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)e...

<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. Howe...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well slides</td>
			<td>Ibidi</td>
			<td>80821</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil Immersol&#160; 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 result...

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

F&amp;#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.

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 ...

The protocol provides a simple procedure to make FRET experiments by sensitized emissions more reproducible and comparable. The main advantage of FRET are measurements in real time so that dynamic processes can be addressed. For laser adjustment using a transmission photomultiplier, place the eight-well slide containing the transfected protoplasts under the microscope.After selecting an empty well, choose line scanning mode and histogram view, decrease the laser intensity to the minimum and adjust the detector gain to detectable background noise. Next, increase the laser intensity in steps of 0.5%while recording the corresponding signal. For laser adjustment using the reflective mode, select an empty well, then apply a reflection filter.Switch on the reflection mode and ensure that the detector wavelength range covers the wavelength of the laser. Choose the line scanning mode and histogram view, decrease the laser intensity to the minimum and adjust the detector gain to detectable background noise. Next, move the objective to the lowest position before moving it back up until the reflection of the coverslip is visible, then increase the laser intensity in steps of 0.5%while recording the corresponding signal.For data evaluation, tabulate and sort the data by signal intensities, then plot the signal intensities against the relative laser power and choose the laser intensities resulting in a similar signal intensity. For adjustment of the photomultipliers, select an empty well and apply a reflection filter, switch to reflection mode and ensure that the detector wavelength range covers the wavelength of the laser. Choose the line scanning mode and histogram view, decrease the detector gain to half the maximum and adjust the laser intensity to detectable background noise.Next, move the objective to the lowest position before moving it back up until the reflection of the coverslip is visible, then increase the detector gain in 50 to 100 volt steps and record the corresponding signal. For data evaluation, plot the intensity against the detector gain for each detector, then choose the individual detector gains to obtain similar sensitivity. For image acquisition, choose the appropriate filters or dichroic mirrors and use the same dichroic mirror for all channels to enable line by line scanning, then select a water immersion objective for the imaging of live cells and choose 12 or 16 bit scanning with moderate scanning speed.Next, define the detection range preferably 470 to 510 nanometers for donor detection and 530 to 600 nanometers for acceptor detection in the case of the FRET pair of enhanced cyan fluorescent protein or ECFP and enhanced yellow fluorescent protein or EYFP. After applying the detector and laser settings as demonstrated previously, 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.After fine tuning with the pinhole diameter while keeping the laser intensities and detector gains constant, perform the FRET measurements taking images of at least 20 cells. To determine the crosstalk corrections, perform FRET measurements with cells expressing the donor fluorophore only and those expressing the acceptor fluorophore only. For calibration of the measurements, perform FRET measurements with cells expressing the donor acceptor fusion.For data evaluation, obtain line profiles of the cells ensuring that each profile contains no more than one cell. Save the profiles as text files. Then using the text file import option in the data section, import the text files into a spreadsheet.Next, read out the maximum values by applying the max function, then list the obtained values in a table having a column each for donor emission ID, FRET emission IF, accepter emission IA, and at least four datasets, donor only, accepter only, donor-accepter fusion, and measurement. The laser adjustment revealed a linear increase in emission with increasing laser intensity. As shown by a steeper slope, the emission of the 514 nanometer line was much higher than the emission of the 458 nanometer line.Varying the detector gains at constant laser power revealed an exponential behavior for both analyzed detectors. The discrepancy and the spectral bleed-through of the donor and the acceptor analyzed with recombinant purified proteins and that analyzed with cells expressing those proteins demonstrates that it is impossible to omit the determination of spectral bleed-through in living cells likely caused by cellular pigments. The FRET efficiency between the labeled vacuolar ATPA subunits, VHA AECFP, and VHA AEYFP decreased with increasing signal intensity.In contrast, the interaction between VHA E1 ECFP and VHA CEYFP was independent of the signal intensity. The choice of appropriate optical components is crucial for the detection of the donor and acceptor. This protocol can also be applied for ratiometric sensors in living cells to increase the reproducibility in these experiments.

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

Research

JoVE Journal

Biochemistry

2.7K visualizações.  Bielefeld University. O protocolo fornece um procedimento simples para tornar os experimentos de FRET por meio de emissões sensibilizadas mais reprodutíveis e comparáveis. A principal vantagem do FRET são as medições em tempo real para que processos dinâmicos possam ser abordados. Para ajuste a laser usando um fotomultiplier de transmissão, coloque o slide de oito poços contendo os protoplastos transfectados sob o microscópio.Depois de selecionar um poço vazio, escolha o modo de varredura de linh...