Report
APPLICATIONSOFFLUORESCENCE RECOVERY AFTER PHOTOBLEACHING Fluorescence recovery after photobleaching (FRAP) is a technique for measuring the diffusion coefficients of fluorescent molecules, revealing transport information for a wide variety of systems. FRAP has been used by analytical chemists to study the diffusion of fluorophors in polymeric matrices, adsorbates on reversed-phase chromatographic surfaces, proteins, and other probes in biological membranes. The technique is based on the high sensitivity of fluorescence spectroscopy which enables the measurement of molecular transport of dilute fluorophors that minimally perturb the media FRAP relies on photobleaching, which is the destruction of absorbing molecules upon irradiation with an intense burst of light The photobleached region of the sample initially exhibits weaker fluorescence because of the lower concentration of absorbers. In time, the area shows a recovery of the fluorescence as unbleached molecules diffuse into the irradiated region. The diffusion coefficients of the absorbers can then be calculated from the rate of recovery of the fluorescence signal. For nearly spherical molecules of radius r in homogeneous media, the StokesEinstein Law relates the diffusion coefficient D to the viscosity T| by the relationship
Determining the diffusion coefficients offluorescentmolecules provides details on molecular-scale structure. geneous solvents of low viscosity in which the solvent molecules are much smaller than the solute of interest. However, for media that are heterogeneous on the molecular scale, such as chemical interfaces and polymeric networks, the diffusion coefficient is sensitive to the molecular-scale structure of the medium rather than to bulk viscosity. Obtaining information about this molecular-scale structure is typically the goal of F RAP measurements. Luis Report describes the evolution, experimental design, and analytical applications of FRAP to heterogeneous media. Development of FRAP
FRAP was invented to investigate how membrane surface proteins move at bioD = kT/6Ttr\r (1) logical interfaces. Lateral movement tiirough membranes had been postulated This relationship applies well in homo- to assist in the transmission of signals by cell receptors, and confirmation of this motion was expected to provide insight into the functional properties of the cell John M. Kovaleski surface (1). Photobleaching techniques M a r y J . Wirth University of Delaware were developed to study this two-dimen600 A
Analytical Chemistry News & Features, October 1, 1997
sional transport, or lateral diffusion. However, the FRAP experiments can be used to study transport at many interfaces because they directly probe the desired sample using a carefully chosen fluorescent probe that minimally perturbs the medium. The first photobleaching experiments measured the diffusion of fluorescencelabeled proteins in micrometer-sized biological cells (2-4). One experiment tagged the integral membrane proteins of human erythrocytes (red blood cells) with fluorescein isothiocyanate (4). After isolating an individual cell under a microscope, one half of the erythrocyte was photobleached. The cell was then passed into a beam of lower intensity, in which fluorescence ws.s monitored as a function of time for both sides of the cell. As the probe molecules redistributed throutrh the membrane the fluorescence intensity increased on the bleached side and decreased on the unbleached side until the cell returned to uniform
The results showed only a slight change in the fluorescence on each side, even after 20 min. Analysis of the data revealed a diffusion coefficient of 3 x 10"22 cm2/s, which is orders of magnitude slower than diffusion in fluid solution. This study confirmed the prediction that erythrocyte membranes are very rigid because of their high saturated fatty acid content. Studies of a wider range of systems could not be undertaken, however, because the experiment entailed moving the sample to different positions, and any reS0003-2700(97)09027-6 CCC: $14.00 © 1997 American Chemical Society
covery that occurred during the repositioning could not be monitored, which prevented the study of rapidly diffusing molecules. A new experimental design in which a spot of known diameter co is bleached and the fluorescence recovery in the bleached spot is continuously monitored using a lower beam intensity was introduced (57). As long as the intensity profile of the laser beam is Gaussian, the diffusion coefficient can be calculated ffom the ttme T required for the fluorescence to recover one-half of its final intensity T1/2= (w /4£>)Y
(2)
in which y depends on the initial amount of fluorophor bleached. The spot-bleaching technique has several advantages over the previous ap-
proach. First, it is a general method that can be applied to both lateral (5, 7) and bulk diffusion (6). Moreover, this approach can measure faster diffusion coefficients because it provides a constant sample position. The data are collected continuously, increasing the number of data points, which gives a more accurate determination of the diffusion coefficient through better curve fitting. Finally, the data allow the determination of the fraction of the probe that is mobile (6), an important consideration for heterogemedia The simplicity of spot photobleaching is alluring, but this method, too, is limited because y must be known accurately to determine the diffusion coefficient. To eliminate y from the diffusion equation, a periodic pattern for bleaching is used instead of a simple spot (8).
Figure 1 illustrates alternating illuminated and dark stripes imaged onto a sample, with photobleaching occurring in the bright stripes. Photobleaching thus imposes a transient periodic pattern in the ground-state concentration. A weak probe beam with the same periodic pattern then excites the fluorescence, and the fluorescence signal recovers as molecules diffuse from dark to illuminated regions. The functionality of the recovery is described by a single exponential with time constant x from which the diffusion coefficient is calculated (8) by x = d /(An D)
(3)
The parameter d is the spacing of the periodic pattern, which, as it changes, varies the recovery time constant for a given diffusion coefficient. In addition to
Analytical Chemistry News & Features, October 1, 1997 6 0 1 A
Report
Figure 1. Images of a periodic pattern projected onto a sample (a) before bleaching, (b) after bleaching, and (c) after complete fluorescence recovery by the diffusing molecules.
neglecting the amount initially bleached, this method allows easy variation of the timescale of the experiment via changes in the spacings of the periodic pattern, thereby accommodating a wide range of diffusion coefficients. Experimental requirements for F R A P
Several criteria must be met to conduct FRAP experiments successfully. The first is selection of a fluorescent probe compatible with the system being studied; the probe and its photobleached product should not perturb the structure or viscosity of the medium. This criterion is tested by verifying that D is independent of the probe concentration. One example of a compatible probe is an amphophilic fluorophor with a polar head group and a long hydrocarbon tail for probing biological membranes (8, 9). These molecules orient themselves parallel to lipids and, ,hus, minimally disturb the membrane. The probe must also undergo irreversible photobleaching on the timescale of the diffusion constant. Reversibility can be tolerated if the timescale is long compared with d/D. However, irreversible photobleaching ensures that the fluorescence recovery is due only to lateral diffusion across the pattern. For pattern photobleaching, the experimental apparatus must have a method of generating bleaching and probe laser beams with the appropriate pattern. Currently, there are two methods for creating 602 A
the periodic pattern. Figure 2a shows a Ronchi ruling, which consists of alternating opaque and transparent lines placed on an optically clear substrate. The imaged grating spacing can be varied by using different grating spacings in a collimated beam (10-12). Alternatively, the entire image can be focused to a known, smaller size using a microscope objective (8, 9,13-15). This method allows smaller regions to be probed. Another more versatile memod of generating the periodic pattern relies on holographic imaging (16-19) (Figure 2b) )n which two laser beams are crossed directly in the sample, thereby creating an interference pattern of known spacing. The size of the fringe spacing, again designated as d, ,s related to the crossing half-angle 6e of the beams and to the excitation wavelength A.e by the Bragg equation d = A,e/(2sin6e)
(4)
The angle is adjusted by changing the position of the movable mirror, as shown in Figure 2b. As a result, the fringe spacing can be continuously changed to any desired value simply by varying the angle at which the beams intersect. This method has generated grating spacings that vary more than two orders of magnitude without changes in optical setup (19), and has allowed the determination of diffusion coefficients spanning more than nine orders of magnitude (19). This wide range of diffusion coefficients is a valuable as-
Analytical Chemistry News & Features, October 1, 1997
set to the analyst for the study of polymeric matrices on both sides of the glass transition temperature. Regardless of the method of generating the pattern, it is essential that the patterns on the bleaching and probe beams are coincident with one another. One way to ensure coincidence is to use the same beam and to change its intensity for bleaching and probing by varying the polarization of the beam electronically with a Pockels cell (10). Thus, when the beam passes through a fixed polarizer, its intensity depends on the voltage applied to the Pockels cell. The single-beam technique affords full electronic control of the data acquisition process and requires fewer optics. Another way to ensure coincidence is to split the laser beam into intense and weak beams and recombine them at a beam splitter. In the space where the beams are separated, a shutter is placed in the intense beam to prevent photobleaching during probing (13,17). The Ronchi ruling is placed in the beam either before it is split or after the beams are recombined. For holographic imaging, the bleaching and probe beams should be recombined before being sent to a second beam splitter, which is needed to create the grating (17). Another experimental consideration is that phenomena other than diffusion can erase the photobleached pattern. For example, convection causes the pattern to translate through the solution, creating oscilla-
tions in the recovery curve (10). Uniform sample temperature combined with minimal photobleaching of the sample reduces this convection. Moreover, desorption and readsorption of the fluorophor at chemical interfaces can also lead to the recovery of the photobleached pattern. Additionally, the probe can undergo reversible photobleaching (19), such as intersystem crossing or a conformational change. To determine if these unwanted effects are occurring, the diffusion coefficient of the system should be measured with at least two different grating spacings. This confirms that the recovery is described by Eq. 3. Recovery curves using two different grating spacings for a system are shown in Figure 3. Both recovery curvesfitwell to Eq. 4 with the same value for the diffusion coefficient, illustrating the dependence of the time constant on the grating spacing as well as verifying that lateral diffusion is responsible for the fluores-
Using FRAP
FRAP has been used as an optical probe for studying the curing of epoxy resins (20). To accomplish this, a small amount offluorescentdye is dissolved into the resin. As the resin cures through polymerization, cross-linking obstructs the path of the dye molecules and thereby lengthens the path of diffusion. FRAP can then be
used to measure the diffusion coefficient of the dye at progressive stages of the curing process, provided that the epoxy cures on a much longer timescale than that of the diffusion measurement. The rate of curing, an important parameter for characterizing the performance of epoxy resins, is determined from the slope of the diffusion coefficient with respect to time. Monitoring the cure rate at various temperatures provides valuable quality control data for the manufacturing process. FRAP has also provided insight into phenomena underlying chemical separations such as the structure of chemically selective media in chromatography, which is typically of microscopic or nanoscopic dimensions. Diffusion through a gel filtration medium limits the speed of polymerbased size-exclusion separations. Typically, macromolecular diffusion is slowed by the cross-linked gel structure, which obstructs movement. Cross-linked polymers can be approximated mathematically as a network of linked fibers which is predicted to dethe diffusion coefficient of a spherical macromolecule of radius r according to the following relationship (21) D/D0 =Aexp(r/B)
(5)
in which D/D0 is the ratio of diffusion coefficients ii the gel and in free solution, and A and B depend on the concentration of fi-
bers. The increase in D/D0 with r arises in theory from larger macromolecules being required to diffuse around more barriers in the polymeric matrix. FRAP was used to test the applicability of Eq. 5 for a set of globular proteins (ribonuclease, chymotrypsinogen, albumin, thyroglobulin, and aldolase) being separated on individual beads of polyacrylamide gel (22). The relationship between protein radius and D/D0 was found to agree well with Eq. 5, indicating that obstruction by the cross-linked gel matrix controls diffusion in this sievelike structure. However further diffusion studies with these proteins in agarose gel revealed that the diffusion coefficients much less than what would be predicted by Eq 5 suggesting a significant difference in the microscopic
