TIRFTotal internal reflection fluorescence microscopy (TIRFM) uses the unique properties of an evanescent wave to selectively excite fluorophores within a restricted region close to the sample surface i.e. the plasma membrane of a cell. By illuminating in this way you can visualise your fluorescent proteins with high contrast, an example of this is in the image showing large dense core vesicles at the plasma membrane of a secretory cell. Total internal reflection is a phenomenon that occurs when incident lights strikes a change in refractive index (from high to low index) at an angle greater that the so-called critical angle. Under these conditions the majority of the light does not travel into the low index material but instead is reflected from this interface. However, a small component of the light travels into the low index material in the form of an evanescent wave travelling perpendicular to the interface. The evanescent wave, a so called near optical effect, decays rapidly (exponentially) as it travels into the sample, typically existing for only 100nm. The benefit is that the evanescent wave only excites molecules close to the sample surface completely restricting out of focus emission typically see in widefield epifluorescence microscopy.

TIRFM

The main advantage of TIRFM over widefield microscopy is that there is no image deconvolution requires as the out of focus light is minimal TIRFM however boasts further advantages notably that cells are able to withstand longer acquisition periods as the laser light is only illuminating a small region, this is important in super-resolution applications such as PALM, where the maximum number of molecules needs to be localised. TIRFM is also ideal for tracking objects at the base of a cell as it’s capable of very fast acquisition (one frame every 30-40ms). Shown below is a single vesicle that has been tracked at the plasma membrane, this vesicle was recorded using TIRFM and analysed using Imaris software. A similar approach can be applied to the tracking of single molecules using sptPALM. ESRIC houses an epifluorescent Olympus IX81 fully motorised microscope for TIRFM imaging. This microscope is controlled by Olympus CellR software and integrated with a Xenon mercury lamp as well as 405 nm, 488 nm, 561 nm and 633 nm diode lasers. The system also has a FRAP device for fluorescence after photobleaching experiments. Super-resolution techniques such as PALM and GDSIM are typically performed on this system under TIRFM illumination. The ability to track fast-moving single molecules in 3D using this system is currently being developed in an on-going collaboration. TIRFM can be combined with Imaris image analysis software to produce high quality images with relevant statistical information regarding your sample. Using Imaris you can also track biological constituents within your sample and plot these tracks over various timescales.