confocal application notes

Confocal Application Notes Vol. 5 February 2006

Leica Microsystems Inc. Telephone (610)-321-0460 410 Eagleview Blvd, Ste. 107 Toll Free 866-830-0735 Exton, PA 19341 Fax (610)-321-0425

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FRET Acceptor Bleaching (AB)

Prepared by Myriam Gastard, PhD Exton Support Group, Leica Microsystems, Inc.

Fluorescence Resonance Energy Transfer (FRET) is a technique allowing study of

proteins interactions, which are in proximities but beyond light microscopy resolution (classically, fluorescently labeled molecules over distances of 1-10 nm). Originally measured by fluorescence spectroscopy, FRET can also be measured by fluorescence microscopy. Since FRET occurs over distances similar to the size of proteins, it can be used to extend the resolution of the fluorescence microscope (typically >250 nm) to detect protein-protein interactions. Hence, it is an ideal approach to determine whether proteins that are co-localized at the level of light microscopy really interact with one another.

Method: FRET involves a non-radiative transferred of energy from an excited state donor fluorophore to a nearby acceptor. The energy transfer efficiency FRETeff is directly related to the distance r separating a given donor and acceptor pair by

FRETeff=1/[1+(r/R0)6]

The resolution of FRET is thus defined by R0 which is typically < 10-70 Å. R0 depend of

the extent of overlap between the donor emission and the acceptor spectra, the absorption coefficient for the acceptor, the quantum yield of the donor, and the relative orientation of the donor and acceptor. In any case, when distances separating donor and acceptor are superior of 2 R0 , no FRET can occur. From this, it is possible to distinguish between 2 proteins being present in the same cytoplasmic compartment for instance, from those undergoing specific protein-protein interactions.

FRET Acceptor Bleaching involves measuring the donor “de-quenching” in the

presence of an acceptor. This can be done by comparing donor fluorescence intensity in the same sample before and after destroying the acceptor by photo bleaching. If FRET was initially present, a resultant increase in donor fluorescence will occur on photo bleaching of the acceptor. The energy transferred efficiency can be quantified as:

FRET eff= (Dpost-Dpre)/Dpost

where Dpos is the fluorescence intensity of the Donor after acceptor photo bleaching, and Dpre the fluorescence intensity of the Donor before acceptor photo bleaching. The FRET eff is considered positive when Dpost > Dpre. Choice of donor-acceptor pair: The FRET AB requires that the acceptor is easily bleached under normal experimental conditions, and that the donor is relatively stable. Also, it requires that on photo bleaching, the acceptor is no longer able to act as an efficient energy transferred acceptor by no longer absorbing an overlapping region with the donor. Few donor-acceptor pair follows these criteria: Cy3-Cy5, CFP-YFP, GFP-Cy3, and GFP-Cy3.5. Fluorescein-Rhodamine will FRET too. But this list is not exhaustive (please refer to our donor/acceptor list at the end of this application note).




FRET wizard in the Leica Confocal Software (LCS, 2.61.1537)

A- Click on Application. B- In the Application menu, choose FRET Acceptor Bleaching.

Fig. 1

Fig. 2




C- Step by step Step 1 of 3: Setting experimental conditions 1. Click on Donor �� (Fig. 3). Open the Beam Path Settings window��. Begin by detecting simultaneously the donor and acceptor. This will enable to access fluorescence intensity relations, PMT gain (the donor PMT should be below saturation), and laser intensity for each fluorophore ��. Use the Continuous �� button for online adjustments. Set the zoom if needed and the appropriate resolution (pixel size).

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Fig. 3




2. Reduce the laser excitation of the acceptor down to 0% �� (fig.4). Define number of frame and/or line averages if needed. Deactivate the acceptor PMT to finish to configure the donor acquisition.

3. Define the Acceptor by clicking on the Acceptor button �� (Fig.5). Reduce to 0% the donor excitation laser �� and deactivate the donor PMT. Activate the acceptor excitation laser, and the acceptor PMT ��. Adjust the PMT gain using the continuous button. Define number of frame and/or line averages if needed.

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Fig. 4

Fig. 5

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4. Proceed with Next . Step 2 of 3: Setting the bleaching for the acceptor fluorophore 1. Activate the acceptor laser and set it up to 100% �� (Fig. 6). 2. Draw your ROI by choosing your ROI shape �� and plot the ROI into the image where you want the bleaching to occur ��. 3. In the Bleach Configuration window,

• you can choose to use the Regulator by checking the box �� (Fig.7a) and entering a number between 0 to 1.0 (= 0 to 100%). The bleaching is stopped if the measured intensity falls below the value X. • if the regulator is not chosen, then you must enter the number of frame you want the bleaching to be applied �� (Fig 7b).

Fig. 6

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Fig. 7a

Fig. 7b




You can then save this configuration if you want to use the same hardware settings later on by clicking on the Save Configuration button . 4. Click on Run �� to execute the bleaching. 5. The experiment then will be running automatically in a precise sequence:

• Acceptor pre • Donor/Acceptor pre • Bleach Acceptor • Acceptor post • Donor/Acceptor post

The experiment progress can be followed �� and stopped if necessary ��. Step 3 of 3: Evaluating the energy transfer efficiency

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1. The result in the ROI_1 will be automatically displayed ��. It corresponds to your entire bleached area, and it would be quite usual to have a FRET eff of 0% in this entire area if the ROI_1 is big enough, or if the events are discrete in size. You can then draw sub-ROIs inside the ROI_1 in order to evaluate more discrete regions in the bleached area, like endosomes, plasma membrane receptors, or nuclear proteins for instance. These ROIs will be displayed as ROI_2, ROI_3, etc… ��. If your ROI_1 included an outside of the cell, or an area without labeling, then it can be used as background and the raw numbers can be subtracted from the ROI of interest for a more accurate result.

2. The ROIs must be SAVED in order to retrieve them once the FRET experiment is closed. For this, right click on your mouse at the level of the ROIs ��, choose Save All �� and give a name in the Save As window, then Click on Save button ��.

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In order to retrieve your ROIs, in your picture window (right screen), right click on your mouse, and then choose ROI and Open. 3. To view your results: you can click on Report. You will be asked to save

the report prior to visualize it, and your data will be displayed in an .xml format.




Once opened in xml format, you can Ctrl+A to select All (or highlight only part of the results that you need), then Ctrl+C to copy. If you open your Word software and Ctrl+V you will be able to edit your results under more “friendly” software. The results will then be presented as followed:

Result

ROI

D pre

D post

A pre

A post

FRETeff (%)

ROI_1

131.43

131.43

133.93

133.93

0.00

ROI_2

73.00

73.00

75.57

75.57

0.00

ROI_3

224.00

224.00

226.53

226.53

0.00

ROI_4

16.50

16.50

19.00

19.00

0.00




Donor-Acceptor pair: For you information, we have listed here the Donor-Acceptor pair which will FRET when doing a FRET AB. Remember that FRET will occur when R0 is <10-70 Å.

R0 (Å) Acceptor Donor

Alexa Fluor 488

Alexa Fluor 546

Alexa Fluor 555

Alexa Fluor 568

Alexa Fluor 594

Alexa Fluor 647

Alexa Fluor 350

50

Alexa Fluor 488

NA 64 70 62 60 56

Alexa Fluor 546

NA 70 71 74

Alexa Fluor 555

NA 47 51

Alexa Fluor 568

NA 82

Alexa Fluor 594

NA 85

Alexa Fluor 647

NA

Donor Acceptor R0 (Å) Fluorescein Tetramethylrhodamin 55

Cy3 Cy5 >50 CFP YFP 50 BFP GFP 40 CFP GFP 48 GFP YFP 57

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