the original articles describing the surface force apparatus (SFA) [10], the atomic force microscope (AFM) colloidal probe [11], the thin film balance (TFB) [12] and total internal reflection microscopy (TIRM) [13] as well as a more recent review [14]. It is, however, important to be aware that the different techniques use different interaction geometries, and the results can be compared only by using the Derjaguin approximation [15,16] ... 

TIRFM TIRM Total internal reflection fluorescence (microscopy)... 

TIRFM TIRM Total Internal Reflection Huo-rescence (Microscopy)... 

Total Internal Reflection Ruorescence (Microscopy) TIRM TIRFM... 

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