Electronic Military & Defense magazine was developed for engineers, program managers, project managers, and those involved in the design and development of electronic and electro-optic systems for military, defense, and aerospace applications.
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Techniques The Process Behind Autofluorescence The term ''autofluorescence'' is used to distinguish the intrinsic fluorescence of cells and tissues from the fluorescence obtained by treating specimens with exogenous fluorescent markers that bind cell and tissue structures. When excited with radiation of suitable wavelength, some cell and tissue components behave as endogenous fluorophores. They pass to an excited state and then decay to the ground state with loss of energy, part of which consists in fluorescence emission. Fluorescence is a type of luminescence (emission of photons or light energy). It is known that when a Figure 2: Light source with different excitation power for autofluorescence signal molecule absorbs a photon (light), the acquired energy promotes the passage of the molecule itself from the ground state to an excited state. Conversely, when a molecule emits light, the energy of the molecule decreases by an amount equal to the energy of the released photon. Although the interaction probability is greatest for single-photon absorption, if two or more lower-energy (longer-wavelength) photons arrive simultaneously, there is some probability that they can excite the molecule as long as (E1 - E0) = hc (1/λ1+ 1/λ2. . . + 1/λn) where 1/λ1 ... 1/λn are the wavelengths of individual photons. The probability of two-photon absorption is smaller than that for single-photon absorption, and the probability of three-photon absorption is much smaller. The absorption probability, however, is nonlinear and increases with the square of photon intensity (I2) for two-photon absorption and as the cube of photon intensity for three-photon absorption (I3). 20 Electronic Military & Defense ■ www.vertmarkets.com/electronics Endogenous Fluorophores The most important endogenous fluorophores are molecules widely distributed in cells and tissues, like proteins containing aromatic amino acids, NAD(P)H, flavins, and lipopigments. Plant cells and tissues contain many other fluorophores, such as chlorophylls, flavonoids, and cell wall components. The pyridine nucleotides and the flavins, which are major endogenous fluorophores emitting in the visible, play important roles in the cellular energy metabolism. Nicotinamide adenine dinucleotide NAD(P)H is a major electron acceptor in the energy metabolism pathways. The reduced form, NAD(P)H, is fluorescent and has an excitation maximum at 340 nm and emission maximum at approximately 450 nm. When NAD(P)H is bound to the proteins, the fluorescence quantum yield increases, and both the excitation and emission maxima are blue-shifted. In the case of flavins, the fluorescent form is the oxidized form, and the reduced form does not fluoresce. The excitation maxima are at 360 and 450 nm, while the emission maximum is approximately 520 nm. On the basis of the different quantum yield of these coenzymes in their oxidized and reduced forms, autofluorescence measures aimed at the monitoring of cell and tissue energy metabolism can be carried out. The aromatic amino acids tryptophan, tyrosine, and phenylalanine are fluorescent molecules. Their excitation maxima are 280, 275, and 260 nm, respectively, while the emission maxima are 350, 300, and 280 nm, respectively. Autofluorescence can rise from structural proteins, in particular collagen and elastin, which can be considered the most important fluorophores in the extracellular matrix. Several excitation and emission maxima, whose molecular origin has not been completely explored, have been observed for collagen and elastin. The fluorescence emission of these proteins is mainly due to the cross-links. Lipopigments are pigments associated with lipid oxidation products. They are generally distinguished in ceroids and lipofuscins, but the two groups are so closely related that some authors consider ceroids as an early stage of lipofuscins. Lipopigments show excitation maxima ranging from 340 to 395 nm. The emission spectrum has a minor peak at approximately 450 nm and a broad major peak centered at approximately 600 nm, which is responsible for the intense orange autofluorescence that characterizes these chromophores. Thus, monitoring of autofluorescence emitted by these molecules in environmental samples can have biodetection significance. By its nature, a bacterial spore is a dormant entity that