## Interactions Between Light and Matter

Light can be regarded as a form of electromagnetic radiation that travels in waves and that can be defined by electric and magnetic vectors that are at right angles both to each other and to the direction of propagation. The relationship between the frequency and wavelength (the distance between wave crests) of the radiation is then expressed by a simple equation:

X X v = c (in which X is the wavelength in meters, v is the frequency in cycles/s, and c is the velocity in meters per second, approx 3 X 108 m/s, the "speed of light").

However, only light that has a frequency between approx 0.4 X 1015 Hz and 0.75 X 1015 Hz (a Hz, or hertz, is one cycle per second) or, alternatively, that has a wavelength between approx 400 nm (near UV) and 700 nm (red) is visible to the human eye. Although for many purposes it is convenient to consider light as a continuous long wave, it really comprises discrete short "wave trains," called "photons," that can also be considered as particles that have electromagnetic energy, but no mass. The electric and magnetic vectors of the wave arise from the force field that surrounds a pair of charges, the oscillating electric dipole, which can be considered as a positive and a negative charge oscillating toward and away from each other so that their separation distance is a sine wave function. The oscillating dipole, therefore, generates a moving wavelike force field that radiates out in all directions with the electric vector in the same plane as the axis of the dipole to produce a single wave train. The process, from most sources, lasts for only approx 10"9 s, and as the force field travels at the speed of light, it would travel approx 3 X 108 X 10"9 m, or 0.3 m, in that time (Fig. 1).

The concept of the oscillating dipole is useful when considering the movement of electrons in atoms and molecules and their interaction with light, because it corresponds to the relative movements of the electrons (negatively charged) and nuclei (positively charged) in matter. To cause a dipole to oscillate, work must be done, but a corresponding (or smaller) amount of energy can then be radiated as light, which if absorbed by other dipoles, can also start them oscillating, so that, in turn, they can also radiate. It is the exchange of energy between oscillating dipoles and the radiation field that governs the interactions between light and matter. The emission of light from matter (e.g., a metal) heated to high temperature is termed incandescence, but light emitted without recourse to heating is known as luminescence, a term that encompasses chemiluminescence (or bioluminescence if it occurs in vivo) and two forms of photoluminescence, which are fluorescence and phosphorescence. Chemiluminescence results when the enthalpy (heat) of a chemical reaction raises an electron in an atom or molecule to a vibrationally excited state, and light is emitted when the electron decays to its ground state. In contrast, photoluminescence results when the energy acquired from the absorption of light (typically, the UV, visible, or near-infrared) causes an electron to be raised to a higher energy state in an unoccupied orbital and (as in chemiluminescence) its return to the ground state is accompanied by light emission. A feature of the exchange of energy between light and matter is that it can occur only in discrete quantities (or packets) (Fig. 2) known as quanta or photons, the energy of which is related to the frequency of the radiation by the Planck-Einstein equation:

Table 1

Examples of the Cellular Parameters That Can Be Measured by Flow Cytometry

Parameter

Huorochromes and/or techniques used"

### Antigens

Presence on cell surface or internally Proximity of different antigens Apoptosis /necrosis / viability Cell division (generation number) DNA Content Base ratio Synthesis

Breakdown (e.g., during apoptosis) Nucleotide sequence Enzyme activity GlutationeIsulphydryl groups Intracellular ion concentrations PH Ca2+

Many fluorocliromes (e.g., fluorescein, phycoerythrin, or QdotsĀ®) can be conjugated to specific antibodies

Fluorescence resonance energy transfer between different fluoroclirome-labeled antibodies

See DNA content, Membrane asymmetry, integrity/permeability, lipid packing, potential Lipophilic dyes, or CFDA-SE, which binds covalently to labeled cells

DAPI, DRAQ5, Hoechst dyes, propidium iodide

Dyes that bind preferentially to A-T or G-C pairs (e.g., chromomycin A and Hoechst 33258)

Incorporation of BrUdR detected with fluorescent anti-BrUdR antibodies

Incorporation of labeled nucleotides at strand breaks (TUNEL)

Fluorescent-labeled oligonucleotides

Huorogenic substrate analogs

Fluorescent bimanes

"Ratio" dyes (e.g., BCECF, SNARF-1) Fluo-3 and "ratio" dyes (e.g., INDO-1)

Membrane

Asymmetry Integrity /permeability

Lipid packing Potential Net charge Receptors Oxidative metabolism Phagocytosis/endocytosis Pinocytosis

Reporter gene expression RNA content

Huorochrome-labeled annexin V

Huorescein diacetate, and nonpermeant DNA dyes (e.g., ethidium bromide, propidium iodide) Merocyanine 540 Rhodamine 123, JC1 Huorescent polyanions

Huorochrome (e.g., fluorescein)-conjugated ligands

Dichlorofluorescein

Huorescent low-molecular weight dextran

### Green fluorescent protein

Acridine orange, pyroninY, thiazole orange aBCECF, 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein; BrUdR, bromodeoxyuridine; CFDA-SE, 5(6)-carboxyfluorescein diacetate succidimyl ester; DAPI, 4', 6-diamidino-2-phenylindole; SNARF-1, carboxy-seminaphthorhodafluor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Fig. 1. The generation of electromagnetic radiation from an oscillating dipole. The dipole (shown by the vertical line with arrows and charge symbols at the left) generates an electromagnetic wave with a magnetic vector (H) in the plane YZ that is at right angles to, and in phase with, the electric vector (E) in the plane XZ. The wavelength (X) is maintained as the wave moves in the direction Z, but its amplitude decreases as the distance from its origin increases.

Fig. 1. The generation of electromagnetic radiation from an oscillating dipole. The dipole (shown by the vertical line with arrows and charge symbols at the left) generates an electromagnetic wave with a magnetic vector (H) in the plane YZ that is at right angles to, and in phase with, the electric vector (E) in the plane XZ. The wavelength (X) is maintained as the wave moves in the direction Z, but its amplitude decreases as the distance from its origin increases.

E = h X v, in which E is the energy (in Joules) in one quantum of radiation that has a frequency of v (cycles/s), and h is Planck's constant, 6.626 X 10-34 J-s (i.e., Joule-seconds); for light in a vacuum, E = h X (c/X) (because v = c/X, from above).

Blue-green light with a wavelength of 488 nm has a frequency of 3 X 108 m/s divided by 488 X 10"9 m, or 6.148 X 1014/s. Consequently, the energy (in Joules) of a single photon/quantum of blue-green light with a wavelength of 488 nm is 6.626 X 10-34 J-s X 6.148 X 1014/s, or 4.07 X 10-19 J. A 488-nm laser operating at 15 mW will, therefore, produce approx 3.68 X 1016 photons or quanta per second (because 1 W is equivalent to 1 J/s). Also, as a cell is illuminated for between 5 X 10"6 to 5 X 10"5 s when it passes through the analyzing beam of a flow cytometer, depending on the ratio of the cross-sectional areas of the cell and beam, it will receive approximately 1010-10n photons or quanta.