TIRFM

Fig. 1 Real-time tracking of cell adhesion [42]. (a) Components of a total internal reflection fluorescent microscope (TIRFM). (b) The cell adhesion process (7) a cell approaches the surface, (2) the cell lands, (3) the cell attaches, and (4) the cell spreads out on the surface. The evanescent field was generated by total internal reflection of a laser beam at the glass-water interface. Cells with fluorescently labeled membranes (dashed lines) were plated on SAMs. Cell membranes within the evanescent field (solid line) were observed by TIRFM. Corresponding TIRFM images are shown below...

We assembled a TIRFM with low magnification to study cell adhesion behavior on SAMs with various functional groups [42]. Figure lb shows a schematic illustration of the cell adhesion process and the corresponding TIRFM images. A suspension of cells with fluorescently labeled cell membranes is applied onto a substrate (Fig. lb-1). At first, no bright spots were observed by TIRFM,... 

TIRFM was used for time-lapse observations of initial cell adhesion to SAMs with different surface functionalities (Fig. 2). After 10 min of plating a suspension of human umbilical vein endothelial cells (HUVECs), a few bright spots were observed on SAMs with COOH and NH2 functionalities this indicated cell adherence. The number of bright spots increased and the spot areas enlarged with incubation time, indicating that HUVECs adhered and spread well on COOH-SAM and NH2-SAM surfaces. Quantitative analysis of the number of adherent cells and cell adhesion areas... 

Fig. 2 TIRFM images of HUVEC adhesion behavior on SAMs with four different types of surface functional groups at the indicated times after first applying the cell suspension. Scale bar 200 pm [42]...

Fig. 4 The effect of proteins on cell adhesion, (a) Kretschmann configuration for SPR. (b) Reflectance (R) as a function of incident angle (9), before (black) and after (red) the adsorption of substances, (c) Left. Time course of SPR angle shift during exposure to culture medium supplemented with 2% FBS (solid line) and the fraction of adherent cells determined by TIRFM (circles) on NH2-SAM. The dashed line is a manual fit to the symbols, included simply as a guide [42]. Right The concentrations of serum proteins in FBS...

Figure 4c shows one example of the time course of an SPR angle shift during exposure of a NH2-SAM to culture medium supplemented with 2% fetal bovine serum (FBS). It also includes the time course of the fraction of adherent cells on the same surface determined by TIRFM observation (Fig. 2). The SPR angle shift rapidly increased, and then leveled off within a few minutes. Cells adhered much more slowly than proteins. Those results indicated that serum proteins in a medium rapidly adsorbed to the surface then, cells interacted with the adsorbed protein layer, as shown schematically in Fig. 5.

FIGURE 6.6. Schematic depiction of the screening assay for monitoring ligand-receptor interactions on a supported fluid lipid bilayer in the presence of a library of soluble inhibitors. Surface specific observation is achieved using TIRFM. 

To discriminate between surface bound protein molecules and those in bulk solution, total internal reflection fluorescence microscopy (TIRFM)41 55 was employed. TIRFM creates an evanescence wave that decays as a function of distance from the surface as ... 

FIGURE 6.9. TIRFM setup for imaging antibody-antigen interactions on bilayer coated chips. 

Figure 2.6 Application of splinted RNA ligation procedure single molecule FRET. (A) Diagram of prism-type total internal reflection fluorescence microscope (TIRFM) for single molecule FRET measurements. (B) Distribution of single-molecule FRET values for dye-labeled telomerase RNA molecules generated by splinted RNA ligation. (C) Dye intensity and FRET traces of a single telomerase RNA molecule Cy3 emission (green), Cy5 emission (red), FRET ratio (blue).

TIRFM total internal reflection fluorescence microscopy TLM thermal lens microscopy... 

Complementary microscopy techniques can be used to follow the morphology and growth of fibrils either on a surface or in aliquots taken from the assembly solution including total internal reflection fluorescence microscopy (TIRFM) (Ban et al., 2004), transmission electron microscopy (TEM), or atomic force microscopy (AFM). 

Fig. 15.1. Schematic representation of amyloid fibrils revealed by total internal reflection fluorescence microscopy, (a) The penetration depth of the evanescent field formed by the total internal reflection of laser light is 150nm for a laser light at 455 nm, so only amyloid fibrils lying parallel to the slide glass surface were observed. (b) Schematic diagram of a prism-type TIRFM system on an inverted microscope. ISIT image-intensifier-coupled silicone intensified target camera, CCD charge-coupled device camera...

Fig. 15.2. Direct observation of (32-m amyloid fibril growth obtained by TIRFM. Adapted from ref. [13] with permission. Incubation times are 0, 30, 60, and 90 min...

Fig. 15.3. Direct observation of AP(l-40) amyloid fibril growth by TIRFM. Realtime monitoring of fibril growth on glass slides. Arrows indicate the unidirectional growth of A (i from a single seed fibril. The scale bar represents 10 pm. Reproduced from [14] with permission...

Considering that TIRFM illumination has a depth of penetration of 150nm and the depth of focus on the objective lens is about 100 nm, the large clusters of seeds formed at first in solution and were not in contact with the substrate. The hazy areas observed at the initial stages, as indicated by the arrows in Fig. 15.4, may represent the clustered seeds or aggregated intermediates formed in solution. Since the thickness of the water medium... 

Figure 6.3. Palytoxin increases glutamate release in cultured cerebellar neurons loaded with FMl-43. Fluorescence was monitored with TIRFM imaging.

The use of an evanescent wave to excite fluorophores selectively near a solid-fluid interface is the basis of the technique total internal reflection fluorescence (TIRF). It can be used to study theadsorption kinetics of fluorophores onto a solid surface, and for the determination of orientational order and dynamics in adsorption layers and Langmuir-Blodgett films. TIRF microscopy (TIRFM) may be combined with FRAP ind FCS measurements to yield information about surface diffusion rates and the formation of surface aggregates. 

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