Application of Fluorescence Total Internal Reflection Fluorescence Microscopy
TIRFM (Total Internal Reflection Fluorescence Microscope), total internal reflection fluorescence microscope, when light enters a medium with a lower refractive index from a medium with a high refractive index, if the incident angle is large enough, all the light is reflected without refraction, but in The interface of the two media produces evanescent waves that can excite the fluorescence within 100nm near the interface to realize the observation of the object surface. The excitation light can be sent through the illuminator of a conventional fluorescence microscope or a special illuminator, and the incident angle of the laser can be controlled. The instantaneous field excitation method is used to prevent the excitation light from entering the detector. The excitation light at the interface between glass and water produces a full internal implemented by reflection. Due to the exponential attenuation of the excitation light, only the sample area very close to the total reflection surface will produce fluorescence reflection, which greatly reduces the interference of background light noise to the observation target, so this technology is widely used in the dynamic observation of cell surface substances.
Schematic diagram of total internal reflection fluorescence microscopy (TIRFM)
①Sample ②Evanescent wave range ③Cover glass ④Oil immersion ⑤Target ⑥Emission beam (signal) ⑦Excitation beam
In order to achieve total internal reflection, a large incident angle is required, for example, the incident angle at the glass-water interface is greater than 61 degrees. This can be achieved by a prism, called prism-based TIRFM, or by an objective lens with a high numerical aperture, which is called objective-type TIRFM. The current commercialized total internal reflection fluorescence microscopes are generally of the objective lens type, with high speed and high precision.
Total internal reflection fluorescence microscopy is widely used in some biological fields because it can realize fluorescence observation in a very thin range (less than 100nm) on the surface of objects. Such as the following applications:
Observation of cell surface images: cell membrane surface structure, cell surface contact, membrane surface dynamics/protein localization.
Single-molecule observation and manipulation: myosin, actin, and Cy3-labeled ATP.
Cell membrane surface movement: such as vesicle engulfment, vesicle exhalation, and vesicle exocrine. the
Observation of calcium sparking phenomenon in cell membrane, ion channel monitoring.
Molecular motor research: rotational motors, cytoskeletal proteins, polymers, G proteins, ring proteins, nucleotide motors.
In addition to the field of biology, it also has good applications in the field of chemistry for the observation of chemical molecular structures.
