Modern clinical and experimental diagnostics heavily rely on molecular imaging techniques. Fluorescently labeled reporter molecules have the ability to monitor therapeutic effects, visualize specific molecular events during disease processes, and offer biological information at the molecular level in living systems. Fluorescent imaging, a crucial molecular imaging technique, has distinct advantages over other imaging tecnologies, including high sensitivity, usage of nonradioactive materials, and easy detection by less expensive instruments.
Particular chemicals known as fluorophores or fluorescent dyes undergo a phenomenon known as fluorescence (Figure 1). When a photon of energy from an external source, such as a laser or incandescent bulb, is absorbed by a fluorophore to produce an excited singlet electronic state (S1), the fluorescence process is started. Only 1 to 10 nanoseconds are spent in the excited state, but during that time, some of the energy of (S1) drains, resulting in a relaxed singlet state relative to the initial fluorescence emission.
A photon of energy is released when the fluorophore returns to the ground state ((S0). The photon's energy is lower than what was initially given and has a longer wavelength as a result of the energy that was lost during the excited-state lifetime. The emission wavelength in absorption spectrophotometry is the same as the excitation wavelength, unlike fluorescence spectroscopy. For instance, the nonfluorescent dye dabsyl (4-(4-Diethylaminophenylazo)-benzenesulfonyl) has an absorption and emission peak at 454 nm. The fluorescent AMC (7-Amino-4-methylcoumarin) dye, on the other hand, exhibits an emission peak at 450 nm and an absorption peak at 350 nm.
Figure 1. Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence (Left). The fluorescent spectrum of AMC (7-Amino-4-methylcoumarin) dye shows an absorption peak at 350 nm and an emission peak at 450 nm (Right).
There are many places in a peptide where molecular chromophors and fluorescent dyes can be inserted, including the (1) N-terminus, (2) C-terminus, and (3) side-chains of amino acids including lysine, glutamic acid, and asparagine.
One of the most common fluorescent markers for peptides is carboxyfluorescein (abbreviated FAM). When it comes to stability, FAM conjugations frequently outperform their FITC cousin. Contrary to FITC conjugates, FAM carboxamide conjugates are often more resistant to hydrolysis.
Fluorescein Excitation and Emission Spectrum
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