Total internal reflection fluorescence (or TIRF) microscopy excites fluorophores in a thin region of the specimen. This way, only fluorescent molecules that are close to the solid (usually a glass coverslip) are efficiently excited.
Breast cancer cells imaged with green fluorescent proteins. (DrimaFilm | Shutterstock)
Principles of TIRF microscopy
TIRF is a microscopy technique that is used to image fluorescent molecules, such as green fluorescent protein (GFP) and fluorochromes, in liquids that are adjacent to a solid with a high refractive index. This results in a small illumination volume, which has several advantages.
Firstly, there is a high signal-to-background ratio and almost no fluorescence collected that is out of focus. Secondly, the light exposure to the cells is much lower in TIRF microscopy. Typically, the fluorophore excitation is only 100-200 nm deep, which is around one-tenth of what is done by confocal fluorescence microscopy techniques.
Theory and method
At its core, the theory makes use of the differences in the refractive index of a liquid and a solid to create a thin electromagnetic field. The solid is usually a glass coverslip, with the cells situated in a culture below.
The electromagnetic field is called the evanescent wave. The evanescent wave is generated on the cell’s side of the coverslip and is basically what causes excitation of the fluorophores.
The evanescent wave is very thin, with its depth depending on the incident illumination angle, wavelength, as well as differences in refractive index between glass and liquid. The relationship between incident illumination angle and refraction can be described using Snell’s laws, which is central to the physics of TIRF microscopy.
When the light meets the interface between glass and liquid, the light is refracted. This causes all, or part, of the light to be confined to the medium with higher refractive index. Total internal reflection can only be achieved when the propagating light is blocked from a medium with a lower refractive index. Therefore, light does not enter the liquid media with the specimen, but penetration of reflected light across the interface creates an evanescent wave which is equal in frequency to the original light.
Total internal reflection occurs as a gradient. With increasing incident illumination angle, it approaches the critical angle which leads to increasing intensity of the reflected beam at the expense of the transmitted (i.e. refracted) beam.
All angles greater than the critical angle result in total internal reflection. Therefore, there is a continuous range going from little reflection to total reflection when the critical angle is exceeded.
What is the prism-based approach in TIRF microscopy?
The main ways to create an evanescent wave are by a prism-based approach and an objective-based approach.
Prism-based methods of TIRF microscopy involve placing a prism on the surface of the glass coverslip. This causes the laser light to be directed towards interface between the coverslip and the liquid media with the specimen.
The prism-based approach causes a larger incident angle, which leads to the evanescent wave being thin and therefore removing reflection disturbance, and collecting only fluorescence from the sample. However, the prism-based approach means the evanescent wave, and therefore excited fluorophores, is located on the other side of the sample to the objective lens. This means the lens needs to focus through the sample to pick up the fluorophores.
Prism-based TIRF microscopy is not preferred when the specimen is thick, such as tissue sections or thick mammalian cells.
What is the objective-based approach in TIRF microscopy?
The objective-based approach has the light source and the objective lens function as the same source. Because the light is delivered and collected by the same source - the lens - an inverted fluorescence microscope is needed. Such microscopes use special excitation paths to deliver the light, which can be customized for specific TIRF optics.
The preferred approach is usually objective-based. This is partly due to convenience because the specimen is easy to access and the incidence illumination angle can be easily altered. However, this approach collects the specular reflection of the incident light in addition to the fluorescence of the sample. Some systems utilize the evidence of specular reflection to verify that TIRF was attained during the microscopy.