Total internal reflection (=TIR) is an optical phenomenon. If light is travelling through a medium with a high refractive index and strikes the interface of an optical medium with a lower refractive index at an angle greater than the critical angle, the incident light will undergo total internal reflection. In biological investigations involving living specimen, these prerequisites are met if a laser beam travels through glass, for example a microscope slide (refractive index n = 1.52), towards an aqueous buffer solution or cell surface (n = 1.33 - 1.38). The beam is totally reflected at the interface between the glass and the medium or the cell surface respectively. But the reflected light generates an electromagnetic wave termed the evanescent wave which enters the low refractive index medium. The intensity of this wave decays exponentially with penetration depth along the z-axis. The penetration depth depends on the wavelength of light, the incident angle and the refractive index of the media. The thickness of the optical section that generates fluorescence under TIR can be adjusted by changing the incident angle and has a range between a few hundred and 50 nm at minimum. That is up to one-tenth of an optical section obtained with a confocal laser scanning microscope. Thus in TIRFM, fluorophores close to the coverslip surface can be selectively excited. As in confocal microscopy background fluorescence is nearly absent in TIRFM, because the fluorophore excitation is restricted to the focal plane of the objective. Therefore TIRFM gives high-contrast images of the cell surface with excellent signal-to-background ratio and is ideally suited for the observation of processes and structures on the cell surface and within or close to the plasma membrane.
In contrast, widefield fluorescence microscopy provides limited spatial resolution in z-direction because background fluorescence from outside the focal plane often impedes the detection of small or weakly fluorescent structures.
TIRFM does not require the rather expensive scanning microscope technique because it generates widefield illumination at the specimen surface. It can be performed relatively cost-efficient with inverted fluorescence microscopes equipped with a special episcopic fluorescence illuminator, special high NA objectives and a laser.
Confocal microscopy has the clear advantage in not being restricted to the optical section directly adjacent to the coverslip/specimen interface. However optical sections obtained by TIRFM can be up to one order of magnitude thinner than by confocal microscopy. Further-more, being a widefield technique, TIRFM comprises the considerable advantages of a higher acquisition speed as compared to scanning microscopy.
The TIRF Microscope
TIRFM is a widefield microscopy technique and consequently modified fluorescence microscope set-ups are used. Lasers are commonly employed as illumination sources because the light is coherent, polarised, intense and well collimated. If a special dual port epi-fluorescence condenser is used the change from widefield illumination with a standard arc lamp to the TIRF laser is rapid and straightforward and does not interfere with the beam alignment.
For TIRFM with inverted microscopes, the incident beam is focused off-axis at the objective back focal plane such that it passes the very periphery of the pupil of a highly refractive objective. The objective’s numerical aperture must be at least 1.38 to exceed the refractive index of a living cell. Thus the incident beam emerges from the front lens into the immersion oil in such a way that it reaches the glass/cell interface in a critical angle and undergoes total internal reflection. The off-axis position of the laser beam determines the incident angle and the depth of the evanescent field accordingly. The further off-centre the alignment is, the larger is the angle and the shallower is the evanescent wave.
Olympus offers a range of special TIRF objectives with very high numerical apertures (NA). There are three objectives available with a NA of 1.45 that can be used with conventional immersion oil and cover slips. With the PLAPON60xO/TIRFM-SP, the PLAPO100XO/TIRFM-SP or the new UAPO150xO/TIRFM-SP a penetration depth of the exciting wave around 100 nm is reached. The UAPO150xO/TIRFM is especially designed for single-molecule detection in TIRFM. The APO100XOHR features an unsurpassed NA of 1.65. To match the extreme high NA special high-refraction cover slips and special immersion liquid (diiodomethane) are required. Therefore the objective offers a unique short penetration depth of about 50 nm and high flexibility regarding the marginal angle available for TIR. The 60x and the 150x objec-tives have a temperature correction collar. Common plan apochromatic objectives like 100x with NA = 1.40 can also be employed for TIRFM, but they are less convenient due to the small angle that is available for the total internal reflection.
Applications
The visualisation of molecular interactions on surfaces is of fundamental interest in cell and molecular biology because many molecular transport and signal transduction processes are transmembrane incidents. Examples are binding and triggering of cells by hormones, neurotransmitters and antigens, cell adhesion to surfaces, electron transport in the membrane, cytoskeletal and membrane dynamics, cellular secretion events and vesicular fusion events with membranes. TIRFM is the perfect tool for visualisation of these processes. The extraordinary small depth of field of TIRFM assures that only surface-bound fluorophores are detected. Fluorophores in the surrounding medium remain invisible though they might be in rapid exchange and be present in large excess. This is different from normal widefield illumination where they would create overpowering background fluorescence.
During many experiments it might be desirable to switch rapidly between TIR illumination to observe the surface and standard epi-illumination to investigate the deeper layers of the specimen as well. For example, a transient process may involve simultaneous but not identi-cal processes in the membrane and the cytoplasm. The Olympus BioSystems dualport illuminator for combined TIR and widefield illumination allows the fast and synchronised alternation of the two techniques. The techniques of TIRFM, DIC and other microscopy techniques as FRET, FRAP and FLIM can also be combined.
