Total Internal Reflection Fluorescence Microscopy (TIRF)
Unlike confocal microscopy which employs pinholes to restrict out of focus information, TIRF microscopy improves the image signal to noise ratio by restricting penetration of the excitation light into the specimen. TIRF microscopy utilizes the properties of an induced evanescent wave of excitation to limit light penetration to the area immediately above the cover slip, roughly a tenth of that of a typical confocal section.
The evanescent wave decreases exponentially (with distance from the cover slip) in intensity and as such, the penetration depth is restricted to around 100nm. In this way, fluorophores located outside of the evanescent wave are not excited and therefore do not contribute as out of focus light. In brief, the evanescent wave is generated only when the incident light becomes totally internally reflected at the glass-media interface. This occurs when the incident light strikes the glass-media interface at a specific angle termed the critical angle. When this angle is equaled, it’s refraction direction then becomes parallel to the interface (totally internally reflected); above the critical angle, incident light is reflected completely (figure 1).
Apart from the restricted depth of excitation light, the principles of TIRF microscopy remain identical to conventional widefield microscopy. TIRF microscopy when in combination with a suitable camera can therefore be used to record processes with high temporal resolution occurring close to the plasma membrane.
Our spinning disk confocal system has been adapted to incorporate TIRF illumination, however, TIRF and confocal measurements cannot be performed at the same time.
