Final answer:
The excitation and emission spectra of fluorescent labels determine the wavelengths of light absorbed and emitted, key characteristics used in labeling antibodies for fluorescence microscopy.
Aromatic amino acids like tryptophan and tyrosine have distinct peak wavelengths and Stokes shifts, aiding in the identification and visualization of antigens. Synchronous spectroscopy is a specific method that utilizes these properties for sensitive detection of fluorophores.
Step-by-step explanation:
The excitation and emission spectra are critical characteristics of fluorescent labels used on antibodies. These spectra represent the wavelengths of light absorbed (excitation) and emitted (emission) by fluorescent molecules.
When a molecule is excited by absorbing light of a specific wavelength, it then emits light of a different, typically longer, wavelength as it returns to its ground state.
For example, the aromatic amino acid tryptophan typically shows a broad emission peak around 350 nm after excitation, whereas tyrosine has a narrower emission peak near 305 nm.
The difference in wavelengths between excitation and emission, known as the Stokes shift, is indicative of how much energy is lost before light is emitted; a larger Stokes shift means more energy is lost.
In a direct fluorescent antibody test, the fluorescent antibody specifically binds to an antigen of interest, which can be a protein, virus, or a bacterial cell, depending on the test's purpose. By attaching fluorescent labels to antibodies, researchers can visualize the location of these antigens under a fluorescence microscope.
The molecules' ability to fluoresce varies in intensity, which can be ranked based on their quantum yield and spectra characteristics. A fluorescing molecule like tryptophan will lose more energy from excitation to emission compared to tyrosine, shown by their respective differences in peak wavelengths.
Synchronous spectroscopy is a technique that takes advantage of these characteristics for sensitive detection of specific fluorophores by using a set difference between excitation and emission wavelengths.