Figure 7.

Typical excitation/detection geometries utilized in single-molecule fluorescence microscopy. A, Epifluorescence: in epi-illumination, a laser beam or broadband source (lamp) is focused at the back focal plane of the objective lens (Obj), resulting in a collimated output beam, which illuminates the whole sample. The emitted light is collected by the same objective and imaged at the focal plane of the tube lens (TL) of the microscope on a wide-field detector (WFD). The role of the dichroic mirror (DM) is to reflect excitation light only and transmit the Stokes-shifted fluorescence light only. An additional emission filter (F) is used in front of the detector to reject background and residual excitation light. B: in Total Internal Reflection (TIR) excitation, a similar approach can be used (bottom part: objective TIR), but the focus beam is shifted off the optical axis, resulting in a tilted incidence on the object plane. At a critical angle, the incident light undergoes total internal reflection, leaving only an evanescent wave on the sample side of the coverslip (CS), with a typical penetration depth of a few hundred nm. An alternative approach (upper part, dashed box: prism TIR) uses a prism (P) to bring a laser beam at the critical angle on the top coverslip, resulting in an evanescent wave on inner, upper side of the sample. In both cases, the fluorescence of molecules within 100 nm of the coverslip (usually adherent molecules only) is collected by the objective and tube lenses as in the case of epifluorescence, with the advantage that little fluorescence (background) is excited away from the coverslip. C: in confocal microscopy, a collimated laser beam is sent into the back focal plane of the objective lens, resulting in a diffraction-limited excitation volume within the sample. This point can be raster-scanned throughout the sample using a scanning stage to move the sample around (XYZ arrows). The emitted light is collected by the objective and tube lenses, and focused on a point detector (PD). Alternatively, a pinhole is placed at the focal point of the TL, and a relay lens images it on a PD, in order to reject out-of-focus light (not shown).

In all drawings, the filled and empty circles between the coverslips represent fluorescent molecules that are excited or not, respectively. The small arrows represent the excitation path, the large arrows indicting the path followed by the emitted light. The Figure is not to scale.