Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.

Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope. Total internal reflection fluorescence (TIRF) microscopy (also called 'evanescent wave microscopy') provides a means to selectively excite fluorophores in an aqueous or cellular environment very near a solid surface (within Յ100 nm) without exciting fluorescence from regions farther from the surface (1). Fluorescence excitation by this thin zone of electromagnetic energy (called an 'evanescent field') results in images with very low background fluorescence, virtually no out-offocus fluorescence, and minimal exposure of cells to light at any other planes in the sample. Figure 1 shows an example of TIRF on intact living cells in culture, compared with standard epi-fluorescence. The unique features of TIRF have enabled numerous applications in biochemistry and cell biology, as follows.(a) Selective visualization of cell/substrate contact regions. TIRF can be used qualitatively to observe the position, extent, composition, and motion of contact regions, even in samples in which fluorescence elsewhere would otherwise obscure the fluorescent pattern (2). A variation of TIRF to identify cell-substrate contacts involves doping the solution 764 surrounding the cells with a nonadsorbing and nonpermeable fluorescent volume marker; focal contacts then appear relatively dark (3,4). Although TIRF cannot view deeply into thick cells, it can display with high contrast the fluorescencemarked submembrane filament structure at the substrate contact regions (5).(b) Visualization and spectroscopy of single molecule fluorescence near a surface (6)(7)(8)(9)(10)(11)(12). The purpose here is to observe the properties of individual molecules without the ensemble averaging inherent in standard spectroscopies on bulk materials, thereby enabling detection of kinetic features and states that otherwise are obscured. Related to single molecule detection is the capability of seeing fluorescence fluctuations as fluorescent molecules enter and leave the thin evanescent field region in the bulk. These fluctuations (which are visually obvious in TIRF) can be quantitatively autocorrelated in a technique called fluorescence correlation spectroscopy (FCS) to obtain kinetic information about the molecular motion (13).

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique utilizes the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within one light wavelength or less of the substrate surface. Demonstrations of this technique are given for two types of cell cultures : rat primary myotubes with acetylcholine receptors labeled by fluorescent a-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appears to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membrane topography, and visualization of reversibly bound fluorescent ligands at membrane receptors.The regions of contact between a tissue culture cell and a solid substrate are of considerable interest in cell biology. These regions are obvious anchors for cell motility (1), loci for aggregation of specific membrane proteins (2-4), and convergence points for cytoskeletal filaments (2, 5, 6). Described here is a fluorescence microscope method for selectively visualizing specific molecules in cell-substrate contact regions while avoiding fluorescence excitation of the cell interior liquid medium and cellular debris. Other potential applications of this method include viewing fluorescence-marked receptors at very low cell surface concentrations, cytoplasmic filaments in thick cells, and fluorescent agonists that bind reversibly to the cell membrane.The new method is an application of total internal reflection fluorescence (TIRF) to cellular microscopy and is an extension to fluorescence of the total internal reflection microscope illumination system introduced by Ambrose (7) to detect light scattered at cell-substrate contacts . TIRF microscopy utilizes a light beam in the substrate that is obliquely incident upon the substrate liquid interface at an angle greater than the critical angle of refraction. At this angle, the light beam is totally reflected by the interface. However, an electromagnetic field called the "evanescent wave" does penetrate into the liquid medium . The evanescent wave propagates parallel to the surface with an intensity I that decays exponentially with perpendicular distance z from the surface: THE JOURNAL OF CELL BIOLOGY " VOLUME 89 APRIL 1981 141-145 ©The Rockefeller University Press -0021-9525/81/04/0141/05 $1 .00 RAPID COMMUNICATIONS where n l = refractive index of the substrate ; n 2 = refractive index of the liquid medium; 0, = the critical angle of incidence = sin-' n2/nt ; 9 = the angle of incidence, 9 > 8,; and X = the wavelength of incident light in vacuum. The decay depth d decreases with increasing 0 . Except for 0 close to 0, (where d -;, oo), d is on the order of X or smaller . l o , the intensity of the evanescent wave at z = 0, is on the order ...

TOTAL REFLECTION FLUORESCENCE 249 for angles of incidence 0 > Oc and light wavelength in vacuum Ao. Depth d is independent of the polarization of the incident light and decreases with increasing e. Except for e � ec (where d-+ (0), d is on the order of Ao or smaller. The intensity at z = 0, 10, depends on both the incidence angle e and the incident beam polarization. lo is proportional to the square of the amplitude of the evanescent electric field E at z = 0. 1 (These expressions are given in the next subsection.) For incident electric field intensities f ll .1-with polarizations parallel and perpendicular, respectively, to the plane of incidence, the evanescent intensities Ig• .1 are Ig = f ll. 4 cos 2 e(2 sin 2 e-n 2) n4 cos 2 e+sin 2 (} _ n 2 and .1 _ J .1. 4 cos2 () 10