What is the importance of fluorescence?
Digital Microscopy Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles, and macromolecular assemblies inside the cell.
How does total internal reflection fluorescence microscopy work?
It allows imaging of fluorescent molecules located close to the glass/water (or glass/specimen) interface. This is achieved by employing an evanescent wave for excitation of the fluorophores instead of direct illumination via light delivered by an arc lamp, LEDs or lasers.
What is TIRF used for?
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.
What is the importance of Dichromatic mirror in epifluorescence microscope?
Besides the excitation and the emission filter, a dichroic mirror is needed for this kind of fluorescence microscope. A dichroic mirror allows light of a certain wavelength to pass through, while light of other wavelengths is reflected. The filters and the dichroic mirror are often plugged in together in a filter cube.
What are the advantages and limitations of fluorescence microscope?
Table 1
| Advantages | Disadvantages |
|---|---|
| • Prolonged exposure to fluorescent light can result in bleaching and loss of fluorescence intensity | |
| • Superior image clarity over fluorescence microscopy | • Unable to produce high definition images of SUVs or oligolamellar liposomes |
| • Can provide a composite 3D image of the sample |
Total internal reflection fluorescence microscopy ( TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices.
When was total internal reflection fluorescence ( TIRF ) developed?
Total internal reflection fluorescence ( TIRF) is a special technique in fluorescence microscopy developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. TIRF microscopy delivers images with an outstandingly high axial resolution below 100 nm.
What is the physical basis of total internal reflection?
Physical Basis of TIRFM The physical phenomenon of total internal reflection (TIR) has been relied upon in such seemingly diverse applications as modern fiber optic data transmission, and in the centuries-old utilization by diamond cutters to enhance the sparkle, or “fire”, of cut gemstones.
What kind of light is used in TIRF microscopy?
It should also be stated that in TIRF microscopy high power laser light, which is stronger than the laser light usually employed in confocal systems, is used to form an evanescent wave with sufficient energy. There are two approaches for achieving total internal reflection in optics: one is prism-based and the other objective-based.