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Results: 4

1.
Fig. 1.

Fig. 1. From: Imaging with total internal reflection fluorescence microscopy for the cell biologist.

Comparison of images obtained using epifluorescence and TIRF. In both cases, the microscope was focused at the adherent plasma membrane and images were acquired with one of three modes of excitation: epifluorescence, TIRF or confocal. (A,B) Actin (LifeAct–GFP) in a migrating MDCK cell. (C,D) Clathrin (clathrin light chain–GFP) in a HeLa cell. (E–G) Caveolin-1 (caveolin-1–EGFP) in MDCK cells. In each case, TIRF clearly eliminates of out-of-focus fluorescence and reveals details at or near the cell surface. Scale bars: 10 μm.

Alexa L. Mattheyses, et al. J Cell Sci. 2010 November 1;123(21):3621-3628.
2.
Fig. 3.

Fig. 3. From: Imaging with total internal reflection fluorescence microscopy for the cell biologist.

The depth of the evanescent field depends on the refractive index of the sample. The depth of the evanescent field (d) is a function of the index of refraction of the sample (n1). Incidence angles were chosen as 1.5° above the critical angle (dotted lines), the midpoint of all possible TIRF angles (dashed lines) and 1.5° less than the maximum incidence angle (solid lines). As the angle of incidence is increased, the sensitivity of d to the sample refractive index decreases in the range for cells. The 1.65 NA objective has a larger range with lower sensitivity to sample refractive index than the 1.49 NA objective.

Alexa L. Mattheyses, et al. J Cell Sci. 2010 November 1;123(21):3621-3628.
3.
Fig. 2.

Fig. 2. From: Imaging with total internal reflection fluorescence microscopy for the cell biologist.

The physical basis of epifluorescence and TIRF illumination. Schematic illustrating the cover-slip–sample interface. (A) Epifluorescence. The excitation beam travels directly through the cover-slip–sample interface. All of the fluorophores in the entire sample are excited. (B) TIRF. The excitation beam enters from the left at incidence angle θ, which is greater than the critical angle, θc (indicated by the dashed line). Angles are measured from the normal. The excitation beam is reflected off the cover-slip–sample interface and an evanescent field is generated on the opposite side of the interface, in the sample. Only fluorophores in the evanescent field are excited, as indicated by the green color. The refractive index of the sample (n1) must be less than the index of refraction of the cover slip (n2) to achieve TIR.

Alexa L. Mattheyses, et al. J Cell Sci. 2010 November 1;123(21):3621-3628.
4.
Fig. 4.

Fig. 4. From: Imaging with total internal reflection fluorescence microscopy for the cell biologist.

TIRF test samples. The differences between epifluorescence and TIRF and the quality of the evanescent field can be examined using a test sample of fluorescent beads (A–D). In epifluorescence (incidence angle θ=0), there is a large fluorescent background and beads appear in focus at both the cover slip z=0 (A) and deeper into the sample z>0 (C). In TIRF (θ>θc), the background is significantly less than in epifluorescence (B) and there are no beads in focus deeper into the sample (z>0) because the excitation field does not extend deep into the sample (D). The region imaged was constant, and identical exposure times and scalings were applied to all four images. DiI deposited on a cover slip reveals (E) interference fringes. For contrast, the same region is shown with elimination of the interference fringes (F). Scale bars: 10 μm.

Alexa L. Mattheyses, et al. J Cell Sci. 2010 November 1;123(21):3621-3628.

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