Posted by Fred Koenig on Dec 15th 2020

Total Internal Reflection Fluorescence (TIRF) Microscopy

What is Total Internal Reflection Fluorescence (TIRF) and How Does it Work?

Total Internal Reflection Fluorescence (TIRF) uses evanescent waves to illuminate and excite selected fluorophores immediately adjacent to the glass-water interface.
The electromagnetic field of the evanescent decays exponentially away from the interface, and so it can only penetrate to a 100nm depth into the sample medium. The TIRF can therefore select surface regions, such as the basal plasma membrane, which is about 7.5nm thick, for visualization. The field of view is at least 300nm wide, so the cytoplasmic zone is also visible. The features and events on the plasma membrane of living cells are thus selectively visualized with high axial resolution.

It is even possible to observe the fluorescence of a single molecule using TIRF. Biophysicists and quantitative biologists can therefore benefit greatly through use of this device and technique. Detecting a single molecule for DNA biomarkers, and SNP discrimination are also possible with this TIRF microscopy.

Cis-geometry (through-objective TIRFM) provides different quality of the effect of total internal reflection. Objective-type TIRFM shares the objective and other optical elements of the microscope. The evanescent wave, in this case, is contaminated with intense stray light comprising roughly 10-15% of the wave, making interpretation of objective-type TIRFM data somewhat problematic.

Trans-geometry (prism- and light guide-based TIRFM) is an alternate option. In this case, the excitation lightpath and the emission channel are separated. Prism-based geometry generates a clean evanescent wave with an exponential decay very close to the theoretically predicted function.

E.J. Ambrose was the first to describe the use of total internal reflection to illuminate cells in contact with a glass surface, back in 1956. By the 1980’s, University of Michigan’s Daniel Axelrod extended the idea and coined the acronym TIRF.

What is a TIRF Microscope?

A total internal reflection fluorescence microscope (TIRFM) is a microscope used for the observation of a thin slice of a specimen, usually less than 200 nanometers thick.

Conventional fluorescence microscopes have been used by molecular and cell biologists to observe molecular events in cellular surfaces, such as cell adhesion, secretion of neurotransmitters, the binding of cells by hormones, and membrane dynamics.

A problem occurs, however, when trying to observe fluorophores that are bound to the specimen surface and are in an equilibrium state with those of the surrounding medium. When excited, the more numerous, non-bound molecules fluoresce in the background, overwhelming the fluorophores that are bound to the surface.

This is where TIRF comes in. It allows for selective excitation of the surface-bound fluorophores, without exciting those non-bound molecules in the background. Because it is able to select just the sub-micron surface, TIRF is the best choice of microscope for single molecule detection.

How to Configure a TIRF Microscope?

During the early stages of TIRFM instrument development and testing, a wide spectrum of optical configurations went under close scrutiny. The result of this intense observation and analysis was a number of designs that can generate the necessary thin, evanescent field at the junction between two materials that differ in regard to refractive index.

Most of the TIRFM designs employ the inverted microscope configuration, as this best allows for the placement of TIRFM optics above the bulky microscopic stage, rather than below it as would otherwise be the necessity. Some models employ an upright microscope configuration to lower the cost of the unit – though these are sometimes the best choice due to experimental conditions as well, even where the budget does allow for a more expensive configuration. An example of such a situation is the observation of cell-substrate contacts with cells growing in monolayer culture on the bottom of a plastic petri dish. This is especially true when the observer wishes to use water immersion objectives with a dipping cone. When examining cells or membrane components attached to the upper portion of a sealed specimen chamber, however, inverted microscopes are more efficient. This is true also when the objective is utilized to illuminate and to retrieve secondary fluorescence emission from the specimen being observed.

Most TIRFM configurations, regardless of the basic design of the microscope, do employ (and rely on) an added prism to direct laser illumination toward the interface in the specimen conjugate plane, where the total internal reflection occurs. The objective can also, simultaneously, direct illumination to capture secondary fluorescence at the interface. This secondary fluorescence is produced by excited fluorophores. This discussion assumes that the example specimen is growing in a monolayer attached to a glass coverslip at the total internal reflection interface and that it consists of cells in tissue culture labelled with one or more fluorescent dyes.

TIRFM instrument configuration typically involves an inverted tissue culture microscope, external laser illumination, a trapezoidal prism block, and a photomultiplier/CCD combination detector system. The laser emits blue light which is expanded by the (aptly named) expander and then split by the beamsplitter. The beamsplitter divides the light between the beam director mirror and the microscope entrance port.

The portion of the light that is reflected from the surface of the director mirror is focused by the focusing lens onto the trapezoidal prism. The prism is positioned just above the specimen chamber and objective, on the stage.The illumination is then directed by the prism to the TIRF interface, at a slightly larger angle than would be critical for total internal reflection. This creates an evanescent field sufficient to excite the fluorophores in the specimen.

The portion of the light that is split and aimed into the microscope port can be directed through the optical train, also to the specimen, but this beam can produce widefield epi-fluorescence excitation.

The secondary fluorescence, that fluorescence that the specimen emits, is collected by the objective and passes through to one of three destinations – the eyepieces, a CCD camera system, or the photomultiplier (PMT). The top of the prism is flat, so in transmitted mode, and using a variety of contrast-enhancing techniques, illumination from the tungsten halogen lamphouse can be used to observe the specimen. The techniques used include Hoffman modulation contrast, darkfield, differential difference contrast, and phase contrast.

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