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Fig. 2. The transition between a higher (E2) and lower (E1) energy state of a valence electron that is associated with the absorption or emission of a photon (or quantum) of light that has the energy E = hv = E2-Er however, the situation is more complex because the electrons of many different atoms occur close together and can interact both with each other and with the nuclei of different atoms. Consequently, although there are a few major electronic singlet states (i.e., ground [S0], first excited [SJ, second excited [S2], etc.), there is also a large number of different vibrational energy levels, or substates (denoted V0, V2, etc.) within each major state. In addition, there is a parallel series of triplet energy states (denoted T0, Tp T2, etc.), each of which is at a slightly lower energy level than the corresponding singlet state. When a fluorochrome is in the dark and at ambient temperatures, the majority of molecules are in the ground state, but when it is irradiated, photon absorption typically excites most molecules to a vibrational energy level within the first excited singlet state (Sx). However, this energy is lost rapidly (in approx 10"12 s) by vibrational relaxation (a nonradiative process) so that the molecules drop to the lowest vibrational substate (V0) in the S1 band. All of the energy transitions that lead to photon emission begin at V0 in one of the excited states (S1, S2, etc.) but can terminate in any of the vibrational substates in the electronic ground state (S0). Thus, there are many different possible transitions between the various ground and excited substates, each of which could give rise to an absorption band. However, intermolecular interactions result in a broadening of the bands so that there is a broad band of wavelengths in which light can provide the energy needed to effect all of the various possible transitions. Similarly, fluorescence occurs when light is emitted at wavelengths that correspond to the energy difference between the lowest vibrational substate (V0) of the excited singlet (S1, S2 etc.) and the various vibrational substates in ground (S0) energy levels (Fig. 3).

The most likely transitions to occur during light absorption usually span a larger energy difference (AE) than those that occur during light emission. Consequently, the photon emitted that produces fluorescence is always of lower

Fig. 3. A Jablonski diagram that illustrates the electronic transitions that occur during light absorption (excitation) and emission by a fluorochrome. (1) The initial absorption of light energy raises the molecule to an electronic excited state (S1 or S2), from which energy can be lost in nonradiative ways, including (2) by vibrational relaxation during a series of transitions between substates in the electronic excited state S1, (3) by internal conversion in going from the S2 to the S1 state, or (4) by external quenching. (5) Molecules with electrons in the lowest excited singlet state (S1) can then undergo the main downward energy transition that produces fluorescence. (6) After the emission of fluorescence, there is also the possibility of energy loss by vibrational relaxation as the molecule goes through the ground substates (S0). (7) Intersystem crossing from the singlet excited state S1 to the triplet state (T1) does not result in the emission of radiation. (8) However, decay (from T1) to the electronic ground state (S0) results in phosphorescence. The various nonradiative vibrational transitions that relax S2 to S1 happen faster than the de-excitation processes between S1 and the ground state S0. The most probable upward energy transition will correspond to the energy of quanta at the peak wavelength of the absorption spectrum and the most probable downward transition to the peak wavelength of the emission spectrum. (After ref. 4.)

Stoke's shift

Wavelength (A,)

Fig. 4. The absorption (or excitation) (solid line) and emission (dotted line) spectra of a fluorochrome. The difference between the peak wavelengths of the absorption (or excitation) and emission spectra is known as the Stokes shift and always occurs in the direction of the longer wavelength (smaller energy).

Wavelength (A,)

Fig. 4. The absorption (or excitation) (solid line) and emission (dotted line) spectra of a fluorochrome. The difference between the peak wavelengths of the absorption (or excitation) and emission spectra is known as the Stokes shift and always occurs in the direction of the longer wavelength (smaller energy).

energy (and therefore longer wavelength) than that originally absorbed, as the process is less than 100% efficient and some energy is always lost in other ways, mainly by vibrational relaxation. The most likely transitions correspond to the wavelengths for maximum absorption and emission, and the difference between is them known as the Stokes shift (Fig. 4). Fluorescence techniques such as fluorescence microscopy and flow cytometry exploit this characteristic by illuminating cells at one wavelength and detecting the light that is emitted at a longer wavelength; consequently, the larger the Stokes shift, the more easily this can be done. Because the probability of an electron returning to a given energy level in the ground state after excitation is similar to it occurring in that position before excitation, the absorbance spectra (or excitation spectra) and emission spectra often overlap and are usually mirror images of one another. The probability of light absorption by a substance is measured as its molar extinction coefficient (£) (conventionally for a 10-mm lightpath) and, of course, varies with wavelength. As an example, the molar extinction coefficient of fluorescein at its wavelength of peak absorbance (495 nm) is 75,000/cm mol/L, but that for PE is 106/cm mol/L. Irrespective of the wavelength used to excite a fluorochrome, its emission spectrum is always constant, but the intensity with which it fluoresce will vary with the excitation wavelength and will reach a maximum when excitation is at the wavelength of maximum absorption. The implications of these points for flow cytometry are, first, that fluorochromes have excitation and emission spectra with quite broad peaks and, second, that they can be excited suboptimally at wavelengths away from those of their peak absorbance, a feature that can be useful in instruments with a single laser.

Light absorption by a fluorochrome is a very fast process (occurring in approx 10"15 s), but the emission of light by fluorescence is considerably slower; generally, however, the more strongly that a fluorochrome absorbs light, the faster will be the subsequent light emission (fluorescence). The intensity of the fluorescent light that can be emitted is an important characteristic of a fluorochrome because it affects the sensitivity of detection. It is proportional to the extinction coefficient and a factor known as the quantum yield (^), which is the ratio of photons (or quanta) emitted to photons (or quanta) absorbed. Numerically, values for ^ can vary from 0 to 1.0 and are a characteristic of the particular fluorochrome. The intensity of fluorescence from a fluorochrome excited in solution is defined by the expression:

F = Io X £ X [C] X x X in which F is the total amount of light emitted, the product (Io£[C]x) is the total amount of light absorbed by the fluorochrome (from Beer's law), Io is the incident light intensity, £ is the molar extinction coefficient, [C] is the molar concentration, x is the path length in centimeters, and ^ is the quantum yield.

Quantum yields are always less than 1.0 because other processes, including internal conversion, quenching of various types, and intersystem crossing, compete with fluorescence to dissipate the energy gained by light absorption. All these processes allow electrons to relax from the excited to the ground state without causing a photon to be emitted and consequently reduce the proportion of excited electrons that decays through fluorescence. During internal conversion, energy can be lost within approx 10"11 s by collision with other (e.g., solvent) molecules or through internal vibrational and rotational modes that result in the production of heat. Quenching is the loss of fluorescence that occurs when energy is transferred by a number of possible mechanisms from a fluorochrome to a nonfluorescent molecule. Most fluorochromes used in flow cytometry are aromatic compounds, which usually have fluorescence lifetimes (t) of 10"8 to 10"9 s, but almost every collision with a quenching molecule such as oxygen results in a loss of the excited singlet state and hence the abrogation of fluorescence. In general, quenching molecules are present in great excess over fluorochrome molecules and collision rates between the two can reach approx 10"8/s when the quencher is present in millimolar concentrations. Intersystem crossing occurs when the excited singlet state is converted into the excited triplet state because the excited electron has changed its spin. The excited triplet state, which is at a lower energy than the excited singlet state, can then decay to the ground state either by phosphorescence (at a longer wavelength than fluorescence) or by internal conversion. In practice, phosphorescence is rarely seen in solution because its lifetime is several seconds and the energy that could give rise to it is more readily lost by collisions with quenchers and by internal conversion. The final process that may diminish the quantum yield is photo-oxidation, which happens when an electron is transferred from a donor molecule that has been excited by absorbing light, to an acceptor molecule that in turn becomes reduced. It occurs because the transferred electron is less tightly bound to the nucleus when in its excited than in its ground state.

The key difference between chemiluminescence and fluorescence is that during chemiluminescence the substrate is irreversibly altered during the lightgenerating reaction, and consequently each molecule of the substrate produces a photon only once. In fluorescence, the process of photon absorption and emission by each molecule involved can occur many times, before the oxidation favored by the excited state results in photobleaching. For example, fluorescein isothiocyanate (FITC) will go through approx 35,000 cycles in which the fluorescence (or excited state) lifetime lasts on average 4 ns (4 X 10"9 s), whereas molecules of Hoecsht 33258 and propidium iodide (PI), when bound to DNA, can go through approx 100 and approx 200 cycles, respectively. Provided that excitation is at an optimal (or near optimal) wavelength, several hundred photons will be emitted from each cell-bound fluorochrome molecule in the time (5-50 ^s) that it takes a cell to pass through the analyzing point of a flow cytometer, but less than 20% of the total will eventually reach the appropriate detector. The intensity of fluorescence is also determined by the lifetime of the excited state (i.e., the time it takes for an excited molecule to decay to the ground state from which further cycles of excitation and, perhaps, emission are possible), which is a characteristic of each fluorochrome and its environment. Many factors that affect the molecular environment (e.g., ionic strength, pH, and solvent polarity) can markedly influence the quantum yield. Thus, it is best that the solution parameters of ionic strength and pH be kept constant and usually within the physiological range, unless they need to be altered for experimental reasons. Fluorochromes can suffer "photobleaching" under high-intensity illumination when highly reactive singlet oxygen is produced by reactions between molecular oxygen and fluorochrome molecules in the excited singlet or triplet states. Photobleaching can occur during flow cytometry, but the resultant fading of the fluorescence is not as serious a problem as in fluorescence microscopy, because the cells are usually illuminated just once, for a very short period, and the signal is collected at the same time.

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