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Introduction

Fluorescence recovery after photobleaching (FRAP) is an imaging technique for the investigation of molecular dynamics and cellular events within living cells. The FRAP elucidates the dynamics and kinetics of the protein-protein interaction and cellular signaling. In a FRAP experiment, the laser beam is focused on a small area of cell membrane labeled with a fluorescent probe, and then the fluorescence intensity excited by the beam is observed as a function of time. For a moment, the optical attenuator is removed to bleach some of the fluorescent probe molecules in the region of interest. This reduces the fluorescence intensity by the bleaching pulse, but the intensity recovers through the diffusion of unbleached molecules from the surrounding. FRAP studies yield qualitative data that provides with an insight into the binding characteristics, relative binding affinity, and the effects of pharmaceutical agents or mutations on protein mobility. Improvements in fluorophores and labeling techniques, and the identification of fluorescent proteins such as green fluorescent protein (GFP), have further advanced the use of FRAP in biomedical science (Carisey., et al., 2011).

Principle

The underlying principle of the fluorescence recovery after photobleaching involves the discontinuation of fluorescence caused by the conversion of the fluorophore to a chemically non-fluorescent compound. Photobleaching needs an incident light and molecular oxygen for most fluorophores. Initially, a series of fluorescence intensity images are collected to record the value for intensity in both the region of interest and the surrounding. Following this, a small area of interest is illuminated with high-intensity light bleaching the fluorophore within that region to create a darker, bleached region in the sample. Photobleached molecules are replaced with nonbleached molecules, diffused from the surrounding, increasing in the fluorescence intensity of the bleached region. FRAP is a quantitative fluorescence technique that is used to measure the dynamics of molecular mobility two-dimensionally by utilizing the irreversibly bleached fluorophores (Bizzarri et al., 2012).

Apparatus

The fluorescence recovery after photobleaching (FRAP) equipment consists of a fluorescence microscope equipped with light sources, filter sets, the photobleaching light source, and fluorescent probes. The fluorescence emission is dependent on the absorption of a specific wavelength restricting the choice of lamps. For this, broad-spectrum mercury or xenon source with a color filter is used. A background image of the sample is captured before photobleaching. Then, the light source is focused onto a small area with laser light of the appropriate wavelength. The fluorescent molecules in this region receive high-intensity illumination which causes their fluorescence lifetime to elapse quickly. This makes the background image of a uniformly fluorescent field with a prominent dark spot. As the diffusion proceeds, the fluorescing probes from the surroundings will diffuse throughout the sample and replace the non-fluorescent probes in the bleached region (Lopez et al., 1988).

Protocol

Cell preparation and transfection

1. Prepare the mEGFP fusion proteins using the standard procedures.
2. Transfect the cells with the fusion protein construct in a six-well plate using Lipofectamine Plus reagent.
3. Coat the dishes with 10 mg/ml fibronectin for 1 hour at room temperature.
4. After 4 hours of incubation, wash the cells with sterile phosphate buffer solution for two times.
5. Trypsinize the cells and rinse them twice with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal calf serum (FCS).
6. Plate the cells (5 × 104 cells/dish) in the glass bottom dish.
7. Incubate the cells for 12-48 hours until the expression of the transfected plasmid reaches its maximum.
8. Replace the Dulbecco’s modified Eagle’s medium with pre-warmed Ham’s F12 medium.
9. Place the dish containing cells in the pre-warmed microscope chamber at least 1 hour before beginning with the FRAP experiment to allow the medium to equilibrate.
   

   Data acquisition

   1. Select the cells expressing the fluorescent protein in the region of interest using the eyepiece/objective of the microscope.
   2. Adjust the microscope parameters for monitoring the fluorescence.
   3. Adjust the focus and select the required desired region of interest.
   4. Start the time-lapse.
   5. Proceed with another cell and acquire multiple cells.

   Intensity measurements

   1. Transfer the collected images of the FRAP experiment to the analysis workstation.
   2. Re-adjust the alignment of the images to avoid any stage drift.
   3. Draw the boundaries of the region of interest using the circular tool.
   4. Obtain the fluorescence intensity values for:
   • The bleached region of interest.
   • Unbleached areas showing the background fluorescence.
   • Unbleached structures similar to the bleached target.
   5. Label the data and store it for the experimental record.

   Protocol for FRAP of dendritic spines in cultured hippocampal neurons

   Neuronal transfection

   1. Culture the hippocampal neurons of rat (embryonic day 18) on poly-d-lysine-coated 35-mm glass-bottom dishes. On 16-18 days in vitro, transfect the neurons using the transfection Kit. Replace the original culture medium with 1.5 ml of Dulbecco’s Modified Eagle Medium (DMEM) half an hour before the transfection.
   2. Mix 10 μg of enhanced green fluorescent protein (pEGFP-N1) plasmid DNA with sterile water and 12.4 μl of 2M calcium solution to make a total volume of 100 μl.
   3. Add 100 μl 2×HBS dropwise to the mixture while vortexing 2×HBS at medium speed.
   4. Leave the mixture at room temperature and let it sit for 20 minutes.
   5. Add the final mixture into the DMEM-incubated neurons.
   6. Incubate the neurons at 37 °C for 1-1.5 hours.
   7. Remove the calcium phosphate-containing medium and wash the cells with DMEM for three times.
   8. Replace the DMEM medium with the culture medium.

   Spine FRAP experiment

   1. After 2-4 days of transfection, replace the culture medium with pre-warmed Tyrode Solution containing NaCl 145, KCl 5, HEPES 10, Glucose 10, Glycine 0.005, CaCl2 2.6, and MgCl2 1.3.
   2. Find the transfected mature dendrite by adjusting the eyepiece of the microscope.
   3. Select the region of interest and capture the images every 1 second for 15 seconds after bleaching. Save the captured images for analysis.

Applications

Visualizing active transport across the cell membrane (Zheng., et al., 2011)

Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique to study binding and diffusion of biomolecules in cells. The study was conducted to estimate diffusion, binding/unbinding rates, and active transport velocities using the FRAP data by capturing intracellular dynamics. The transport and localization of mRNA molecules in Xenopus laevis oocytes were observed. The results suggested that the RNA movement in both the animal and vegetal directions could impact the duration and the time of RNA localization in Xenopus oocytes. It was also presented that the initial model conditions extracted from FRAP postbleach intensities prevent the underestimation of diffusion, which could arise from instantaneous bleaching. The FRAP technique is a broadly applicable tool to analyze the systems where intracellular transport is a critical molecular mechanism.

Demonstrating the assembly dynamics of E.coli FtsZ-ring (Stricker et al., 2002)

FtsZ is an important cytoskeletal component of the bacterial cell division machinery. It assembles into a ring called the Z-ring which contracts at septation. FtsZ is involved in guanosine triphosphate (GTP) hydrolysis and in vitro assembly. In the FRAP experiment, green fluorescent protein-labeled FtsZ was used to show the dynamics of E. coli Z-ring which continually remodels itself with a half-time of 30 s. It was found that the ZipA, a membrane protein involved in cell division, is also dynamic. The Z-ring of the mutant ftsZ84 showed a 9-fold slower turnover in vivo, which in the wild-type has 1/10 the guanosine triphosphatase activity in vitro. The study indicated that the assembly dynamics could be determined by GTP hydrolysis. The FRAP technique was found to be a powerful imaging tool to observe the dynamics of membrane proteins in bacterial species.

Detection of the lateral mobility in membranes (Yguerabide., Schmidt., & Yguerabide., 1982)

The fluorescence recovery after photobleaching (FRAP) can demonstrate the lateral diffusion coefficients of membrane components. The method was developed for precise analysis of FRAP data to minimize uncertainties arising from the photobleaching in the experiment.  For this, thin, multi-bilayer films containing a fluorescent probe were used with a molar-lipid-to-probe ratio of 500:1. The thin, clear multi-bilayer film was hydrated using the fully hydrated agarose gel, and the cover glass was sealed onto a microscope slide for the observation of FRAP experiment. The results indicated that the recovery of fluorescence could be represented over a broad range of percent bleach and recovery time. It was also shown that the linear reciprocal plot provides the researchers with a simple method to detect flow or multiple diffusion coefficients and to establish different parameters such as data precision, differences in multiple diffusion coefficients, the magnitude of flow rate compared to lateral diffusion. It was concluded that the FRAP method is applicable to obtain data from biological cells, tissues, and living systems when the optics are properly aligned, and the cellular movement is masked.

Analyzing the thylakoid membrane proteins (Rayan et al., 2010)

The diffusion of grana thylakoid membrane chlorophyll-protein complexes was observed using the fluorescence recovery after photobleaching (FRAP). The samples were obtained from isolated spinach (Spinacia oleracea) grana membranes. Grana are the clusters of thylakoid membrane domains and are protein hubs with around 70% to 80% of the membrane occupied by proteins. It was found that around 75% of chlorophyll-protein complexes remain immobile up to 9 minutes, while the remaining proteins diffused rapidly after photobleaching. These “rapidly” diffusing proteins were the mobile proteins, which exchange between the grana and stroma lamellae. The FRAP technique is a promising imaging technique for the observation of protein diffusion across the cell membrane.

Precautions

• The cells should be properly aligned before the experiment because living cells often move during the experiment.
• Bleaching event reduces the total amount of excitable fluorochromes in the cell, so it is recommended to measure a control region to have background data for the overall loss in fluorescence.
• In some experiments the final FRAP result is determined by the size of the region of interest; therefore, it is important to include a control to avoid this.
• It is essential to check the recovery rate for different bleaching intensities because photo-induced cross-linking may occur.
• If the study involves living cells then the removal of oxygen cannot be done; therefore there is a need to carefully control the dose of light received by the sample in fluorescence imaging to avoid bleaching.
• If long-term experiments are planned, then it is advisable to use Photoactivatable GFP because proteins may degrade or denature at extended periods.

Strengths and limitations

• Fluorescence recovery after photobleaching (FRAP) is a standard method that unveils subtle dynamic and biochemical properties of intracellular components and cellular events.
• The FRAP imaging provides valuable information about the diffusion and mobility of cellular protein population.
• The photobleaching effect of the FRAP provides with high fluorescence signal enabling the detection of strongly associated components.
• FRAP allows the analyses of binding dynamics of the protein molecules.
• The data obtained from the FRAP studies are qualitative and provides an insight into the binding characteristics, binding affinity, and the effect of drugs or mutations on protein mobility and diffusion.

References

1. , M. Stroud., R. Tsang., & Ballestrem., C. (2011). Fluorescence recovery after photobleaching. Methods Mol Biol, 769, 387-402.
2. , L. Dupou., A. Altibelli., J. Trotard., & Tocanne., J. F. (1988). Fluorescence recovery after photobleaching (FRAP) experiments under conditions of uniform disk illumination. Biophys J, 53(6), 963-70.
3. , R. S. Petralia., Y. Wang., & Kachar., B. (2011). Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons. J Vis Exp, 50, 2568.
4. Rayan., J. Guet., N. Taulier., F. Pincet., & Urbach., W. (2010). Recent applications of fluorescence recovery after photobleaching (FRAP) to membrane bio-macromolecules. Sensors (Basel), 10(6), 5927-48.
5. J Yguerabide., J. A. Schmidt., & Yguerabide., E. E. (1982). Lateral mobility in membranes as detected by fluorescence recovery after photobleaching. Biophys J, 40(1), 69-75.
6. Stricker., P. Maddox., E. D. Salmon., & Erickson., H. P. (2002). Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching. Proc Natl Acad Sci U S A, 99(5), 3171-5.
7. Bizzarri., F. Cardarelli., M. Serresi., & Beltram., F. (2012). Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange. Anal Bioanal Chem, 403(8), 2339-51.