TIRF Microscopy

Total Internal Reflection Fluorescent (TIRF) Microscope

A stringent test for TIRF. A fibroblast co-expressing vinculin and myosin II regulatory light chain (both tagged with GFP) was imaged using the TIRF system. Wide field image is pseudo-colored green while TIRF image is pseudo-colored red. As shown, TIRF is capable of separating the two green-labeled proteins based solely on their relative intracellular positions. Picture by Teng-Leong Chew, NU Cell Imaging Facility.

Olympus Arc-delivered TIRFM
The facility is equipped with an arc lamp-delivered TIRF microscope from Olympus. The system is fitted with excitation and emission filters wheels, allowing users to image red, green, cyan, and yellow fluorescent emitters. 60X and 100X (N.A. 1.45) lenses are available for TIRF imaging. Unlike the N.A.1.65 100X objective lens, our 100X TIRF objective does not require special immersion oil or special coverslips. The rapid switching between TIRF and wide field is accomplished by a modified ARCEVA crescent for automated insertion/removal in the illuminating path. The system is operated on a MetaMorph-driven platform. To facilitate imaging of extreme low-light samples under TIRF mode, we have equipped the system with a Hamamatsu Electron Multiplier C9100 CCD camera. This 14-bit camera provides a gain factor of up to 2000 and maintain a speed of 30 frames per second, thus perfectly suited for high speed, ultra-dim, TIRF imaging.

How does TIRF work?
Various mechanisms are often employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. A light beam, incident on an interface with different refractive indexes for the two phases, is totally reflected if the incident angle exceeds a critical angle qc. While the light is totally reflected, a portion of the radiation exists in the distal phase called the evanescent wave. The evanescent wave will continue to travel into the medium of higher refractive index, but its strength will decay exponentially.

The TIRF system takes advantage of the evanescent wave to specifically illuminate only a range of 100-200 nm from the coverslip, thus serving as an extremely powerful tool to study cell-substrate contact and adsorption of macromolecules to surfaces. The TIRF system can focus on an optical section at least 5 times thinner than any existing confocal microscope, as fluorophores above the 200nm range will not be excited.

In general, total internal reflection illumination has potential benefits in any application requiring imaging of minute structures or single molecules in specimens having large numbers of fluorophores located outside of the optical plane of interest. Since the excitation light is completely reflected away from the detection, one can easily discriminate the fluorescence signal from the excitation light and achieve very low sensitivities and detection limits. This capability thus allows users to (1) monitor the interaction between intracellular protein and the substratum, especially in cases where the intracellular protein is of very high abundance in the cytoplasm, which will inevitably generate very high internal noise using conventional epi-illumination. (2) Another important application of TIRF is in the characterization of force exerted on the substratum during cell motility. The currently available fluorescent scopes in the facility do not allow users to accurately measure the displacement of gel-embedded fluorescent beads, due to significant light scattering problem. The TIRF system will completely eliminate this hindrance, thus allowing users to accurately calculate traction force during cell movement. (3) By varying the illumination incidence angle, and consequently the penetration depth of the evanescent wave, fluorophores can be distinguished by depth on a nanometric scale. By rapid variation in the evanescent field depth, target vesicles or other structures can be tracked at different depths and their positions accurately determined.

It is important to note that TIRF does not work well with fixed and mounted samples. Most mounting agent seek to generate a refractive index close to that of the coverslips. While this works for other fluorescent microscopes, it actually hinders the occurrence of total internal reflection. The system is designed, therefore, to work exclusively with aqueous samples.