Total internal reflection fluorescence

Definition

Total internal reflection fluorescence (TIRF) is a sophisticated microscopy technique that exploits the phenomenon of total internal reflection to excite fluorescent molecules near a surface, allowing for high-resolution imaging of biomolecules and interactions at the nanoscale. This method significantly reduces background fluorescence from the bulk solution, leading to enhanced signal clarity and the ability to observe single molecules in real time.

5 Must Know Facts For Your Next Test

1. TIRF can achieve spatial resolution on the order of 100 nanometers, making it ideal for studying molecular interactions at cell membranes.
2. The method relies on using an incident light beam at an angle greater than the critical angle, which generates an evanescent wave that penetrates a few hundred nanometers into the sample.
3. Because TIRF selectively excites fluorophores close to the surface, it minimizes background noise from fluorescent signals coming from other regions.
4. TIRF is particularly useful for studying live cells and dynamic processes such as membrane trafficking, receptor-ligand interactions, and protein conformational changes.
5. In addition to biology, TIRF is also used in materials science and nanotechnology to investigate surface properties and interactions at the nanoscale.

Review Questions

• How does total internal reflection fluorescence improve the observation of single molecules compared to traditional fluorescence microscopy?
  • Total internal reflection fluorescence enhances single-molecule observation by significantly reducing background noise from bulk solution fluorescence. By using an incident light angle that exceeds the critical angle, TIRF creates an evanescent wave that selectively excites fluorescent molecules located very close to a surface. This results in clearer images of individual molecules or interactions without interference from signals originating further away.
• Discuss the principles of evanescent waves and their role in total internal reflection fluorescence.
  • Evanescent waves play a crucial role in total internal reflection fluorescence by facilitating the excitation of fluorescent molecules without illuminating the entire sample. When light travels from a medium with a higher refractive index to one with a lower refractive index at an angle greater than the critical angle, it reflects entirely at the interface, generating an evanescent wave. This wave extends a short distance into the lower refractive index medium and is responsible for exciting fluorophores situated near the surface, enabling high-resolution imaging.
• Evaluate how total internal reflection fluorescence can be applied in live-cell imaging and what advantages it offers over other imaging techniques.
  • Total internal reflection fluorescence is highly advantageous for live-cell imaging as it provides exceptional spatial resolution while minimizing photobleaching and photodamage. This technique allows researchers to monitor dynamic processes like protein interactions and membrane dynamics in real time with minimal interference from background signals. By focusing on only the molecules close to the cell surface, TIRF provides detailed insights into cellular mechanisms and behaviors that other imaging methods may miss due to excessive background fluorescence or lower spatial resolution.

Related terms

Fluorescence microscopy: A type of microscopy that uses fluorescence to generate an image, where fluorescent molecules are excited by light and emit light of a longer wavelength.

Evanescent wave: A decaying electromagnetic wave that occurs during total internal reflection, which can excite nearby fluorescent molecules without exciting those farther away.

Single-molecule imaging: A technique that allows researchers to observe and analyze individual molecules in real time, providing insights into molecular dynamics and interactions.