The experiments were performed at the Light Microscopy Technology Platform (LiMiTec) of the Faculty of Biology, Bielefeld University. This work has been funded by Bielefeld University.

NOTE: For the following protocol, transient transfection of protoplasts was performed, as described previously12. A brief description is given below.
1. Transient transfection of protoplasts
<ol>
	<li>Cut ~4 g of healthy leaves of Arabidopsis thaliana ecotype Columbia into 1 mm slices and transfer them to 20 mL of enzyme solution (1.5% cellulase; 0.4% macerozyme; 0.1% bovine serum albumin Fraction V; 0.4 M mannitol; 20 mM KCl; 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7; 10 mM CaCl2).</li>
	<li>Vacuum-infiltrate the leaf slices followed by an incubation with agitation for 2 h at room temperature. Harvest the cells by centrifugation for 3 min at 100 &#215; g.</li>
	<li>Wash the protoplasts with W5-solution (154 mM NaCl; 125 mM CaCl2; 5 mM KCl; 2 mM MES, pH 5.7) and resuspend them in MMG solution (0.4 M mannitol; 15 mM MgCl2; 4 mM MES, pH 5.7).</li>
	<li>Perform the transfection in an 8-well slide by osmotic shock in the presence of polyethyleneglycol (PEG) 4000. Mix 20 &#181;L of the protoplast suspension with 5 &#181;L of plasmid DNA (5 &#181;g/&#181;L) and 25 &#181;L of PEG solution (0.2 M mannitol, 0.1 M CaCl2, 40% PEG 4000).</li>
	<li>Reverse the osmotic shock by gentle readjustment of the osmotic conditions. 
	&#8203;NOTE: Besides the sample of interest, the expression of donor alone and acceptor alone is required to determine the spectral bleed-through of the donor and the acceptor, respectively. A fusion protein of the donor and the acceptor must be expressed too for calibration purposes. The fluorescent protein expression was under the control of a cauliflower mosaic virus 35S promoter (pCaMV35S). For all measurements, two confocal laser scanning microscopes (LSM1 and LSM2) were used. LSM1 has two types of detectors: for FRET measurements, the donor signal was detected by a GaAsP-detector, while FRET and acceptor emission were recorded with a photomultiplier. LSM2 has two photomultipliers, which were used for the detection of donor, FRET, and acceptor emission.</li>
</ol>
2. Laser-adjustment
NOTE: Here, 458 nm and 514 nm lines of an argon-ion laser have been applied for FRET analysis between enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-labeled proteins. For reproducible data acquisition, both lines were adjusted to similar intensity. This was achieved by either a transmission photomultiplier or the reflection mode.
<ol>
	<li>Laser adjustment with a transmission photomultiplier
	<ol>
		<li>Use an empty well for adjustment.</li>
		<li>Choose line-scanning mode and histogram view.</li>
		<li>Decrease the laser intensity to the minimum, and adjust the detector gain to detectable background noise.</li>
		<li>Increase the laser intensity in steps of 0.5% and record the corresponding signal.</li>
		<li>Apply the routine for both laser lines.</li>
	</ol>
	</li>
	<li>Laser adjustment with reflection mode
	<ol>
		<li>Use an empty well for adjustment.</li>
		<li>Apply a reflection filter, switch on the reflection mode, if available.</li>
		<li>Ensure that the detector wavelength range covers the wavelength of the laser.</li>
		<li>Choose the line-scanning mode and histogram view.</li>
		<li>Decrease the laser intensity to the minimum, and adjust the detector gain to detectable background noise.</li>
		<li>Move the objective to the lowest position.</li>
		<li>Move the objective up until the reflection of the coverslip is visible.</li>
		<li>Increase the laser intensity in steps of 0.5% and record the corresponding signal.</li>
		<li>Apply the routine for both laser lines.</li>
	</ol>
	</li>
	<li>Data evaluation
	<ol>
		<li>Tabulate the data and sort the data by signal intensities.</li>
		<li>Plot the signal intensities against the relative laser power.</li>
		<li>Choose laser intensities that result in similar signal intensity.</li>
	</ol>
	</li>
</ol>
3. Adjustment of photomultipliers
NOTE: After laser adjustment, the photomultipliers were adjusted to individual gains to obtain similar sensitivity. This calibration was done with the 514 nm laser line, which is in the center of the wavelength range of interest.
<ol>
	<li>Use an empty well for adjustment.</li>
	<li>Apply a reflection filter, and switch to reflection mode if available.</li>
	<li>Ensure that the detector wavelength range covers the wavelength of the laser (514 nm).</li>
	<li>Choose the line scanning mode and histogram view.</li>
	<li>Decrease detector gain to half the maximum, and adjust the laser intensity to detectable background noise.</li>
	<li>Move the objective to the lowest position.</li>
	<li>Move the objective up until the reflection of the coverslip is visible.</li>
	<li>Increase the detector gain in steps of 50 to 100 V and record the corresponding signal.</li>
	<li>Apply steps 3.1 to 3.8 for both detectors.</li>
	<li>Data evaluation
	<ol>
		<li>Plot the intensity against the detector gain for each detector.</li>
		<li>Choose the individual detector gains to obtain similar sensitivity.</li>
	</ol>
	</li>
</ol>
4. FRET image acquisition
NOTE: Start with the sample of interest for setting up image acquisition.
<ol>
	<li>Choose the appropriate filters/dichroic mirrors, e.g., a double dichroic mirror MBS 458/514 for the FRET-pair ECFP/EYFP. Use the same dichroic mirror for all channels to enable line-by-line scanning. Select a water immersion objective for the imaging of living cells. Choose 12 bit- or 16-bit scanning and moderate scanning speed.</li>
	<li>Define the detection range, preferably 470-510 nm for donor detection and 530-600 nm for acceptor/FRET detection in the case of ECFP/EYFP. When using a 445 nm or 440 nm diode laser, use 450 to 510 nm as the detection range. In the case of an acousto-optic beam splitter (AOBS), define donor detection in the range of 450 to 500 nm to prevent unwanted acceptor detection.</li>
	<li>Apply the detector setting according to 3.10.2.</li>
	<li>Apply the laser setting according to 2.3.2. Revise the laser intensity based on the obtained laser power table, if required. Ensure that the signal-to-noise ratio covers the entire dynamic range of the detectors (intensity ranging from 0 to 4095 for 12-bit scanning).</li>
	<li>Keep laser intensities and detector gains constant. Use the pinhole diameter for fine-tuning. 
	NOTE: Keep in mind that changes in the pinhole diameter affect spatial resolution.</li>
	<li>Perform the measurements (take images of at least 20 cells).</li>
</ol>
5. Determination of crosstalk corrections
NOTE: Cells expressing only the donor or the acceptor are required to determine donor spectral bleed-through (DSBT) and acceptor spectral bleed-through (ASBT), respectively. Keep the same settings described in section 4.
<ol>
	<li>Perform FRET measurements with cells expressing the donor fluorophore.</li>
	<li>Perform FRET measurements with cells expressing the acceptor fluorophore.</li>
</ol>
6. Calibration of the measurements according to Beemiller et al.13
NOTE: Cells expressing a donor-acceptor fusion of known FRET efficiency are required. Here, an ECFP-5 aa-EYFP-fusion with a FRET efficiency of 0.46 has been used4. Keep the same settings described in section 4.
<ol>
	<li>Perform FRET measurements with cells expressing the donor-acceptor fusion</li>
</ol>
7. Data evaluation
<ol>
	<li>Obtain line profiles of the cells, ensuring that each profile contains no more than one cell. Save the profiles as text files.</li>
	<li>Import the text files into a spreadsheet using the text file import option in the Data section.</li>
	<li>Read out the maximum values by applying the Max function.</li>
	<li>List the obtained values in a table, have a column each for donor emission ID, FRET emission IF, acceptor emission IA, and at least four data sets: donor only, acceptor only, donor-acceptor fusion, and measurement. 
	NOTE: Excitation of the donor also results in direct excitation of the acceptor and causes ASBT that is described by the &#945; value.</li>
	<li>Calculate the ASBT &#945; values with the acceptor-only dataset using equation (1). 
	<img alt="Equation 1" src="/files/ftp_upload/62758/62758eq01.jpg" /> (1) 
	NOTE: Use the median of all &#945;-values in the following equations. The donor shows a broad emission spectrum that results in emission crosstalk with the sensitized emission of the acceptor. This DSBT is given by the &#946; value.</li>
	<li>Calculate the donor spectral bleed-through &#946; values with the donor-only dataset using equation (2). 
	<img alt="Equation 2" src="/files/ftp_upload/62758/62758eq02.jpg" /> (2) 
	NOTE: Use the median of all &#946; values in the following equations. The calibration factor &#958; describes the linear relationship of FRET-derived donor quenching and sensitized emission of the acceptor. Use the medians of 7.5 and 7.6 in the following equations.</li>
	<li>Calculate the calibration factors &#958; with the donor-acceptor fusion dataset and its FRET efficiency E (0.46) using equation (3). 
	<img alt="Equation 3" src="/files/ftp_upload/62758/62758eq03.jpg" /> (3) 
	NOTE: Use the median of all &#958; values in the following equations.</li>
	<li>Calculate the FRET efficiencies of the protein pair of interest using equations (4) and (5). 
	<img alt="Equation 4" src="/files/ftp_upload/62758/62758eq04.jpg" /> (4) 
	<img alt="Equation 5" src="/files/ftp_upload/62758/62758eq05.jpg" /> (5)</li>
	<li>Estimate the effects of expression strength and/or donor-acceptor ratio: plot the sum of ID, IF, and IA against the FRET efficiencies. Perform a linear regression; note that the steeper the graph and the higher R2 is, the higher is the impact of the expression level or the greater is the difference of donor and acceptor abundance.</li>
</ol>

<ol>
	<li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescent Spectroscopy. Third Edition. , (2006).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F&#246;rster+resonance+energy+transfer-+FRET+what+it+is,+why+do+it,+and+how+it's+done.">F&#246;rster resonance energy transfer- FRET what it is, why do it, and how it's done.</a> Laboratory Techniques in Biochemistry and Molecular Biology. 33, 1-57 (2009).</li><li>Vogel, S. S., Nguyen, T. A., vander Meer, B. W., Blank, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+heterogeneity+and+dark+acceptor+states+on+FRET:+implications+for+using+fluorescent+protein+donors+and+acceptors.">The impact of heterogeneity and dark acceptor states on FRET: implications for using fluorescent protein donors and acceptors.</a> PLoS ONE. 7, 49593 (2012).</li><li>M&#252;ller, S. M., Galliardt, H., Schneider, J., Barisas, B. G., Seidel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+F&#246;rster+resonance+energy+transfer+by+monitoring+sensitized+emission+in+living+plant+cells.">Quantification of F&#246;rster resonance energy transfer by monitoring sensitized emission in living plant cells.</a> Frontiers in Plant Science. 4, 413 (2013).</li><li>Gadella, T. W. J., vander Krogt, G. N., Bisseling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+FRET-microscopy+in+living+plant+cells.">GFP-based FRET-microscopy in living plant cells.</a> Trends in Plant Science. 4, 287-291 (1999).</li><li>Van Rheenen, J., Langeslag, M., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+confocal+acquisition+to+optimize+imaging+of+fluorescence+resonance+energy+transfer+by+sensitized+emission.">Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission.</a> Biophysical Journal. 86, 2517-2529 (2004).</li><li>Seidel, T., Golldack, D., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+C-termini+of+V-ATPase+subunits+by+in+vivo-FRET+measurements.">Mapping of C-termini of V-ATPase subunits by in vivo-FRET measurements.</a> FEBS Letters. 579, 4374-4382 (2005).</li><li>Seidel, T., Schnitzer, D., Golldack, D., Sauer, M., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+iso-enzymes+of+plant+V-ATPase+as+revealed+by+in+vivo-FRET.">Organelle-specific iso-enzymes of plant V-ATPase as revealed by in vivo-FRET.</a> BMC Cell Biology. 9, 28 (2008).</li><li>Schnitzer, D., Seidel, T., Sander, T., Golldack, D., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+energization+state+affects+peripheral+stalk+stability+of+plant+vacuolar+H+-ATPase+and+impairs+vacuolar+acidification.">The cellular energization state affects peripheral stalk stability of plant vacuolar H+-ATPase and impairs vacuolar acidification.</a> Plant Cell Physiology. 52, 946-956 (2011).</li><li>Roshchina, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+autofluorescence:+application+to+the+study+of+plant+living+cells.">Vital autofluorescence: application to the study of plant living cells.</a> International Journal of Spectroscopy. 2012, 124672 (2012).</li><li>Holtorf, S., Apel, K., Bohlmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+constitutive+and+inducible+promoters+for+the+overexpression+of+transgenes+in+Arabidopsis+thaliana.">Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.</a> Plant Molecular Biology. 29, 637-646 (1995).</li><li>Seidel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+and+FRET-analysis+of+subunits+c+and+a+of+the+vacuolar+H+-ATPase+in+living+plant+cells.">Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells.</a> Journal of Biotechnology. 112 (1-2), 165-175 (2004).</li><li>Beemiller, P., Hoppe, A. D., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phosphatidylinositol-3-kinase-dependent+signal+transition+regulates+ARF1+and+ARF6+during+FC&#947;+receptor-mediated+phagocytosis.">A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during FC&#947; receptor-mediated phagocytosis.</a> PLoS Biology. 4, 162 (2006).</li><li>Lambert, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FPbase:+a+community-editable+fluorescent+protein+database.">FPbase: a community-editable fluorescent protein database.</a> Nature Methods. 16, 277-278 (2019).</li></ol>

We ensure that all authors have disclosed any and all conflicts of interest and have no competing financial interests.

Donor quenching and sensitized acceptor emission are characterized by a linear relationship that allows for either donor- or acceptor-based calculation of FRET. The corresponding factors of linearity are called either G factor (donor to acceptor) or xi (acceptor to donor), which are reciprocal values4. Measuring FRET between fluorescent proteins by fluorescence microscopy often requires corrections for DSBT and ASBT due to the broad absorption and emission spectra of the fluorescent proteins. However, many corrections depend on a measurable ASBT, which is a factor in the denominator of the equations in protocol section 7, and an &#945; = 0 results in undefined equations5,6.
An adjustment of laser intensities is recommended to avoid such effects. This enables the use of acceptor-based calculations and easy detection of inappropriate donor-acceptor ratios. In this protocol, the equation of Beemiller and co-workers has been applied, which has the advantage of simple calibration by a single reference construct of known FRET efficiency. The adjustment of the detectors is critical, especially when two different types of detectors are applied. Previous data obtained with two identical photomultipliers and identical gains resulted in a constant &#946; = 0.61 for multiple experiments over years7,8,9 and demonstrate that detector adjustment is advantageous for reproducibility. If the detectors do not allow for a reliable adjustment, a sequential scan with a single detector in a frame-by-frame mode might be an option to avoid bias from detector properties.
Measurements in plant cells are affected by many pigments, which result in excitation light absorption and background autofluorescence. The cellular pigments are mainly excited by light in the UV to blue range4,10. This explains the discrepancy between the measurements in cells and those with purified proteins. The purified proteins might serve as a proof of adjustment because the expected values for ASBT &#945; and DSBT &#946; can be derived from the acceptor absorption spectrum and the donor emission spectrum, respectively (Figure 6). The values of ASBT are similar in cells (&#945; = 0.094) and with purified fluorophores (&#945; = 0.114) and close to the expected value of 0.096 due to the low cellular absorption at 514 nm. The DSBT of &#946; = 0.498 with purified proteins is also close to the expected value of 0.406. The spectral bleed-through further depends on the setup; diode lasers of 445 or 440 nm reduce the direct excitation of EYFP and thereby reduce the ASBT. AOBSs are characterized by a smaller transmission gap than dichroic mirrors. Thus, the detection of EYFP is enhanced in the range of 500 to 510 nm and might result in additional ASBT into the donor channel. However, spectral properties of EYFP point to less than 1% emission of the maximum peak, considering both less effective excitation and low emission intensity. The filter gap is much wider with dichroic mirrors, resulting in an additional suppression of such acceptor crosstalk.
Using the values from purified proteins does not reflect the situation in the cell. The same is true for the calibration by the factor &#958;. Although the calibration factor has been similar for both LSM, it was much higher in measurements with the recombinant protein, revealing the impact of cellular pigments. It should be mentioned that in these measurements, the background noise was negligible but detectable and did not affect the observed intensities. In contrast to measurements of the donor lifetime, sensitized emission experiments are sensitive towards the donor-acceptor ratio so that an excess of donor results in decreased FRET efficiency.
However, the protein expression has been under the control of the CaMV35S-promoter in the present experiments. This promoter is frequently used for the overexpression of proteins in plants and might result in unphysiologically high amounts of proteins13. Under such conditions, the probability of interaction increases, and false-positive results might occur with higher protein concentrations. Plotting the emission intensities against the FRET efficiency then results in an increasing FRET efficiency with increasing emission intensity and allows for evaluation of the FRET data with respect to the expression level.
It is recommended to separate high-resolution colocalization experiments from FRET measurements. For colocalization, optimal settings are chosen for each fluorophore, while for FRET recording, emission intensities and fine-tuning by the pinhole diameter interfere with optimal image conditions. Nevertheless, the separation of organelles and information on subcellular structures are still encompassed in FRET measurements.

F&#246;rster resonance energy transfer ((F)RET) is typically observed by fluorescence spectroscopy, although the process itself is not limited to occur between fluorophores. The underlying dipole-dipole coupling simply requires a light-emitting donor molecule and a light-absorbing acceptor. This is derived from the required spectral overlap integral J of the normalized donor emission and acceptor absorbance spectra1. However, because RET competes with fluorescence, the energy transfer becomes measurable by alterations in fluorescence emission: RET induces donor quenching and sensitized acceptor emission.
Fluorophore-based RET has been termed fluorescence resonance energy transfer (FRET) to separate it from bioluminescence resonance energy transfer (BRET). RET depends strongly on the distance between donor and acceptor, which is widely in the range of 0.5-10 nm2 and thus, in the same range as the dimensions of proteins and their complexes. Second, RET depends on the dipole-dipole orientation kappa squared. Combined with the fact that rotational freedom of protein-bound fluorophores can be neglected due to the molecular weight and the slow rotational relaxation, RET allows for the analysis of conformational alterations3.
The so-called F&#246;rster radius is based on the spectral overlap integral and the wavelength range of the overlap, so that red light-absorbing chromophores result in longer F&#246;rster radii than blue light-absorbing dyes. As the dynamic range of FRET measurements is limited by 0.5 &#215; R0 and 1.5 &#215; R0, the FRET pair ECFP-EYFP has a dynamic range of 2.5-7.3 nm due to its R0 of 4.9 nm4.
The brightness of a fluorophore is given by the product of its molar extinction coefficient and its quantum yield. For FRET measurements, it is advantageous to choose fluorophores of nearly similar brightness. This enhances the detection of donor quenching and sensitized acceptor emission. It also favors the calibration of the microscopy system. Looking at the frequently used FRET pairs of cyan and fluorescent proteins, the lower brightness of the cyan fluorescent proteins becomes obvious (Figure 1A).
However, the lifetime of the acceptor must be lower than the lifetime of the donor, ensuring the availability of the acceptor for energy transfer. If the lifetime of the acceptor exceeds the lifetime of the donor, the acceptor might still be in the excited state when the donor is excited again. Advanced cyan fluorescent proteins such as mTurquoise show an extended lifetime and thus contribute to an increased probability of FRET (Figure1B). The probability of FRET also depends on the molar extinction coefficient of the acceptor.

<table><tbody><tr><td>8-well slides</td><td>Ibidi</td><td>80821</td><td></td></tr><tr><td>Immersion oil Immersol&amp;nbsp; W2010</td><td>Zeiss</td><td>444969-0000-000</td><td>refraction index of water</td></tr><tr><td>LSM 1: AxioObserver with LSM 780 scan head, confocal laser scanning microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>LSM 2: AxioObserver with LSM 5 scan head, confocal laser scanning microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

f&#246;rster resonance energy transfer measurements in living plant cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

Adjustment of the confocal laser-scanning microscope 
The laser adjustment revealed a linear increase of emission with increasing laser intensity (Figure 2 and Table 1). As expected for argon-ion lasers, the emission of the 514 nm line was much higher than the emission of the 458 nm line, as evidenced by a steeper slope. For subsequent experiments, laser power of 4.5% and 6.5% was chosen for the 514 nm line and the 458 nm line, respectively. This resulted in almost equal emission intensity of 1123 (514 nm) and 1141 (458 nm).
Varying the detector gains at constant laser power revealed an exponential behavior for both analyzed detectors. Similar emission intensities were obtained for a gain of 700 V (Figure 3). Although the adjustment of laser lines benefited from the linear behavior, the adjustment of the detectors is likely affected by the exponential behavior, resulting in marked differences with minor changes in the gain at higher voltages. Unfortunately, this higher range is of interest for measurements in living cells, as increasing the laser power is cytotoxic and promotes photobleaching.
Determining spectral bleed-through 
At first, the spectral bleed-through of the donor and the acceptor were analyzed with recombinant purified ECFP and EYFP; DSBT &#946; = 0.498 and ASBT &#945; = 0.100. The same was done with cells expressing either ECFP or EYFP. With LSM 2, the estimated DSBT was &#946; = 1.602 &#177; 0.207 (mean &#177; SD) and ASBT was &#945; = 0.119 &#177; 0.018. The median values were &#946; = 1.506 and &#945; = 0.120. This discrepancy between the data obtained in living cells and the data obtained with recombinant protein demonstrates that it is impossible to omit the determination of spectral bleed-through in living cells. This is likely caused by cellular pigments.
The images reveal the higher brightness of EYFP in comparison to ECFP (Figure 4 and Table 2). Compensating the differences in brightness by different laser settings enhances the dynamic range of the donor and the FRET channel. Providing the relative output by measuring the laser intensities, as done for the adjustment, still increases the reproducibility of the measurements.
For LSM 1, &#946; = 0.171 &#177; 0.044, &#945; = 0.094 &#177; 0.031 with medians of &#945; = 0.084 and &#946; = 0.180. The determination of the ASBT depends on the laser adjustment and a single detector, and thus, the determination of ASBT performed well. The DBST involves two detectors, resulting in vastly different results for both microscopes. It should be remembered that LSM 2 is equipped with two photomultipliers, whereas LSM 1 uses a GaAsP detector and a photomultiplier, two different types of detectors with different spectral properties and sensitivity. Accordingly, the adjustment works better with two identical detectors.
Calibration of the measurement 
For calibration, the median values of &#945; and &#946; were applied, and a fusion of ECFP and EYFP was used as the standard of known FRET efficiency (E = 0.46). The calibration of recombinant and purified ECFP-EYFP resulted in a &#958; value of 13.44, while the in vivo measurements revealed a &#958; value of 1.525 &#177; 1.844. The median was 0.798, revealing extreme outliers together with the high standard deviation. For LSM 1, the values were &#958; = 1.978 &#177; 0.807 with a median of &#958; = 1.883. For LSM 2 and LSM 1, the datasets obtained for the calculation of &#958; (Table 3) were statistically identical, as proven by Student&#39;s t-test (p &#62; 0.2).
FRET measurement 
As a proof of concept, the FRET measurement was repeated with the donor-acceptor fusion. The measured FRET efficiency was 0.47 &#177; 0.07 for the purified protein and 0.47 &#177; 0.06 in living cells with LSM 2 (Table 4). The median of the measurements in living cells was E = 0.45. For LSM 1, the FRET efficiency was 0.46 &#177; 0.09 with a median of E = 0.45 (Table 4). These data demonstrate the effective calibration with a FRET construct of known FRET efficiency.
Effects of expression level and donor-acceptor ratio 
For the analyzed data set, the FRET efficiencies were not influenced by the expression level. Due to the fusion of the donor and the acceptor, the ratio was also constant. The inclusion of previous datasets revealed a dependency on the expression level in one case (Figure 5). The FRET efficiency between the labeled vacuolar ATPase (V-ATPase) subunits, VHA-A-ECFP and VHA-a-EYFP, decreased with increasing signal intensity. In contrast, the interaction between VHA-E1-ECFP and VHA-C-EYFP was independent of the signal intensity. In the case of VHA-A and VHA-a, the three copies of VHA-A are increasingly replaced by VHA-A-ECFP, which results in a donor excess in comparison to the single copy of VHA-a in the complex. Although VHA-E1 might be present in the form of three copies, the high solubility of VHA-C might abolish this effect in this example. Here, the FRET efficiencies have been comparatively low. This simple approach allows for the testing of expression and ratio artifacts.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62758/62758fig01.jpg" /> 
Figure 1:&#160;Overview of fluorescent protein FRET pairs with cyan-emitting donors. (A) The F&#246;rster radii of the pairs and the brightness of donor and acceptor are given for well-characterized FRET pairs. (B) Comparison of donor and acceptor lifetimes. Grey asterisks indicate a multiexponential decay. Abbreviations: FRET = F&#246;rster resonance energy transfer; CFP = cyan fluorescent protein; GFP = green fluorescent protein; YFP = yellow fluorescent protein; mX = monomeric X; EX = enhanced X; SX = super X. <a href="https://www.jove.com/files/ftp_upload/62758/62758fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62758/62758fig02.jpg" /> 
Figure 2:&#160;Laser adjustment. The emission of the lasers was recorded by the reflection of the coverslip and plotted against the relative laser intensity as modulated by the acousto-optic tunable filter of the LSM. Emission of the 458 nm line (A) and the 514 nm line (B) of an argon-ion laser is shown. Abbreviations: LSM = laser scanning microscope; r.u. = relative units. <a href="https://www.jove.com/files/ftp_upload/62758/62758fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62758/62758fig03.jpg" /> 
Figure 3:&#160;Detector gain and emission intensity. The resulting emission intensities were recorded in the reflection mode for detector gains ranging from 300 to 750 V and 300 to 750 V for detector 1 (A) and detector 2 (B), respectively. Abbreviation = r.u. = relative&#160;units. <a href="https://www.jove.com/files/ftp_upload/62758/62758fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62758/62758fig04.jpg" /> 
Figure 4:&#160;Spectral bleed-through. Images were obtained in the donor, the FRET, and the acceptor channels. (A) Images obtained with purified fluorescent proteins; scale bars = 100 &#181;m; (B) corresponding images for cells expressing the fluorescent proteins; scale bars = 10 &#181;m. (C) Graphs of the obtained SBT values; mean &#177; SD is given. Abbreviations: FRET = F&#246;rster resonance energy transfer; SBT = spectral bleed-through; ECFP = enhanced cyan fluorescent protein; EYFP = enhanced yellow fluorescent protein; LSM = laser scanning microscope; ASBT = acceptor SBT; DSBT = donor SBT. <a href="https://www.jove.com/files/ftp_upload/62758/62758fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62758/62758fig05.jpg" /> 
Figure 5:&#160;Effects of expression level and/or donor-acceptor ratio. The sum of the emissions in the donor, FRET, and acceptor channels has been plotted against the FRET efficiency. This has been done for VHA-E1-ECFP and VHA-C-EYFP (A), VHA-A-ECFP and VHA-a-EYFP (B), and the ECFP-EYFP fusion (C). Linear regression was performed; the equation and R2 are given. Abbreviations: FRET = F&#246;rster resonance energy transfer; ECFP = enhanced cyan fluorescent protein; EYFP = enhanced yellow fluorescent protein; VHA = vacuolar ATPase subunit. <a href="https://www.jove.com/files/ftp_upload/62758/62758fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62758/62758fig06.jpg" /> 
Figure 6:&#160;Donor and acceptor bleed-through of ECFP and EYFP. The spectra of the fluorescent proteins were obtained from the FP database14. (A) During sensitized emission experiments, the vast portions of the donor and the acceptor are detected by individual photomultipliers, named PMT1 and PMT2, respectively. ECFP emission is also detectable by the acceptor detector upon excitation with 458 nm. The calculated donor spectral bleed-through into PMT2 is 40.6% of the emission detected by PMT1. (B) The emission spectrum of EYFP demonstrates that EYFP shows direct excitation at 458 nm, which is frequently applied for the excitation of cyan donors. The expected excitation efficiency is 9.6% of the excitation at 514 nm. Abbreviations: FP = fluorescent protein; ECFP = enhanced cyan fluorescent protein; EYFP = enhanced yellow fluorescent protein; PMT = photomultiplier. <a href="https://www.jove.com/files/ftp_upload/62758/62758fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1:&#160;Laser power and signal intensity. The signal intensities of the 458 nm line are given in blue, the signal intensities of the 514 nm line in green. The intensities applied for the experiment are highlighted in grey. <a href="https://www.jove.com/files/ftp_upload/62758/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2:&#160;Signal intensities for SBT determination. Recorded maxima and calculated &#945; and &#946; values are given. Abbreviations: SBT = spectral bleed-through; LSM = laser scanning microscope; FRET = F&#246;rster resonance energy transfer; ASBT = acceptor SBT; DSBT = donor SBT. <a href="https://www.jove.com/files/ftp_upload/62758/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3:&#160;Signal intensities for calibration. Recorded maxima and calculated &#958; values are given. Corrected values correspond to the FRET signal intensities minus SBT. Abbreviations: SBT = spectral bleed-through; LSM = laser scanning microscope; FRET = F&#246;rster resonance energy transfer. <a href="https://www.jove.com/files/ftp_upload/62758/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4:&#160;Signal intensities for FRET measurements. Recorded maxima and E values are given. Corrected values correspond to the FRET signal intensities minus SBT. Calibrated E values were calculated with calibration by &#958;. Abbreviations: SBT = spectral bleed-through; LSM = laser scanning microscope; FRET = F&#246;rster resonance energy transfer; cal. = calibrated. <a href="https://www.jove.com/files/ftp_upload/62758/Table 4.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about FÃ¶rster Resonance Energy Transfer Measurements in Living Plant Cells at JoVE.com

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

frster-resonance-energy-transfer-measurements-in-living-plant-cells

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.8K Views.  Bielefeld University. A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo Förster resonance energy transfer measurements, followed by data evaluation.

Video: Förster Resonance Energy Transfer Measurements in Living Plant Cells

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Bioengineering

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 F&#246;rster Resonance Energy Transfer (FRET) Biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.

Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips.

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental ...

Developmental Biology

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Biology

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within &#60;10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone ...

F&#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred&#8212;and consequently the overall emission of sample&#8212;depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry techniques to obtain detailed information of biomolecular structures and interactions from this &#8220;spectroscopic ruler.&#8221;
This video covers the principles and concepts of FRET analysis. The procedure focuses on preparing samples for FRET and ways to present and interpret data. Finally, the applications include monitoring conformational and cellular processes by labeling parts of a cell or protein, monitoring enzyme reactions that alter protein structures, and using FRET to monitor aggregation of monomers expressed by cells.
<div class="chapter_text" id="script_0">
F&#246;rster Resonance Energy Transfer, or FRET, is a non-radiative transfer of energy between light-emitting molecules, and is often used to investigate close-range biochemical interactions. FRET only occurs when fluorescent molecules are spaced within 10 nm of each other. FRET analysis can be combined with other techniques to obtain detailed structural information. This video will introduce the underlying principles of FRET, summarize a protocol and data presentation, and discuss some biochemical applications.
</div>
<div class="chapter_text" id="script_1">
A photoluminescent molecule such as a fluorophore is excited by absorbing electromagnetic radiation at a wavelength in its absorption spectrum. As it relaxes, it emits light at a wavelength within its emission spectrum. For more information about fluorescence, see JoVE&#39;s video on fluorescence microscopy. Different fluorophores absorb and emit light at different wavelengths, which frequently overlap. If the emission spectrum of a fluorophore overlaps significantly with the absorption spectrum of another fluorophore, the &#8220;donor&#8221; will release a virtual photon, which is absorbed by the &#8220;acceptor&#8221;. When an excited donor is within 10 nm of an acceptor, energy is transferred from donor to acceptor by dipole-dipole interactions. The release of energy by emission of light from the donor correspondingly decreases. Meanwhile, the excited acceptor emits light at its emission wavelength. The FRET response is evaluated in terms of efficiency, or the percentage of energy released from the donor by FRET rather than by fluorescence or other radiative processes. The efficiency depends strongly on the distance between the donor and acceptor, which allows FRET to act as a &#39;molecular&#39; or &#39;spectroscopic&#39; ruler.
In biochemistry, FRET is often used qualitatively to observe conformational changes in molecules by monitoring fluorophores as they move in and out of FRET range of each other. Similarly, cellular functions can be studied with molecules containing a FRET pair. If the labeled molecule is cleaved by enzyme activity, FRET stops and the observed fluorescence wavelength changes.
Now that you understand the principles behind FRET, let&#39;s look at an overview of a protocol and a few ways to present and interpret the data.
</div>
<div class="chapter_text" id="script_2">
Prior to the experiment, the biomolecules of interest, typically DNA or proteins, are engineered with fluorescent tags, using molecular biology techniques.&#160;Common ways to introduce the modified genetic material into the cells include transfection and electroporation.
Then, the cells are prepared for FRET visualization on a fluorescence microscope.&#160;For instance, the molecules may be immobilized on a slide for single-molecule FRET, or samples are loaded into wells for high-throughput screening.
Then, the excitation lasers, microscope, and associated equipment are prepared. (A) FRET experiments often involve powerful lasers; (B) so appropriate PPE and safety procedures should be used.&#160;The sample is then placed in the instrument and illuminated with the excitation laser.
</div>
<div class="chapter_text" id="script_3">
For experiments monitoring cell behavior, color images showing differences or changes in emission intensity are used. Donor and acceptor emission intensities are plotted together to track FRET response over time.
FRET data can also be fitted to various functions for more complex analyses. Depending on the experiment, data may be presented in multiple ways to best represent the results, making FRET a flexible experimental tool.
</div>
<div class="chapter_text" id="script_4">
Now that you&#39;re familiar with the basics of running and analyzing a FRET experiment, let&#39;s look at some applications of FRET in biochemistry research.
FRET can be used to study conformational changes or cellular processes by labeling parts of the protein or cell predicted to move within 10 nm of each other with a FRET pair. For example, protein sensors are prepared by labeling receptors with a pair of fluorophores. The FRET response is monitored live by confocal microscopy. Variation of emission wavelength and intensity indicate conformational changes.
FRET can also be used by preparing molecules with an active FRET pair and observing changes in the response. When the substrate is cleaved, FRET is disrupted, causing an increase in donor emission and a decrease in acceptor emission. The emissions are analyzed to determine contributions by donor, acceptor, and FRET. Once the direct emission factors are calculated for the cyan and yellow fluorescent proteins, the concentration and kinetic parameters of the substrate can be determined.
Cells designed to express monomers containing either of a FRET pair function as &#39;sensors&#39; for interactions between those monomers. If aggregation of those monomers is induced, a FRET response is observed. This can be used to investigate protein aggregation triggered by &#39;seeding&#39; of misfolded proteins. Here, cells were transduced with aggregates of the protein of interest, incubated, and analyzed with flow cytometry.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on F&#246;rster Resonance Energy Transfer, or FRET. This video contained the underlying principles of FRET, preparation and analysis of a FRET experiment, and a few biochemical applications.
Thanks for watching!
</div>

F&amp;#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent ...

Förster Resonance Energy Transfer Measurements in Living Plant Cells

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Rozdziały w tym wideo

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor

A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Förster Resonance Energy Transfer (FRET)