Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.

The method of fluorescence recovery after photobleaching has been used to measure the temperature dependence of the lateral diffusion coefficients (D) of two fluorescent lipid analogues in phospholipid multibilayers of various compositions. The probes employed were 3,3-dioctadecyloxocarbocyanine (diO-C18(3) and N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE). In fluid egg phosphatidylcholine multibilayers at 25 degrees C, D was about 4 X 10(-8) cm2/s for NBD-PE and 1.5 X 10(-7) cm2/s for diO-C18(3) and was moderately temperature dependent (2-fold change over 10 degrees C). Equimolar cholesterol reduced D for NBD-PE in these multibilayers by a factor of 2. A greater than 100-fold decrease in D was detected in dimyristoylphosphatidylcholine multibilayers at approximately 23 degree C, which coincides with the gel-to-liquid-crystalline transition temperature, Tm (D 5 X 10(-8) cm2/s at T greater than Tm to D less than 5 X 10(-10) cm2/s at T less than Tm). Equimolar cholesterol abolished this transition behavior, raising D below Tm and decreasing D above Tm. These results confirm and extend previous studies of lateral diffusion employing magnetic resonance and other optical techniques and give additional confidence in the fluorescence methods.

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGS-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 X 10(-11) to 2 X 10(-10) cm2/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.

The diffusion of macromolecules introduced into the cytoplasm of human fibroblasts by erythrocyte-mediated microinjection was measured by the fluorescence recovery after photobleaching technique. The apparent diffusion coefficients for fluorescein-labeled IgG and fluorescein-labeled bovine serum albumin were ==10-8 cm /sec at 22°C, consistent with the kinetics of spreading of the fluorescent probe following microinjection and -1/70 the values in aqueous buffer. The diffusion of labeled bovine serum albumin was shown to be strongly dependent on temperature and, in fact, similar to that expected in a 61% aqueous sucrose solution. However, the marked reduction in diffusion at 5°C could be fully reversed by incubation with 0.1 mM colchicine.These findings suggest that cytoplasmic. diffusion rates are reduced relative to rates in aqueous media as a result of increased aqueous phase viscosity or the impedence provided by structural elements. Several simple models to account for the data are presented.The organization ofthe cytoplasm is a relatively unexplored area of modern cell biology. Measurement of the translational diffusion of probe molecules placed within the cytoplasm should offer information concerning the resistance to diffusion that the cytoplasm provides, as well as indicate the rates at which various components are transported within the cell. Thus far, the rotational diffusion of small probe molecules has been measured by spin-label techniques (for review, see ref. 1) and by fluorescence polarization (2, 3). Cytoplasmic diffusion rates have been calculated for a variety of radiolabeled probes following pipette microinjection, fixation, and autoradiography (4). However, translational diffusion coefficients of molecules in the cytoplasm ofliving cells have not been measured. In the last several years, biophysical and biological techniques have been developed that make this measurement feasible.We have introduced fluorescein-labeled macromolecules into the cytoplasm ofliving human fibroblasts by using the technique oferythrocyte-mediated microinjection (5) and then measured the apparent translational diffusion of these probe molecules within single cells by using the fluorescence recovery after photobleaching (FRAP) method (6, 7). The impedence provided by cytoskeletal and membraneous structures within the cell, superimposed on the intrinsic viscosity of the cytoplasmic aqueous phase, might be expected to reduce the translational mobility from the values measured in simple aqueous solutions. Such is the case as demonstrated here, illustrating that these transport measurements can be used to provide insight into the structure of the cytoplasm. MATERIALS AND METHODS

Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported . These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton . On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) -3 x 10-9 cm'/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane . In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D :Z 10-10 cm2/s with substantial fractions virtually nondiffusible (D < 10-' 2 cm2/s) . Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction . This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane .The lateral diffusion of plasma membrane components has become routinely accessible to measurement by fluorescence photobleaching recovery (FPR) (1) . Taken together, data accumulated for vertebrate cells in culture establish several distinctive common characteristics of lateral diffusibility of cell surface components (2--6) : (a) A substantial fraction of the population of each cell surface protein is not detectably diffusible over distances of several micrometers during an experimental interval ofhours . Thus the diffusible fraction (R) is said to be <100%, and it is often <75%. (b) Diffusion coefficients (D) of the diffusible fraction ofcell membrane proteins do not generally exceed 1 x 10-1°cm 2/s and are frequently much smaller. (Implied diffusive transit time across a 10-8m cell: 1/2 h.) (c) Plasma membrane lipid diffusibility is virtually complete over distances of several micrometers (R = 100%, except possibly in a few compartmentalized cell types) with diffusion coefficients of the order of 1 x 10-e cm2/s, i.e., usually >100 times faster than membrane proteins. (Implied diffusive transit time across a 10-N.m cell: 1/2 min.)Certain proteins are virtually nondiffusible in toto (R = 0%) on the cell surface. For example, the acetylcholine receptor (AChR) localized at neuromuscular junctions and in patches on myotubes (7,8), and frbronectin on the surface of cultured fibroblasts (9) .In contrast with the cell surface, protein molecules that are reconstituted into phospholipid model membranes diffuse nearly as fast as lipid molecules and with no immobile fraction.