Total internal reflection fluorescence microscope

TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide.

In the 1980s, the introduction of TIRFM further decreased background and exposure time by only illuminating the thin section of the sample being examined.

There is great complexity and precision required in imaging this system which meant that the prism method was not used by many biologists but rather limited to use by physicists.

[2] In this mechanism, one can easily switch between standard widefield fluorescence and TIRF by changing the off-axis position of the beam focus at the objective's back focal plane.

Due to the fact of sub-micron surface selectivity, TIRFM has become a method of choice for single molecule detection.

Some of these applications include: With the ability to resolve individual vesicles optically and follow the dynamics of their interactions directly, TIRFM provides the capability to study the vast number of proteins involved in neurobiological processes in a manner that was not possible before.

[2] TIRFM provides several benefits over standard widefield and confocal fluorescence microscopy such as: The idea of using total internal reflection to illuminate cells contacting the surface of glass was first described by E.J.

A TIRFM uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface.

The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium.

Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells.

TIRF can also be used to observe the fluorescence of a single molecule,[6][7] making it an important tool of biophysics and quantitative biology.

Prism-based geometry was shown to generate clean evanescent wave, which exponential decay is close to theoretically predicted function.

Typically objective-based TIRFM are more popularly used, however have lowered imaging quality due to stray light noise within the evanescent wave.

An observed phenomena accompanying total internal reflection is the evanescent wave, which spatially extends away perpendicularly from the interface into medium 2, and decays exponentially, as a factor of wavelength, refractive index, and incident angle.

For complex fluoroscope microscopy techniques, lasers are the preferred light source as they are highly uniform, intense, and near-monochromatic.

In practice, a lightbox will generate a high intensity multichromatic laser, which will then be filtered to allow the desired wavelengths through to excite the sample.

In the context of TIRFM, only fluorophores close to the interface will be readily excited by the evanescent field, while those past ~100 nm will be highly attenuated.

The objective lens numerical aperture (NA) specifies the range of angles over which the system can accept or emit light.

Adjusting the distance between the objective and BPF can yield different imaging magnification, as the incident angle will become less or more steep.

The beam must also be focused at the BPF because this ensures that the light passing through the objective is collimated, interacting with the cover slip at the same angle and thus all totally internally reflecting.

[15] This maximizes the amount of exciting radiation passing through the filter and emitted fluorescence beam that is detected by the detector.

CCD cameras have photon detectors, which are thin silicon wafers, assembled into 2D arrays of light-sensitive regions.

The detector arrays capture and store image information in the form of localized electrical charge that varies with incident light intensity.

[18] Most fluorescence imaging techniques exhibit background noise due to illuminating and reconstructing large slices (in the z-direction) of the samples.

Since TIRFM uses an evanescent wave to fluoresce a thin slice of the sample, there is inherently less background noise and artifacts.

Diffraction of light on the sample slide can spread the fluorescence signal and result in blurring in the convoluted images.

Similarly, if there is a misalignment between the objective lens, filter, and detector, the excitation or emission beam may not be in focus and can cause blurring in the images.

[14] Photobleaching can occur when the covalent or noncovalent bonds in the fluorophores are destructed by the excitation light and can no longer fluoresce.

The deconvolution technique is simply using an inverse fourier transform to obtain the original fluorescence signal and remove the artifact.

To obtain better image resolution and quality, researchers have used statistical techniques to model the probability where photons may be distributed on the detector.