Fluorescence Recovery After Photobleaching (FRAP)

Abstract

Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscope technique to measure molecular diffusion and transport. FRAP is a valuable technique in cell biological research and evolved conjointly with microscope and fluorescent probes advancements. Although developed in the 1970s, the discovery and further development of fluorescent proteins revolutionised FRAP. After the discovery of green fluorescence protein and its application as a noninvasive and genetically coded protein‐tag, in vivo studies of protein dynamics and interactions became possible. FRAP is based on irreversibly bleaching a pool of fluorescent probes and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot. Although measurements are straightforward, quantitative FRAP requires careful experimental design, solid controls, data collection, and analysis. Over the past years, several FRAP‐related techniques have been tailored to suit particular cell biological questions, including inverse FRAP, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching.

Key Concepts:

  • Fluorescence recovery after photobleaching (FRAP) is a method to qualitatively and quantitatively study biomolecule dynamics in living cells.

  • FRAP is based on irreversibly bleaching a pool of fluorescent probes with high intensity light and monitoring the recovery in fluorescence due to movement of surrounding intact probes into the bleached spot.

  • FRAP experiments are often conducted on confocal microscopes. To derive quantitative results from such experiments, several parameters and controls need to be considered and utilised in the analysis.

  • There are several FRAP‐related methods that have been developed for specific applications and biological questions.

  • FRAP is a versatile and popular method in modern biomedical research. Its application is broad and is increasingly applied in pharmacological, therapeutic and diagnostic areas.

Keywords: FRAP; photobleaching; fluorescence; diffusion; protein dynamics; protein interaction; microscopy; confocal microscopy; fluorescent protein; GFP

Figure 1.

Fluorescence principle. (a) Classical Bohr model: Absorption of a light quantum (blue) causes an electron to move to a higher energy orbit. After residing in this ‘excited state’ for a particular time, the fluorescence lifetime, the electron falls back to its original orbit and the fluorochrome dissipates the excess energy by emitting a photon (green). (b) Jabłoński diagram: When a quantum of light (a single photon) is absorbed, the electron moves from the ground state GS0 (electronic singlet) to a higher excited state (1), relaxes quickly to a lower vibrational excited state (2) and thereby loses energy. When returning to the ground state it dissipates the remaining energy by emitting a photon with a longer wavelength (3), that is, fluorescence emission. (4) Nonfluorescent energy loss via, for example, collision with solvent molecules. (5) Intersystem crossing and delayed fluorescence. (6) Photobleaching through oxidising species formation (ROS: reactive oxygen species). (c) Stokes shift exemplified for the fluorescent fatty acid cis‐parinaric acid. Modified from Ishikawa‐Ankerhold HC et al. (). © MDPI.

Figure 2.

Schematic representation of a FRAP experiment. (a) A region of interest (ROI) is selected, bleached with an intense laser beam, and the fluorescence recovery in the ROI is measured over time. Below: actual experiment in a myoblast cell line (myo3) homogenously expressing GFP‐Myosin III before bleaching. (b) From the initial (pre‐bleach) fluorescence intensity (Ii), the signal drops to a particular low value (I0) as the high‐intensity laser beam bleaches fluorochromes in the ROI. Over time, the signal recovers from the post‐bleach intensity (I0) to a maximal plateau value (I). From this plot and eqn (2) and eqn (3), the mobile fraction (Mf), immobile fraction (IMf), and I½ can be calculated. Light blue line: reference photobleaching curve to correct for fluorescence loss during data acquisition. The information from the recovery curve (from I0 to I) can be used to determine the diffusion constant and the binding dynamics of fluorescently labelled proteins. A more absolute way of obtaining the half‐life and immobile/mobile fractions, which is also suitable for automation, is through nonlinear curve fitting of the experimental data points using the simple exponential eqn (4). (c) The curve directly shows the extent of the mobility observed. Modified from Ishikawa‐Ankerhold HC et al. (). © MDPI.

Figure 3.

Example of ROI selection and intensity correction in a FRAP experiment in Dictyostelium discoideum cells expressing GFP‐cofilin (actin‐binding protein). (a) A region of interest ROI1 (circle) in the cytosol is bleached with high‐intensity laser light. After bleaching, the cell shows a dark area where the fluorochromes were irreversibly damaged. The fluorescence in the photobleached region is monitored and recovers through replacement with intact fluorochromes from the surrounding area. Note that the total amount of fluorescence has decreased during monitoring (ROI2), because a subfraction of fluorochromes were irreversibly damaged by bleaching. ROI2 (dotted line) represents the total fluorescence intensity in the cell during monitoring, and ROI3 (rectangle) is used for measuring the background intensity. (b) Intensity profiles in the various ROIs.

Figure 4.

FRAP‐related methods. (a) FRAP: A region of interest (ROI) is selected, bleached with an intense laser beam, and the fluorescence recovery in the ROI is measured over time. (b) iFRAP: A ROI is selected to remain intact, while the rest of the cell is bleached. This is particularly useful when studying dynamic movement in organelles, such as the nucleus. (c) FLIP: Repetitive bleaching of a selected ROI during the entire monitoring period and the fluorescence intensity in regions outside the selected bleached area is measured. The decline in fluorescence intensity in the surrounding regions is due to bleaching of fluorochromes that move through the ROI during the repetitive bleaching process. The drop in fluorescence intensity outside the bleached region is caused by a steadily increasing population of bleached, nonfluorescent molecules within the cell and thus provides quantitative data on their molecular mobility. (d) FLAP: A protein is tagged with two fluorescent labels: one is photobleached and the other acts as a reference. The use of a reference fluorochrome allows the tracking of the distribution of the labelled molecules by simple image differencing (DI) and thus enables measurement of fast relocation dynamics.

Figure 5.

Example of a FLIP experiment. (a) ROI selection and monitoring in a FLIP experiment in Dictyostelium discoideum cells expressing GFP‐cofilin (actin‐binding protein). A region of interest ROI1 (circle) is bleached repetitively with high‐intensity laser light. The fluorescence in the region outside the photobleached area (ROI2) is monitored and conclusions on compartment interconnectivity or immobile proteins can be drawn from the general loss in fluorescence and in those areas that remain unaffected. Corrections are made by evaluating the fluorescence intensity in a control cell (ROI4),and ROI3 and ROI5 (rectangles) are used for determining the background. (b) FLIP curve. From the initial (pre‐bleach) fluorescence intensity (Ii), the signal drops exponentially to a minimal plateau value (I), because mobile fluorochromes enter ROI1as the laser beam bleaches ROI1. From this plot and eqn (10) and eqn (11), the mobile fraction (Mf), immobile fraction (IMf) and I½ can be calculated.

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Further Reading

Müller‐Taubenberger A and Ishikawa‐Ankerhold HC (2013) Fluorescent reporters and methods to analyze fluorescent signals. Methods in Molecular Biology 983: 93–112.

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Ishikawa‐Ankerhold, Hellen, Ankerhold, Richard, and Drummen, Gregor(Oct 2014) Fluorescence Recovery After Photobleaching (FRAP). In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003114]