Setting up ‘Sensitized Emission’ based FRET experiments on Zeiss LSM780 confocal microscope

[Credits: Manoj V Mathew, Centre for Cellular and Molecular Platforms (CCAMP), Bangalore, India.
Experiments were conducted at the Central Imaging and Flow Cytometry Facility (CIFF), National Center for Biological Sciences (NCBS), Bangalore, India.]

Optical microscopes are resolution limited. However, there are ways to obtain sub-resolution information or measure sub-resolution distances in resolution limited images. One such technique is FRET.

FRET stands for Förster Resonance Energy Transfer. It involves two sets of molecules-one called 'Donor' and the other called 'Acceptor'. When a 'Donor-Acceptor' pair of molecules is in close proximity to each other the 'Donor' molecule can transfer its energy to the 'Acceptor' molecule. 

This happens non-radiatively via a dipole-dipole interaction mechanism. A 'Donor' molecule gets excited by excitation with its excitation wavelength. It transfers its energy to an 'Acceptor' molecule in its close proximity. The 'Donor' molecule then relaxes to the ground state without emitting a photon. The energy transfer results in the 'Acceptor'  molecule getting excited to its excited state and subsequently relaxing to its ground state radiatively (spontaneously emitting a photon).

These interactions are non-linearly (to the 6th power) dependent on the distance between the 'Donor' and the 'Acceptor'. They are significant only at very short distances between the 'Donor' and 'Acceptor', usually of the order of 10nm. By looking at FRET signals in diffraction-limited fluorescence images one can determine regions where the 'Donor-Acceptor' pairs are in very close proximity (<10nm) and measure the distance to an accuracy of the order of 1nm.

For FRET to occur there are some conditions the 'Donor' and 'Acceptor' molecules need to meet. For example, the 'Donor' emission spectrum should overlap with the absorption spectrum of the 'Acceptor'. One widely used FRET pair is ECFP and EYFP which satisfies this condition. Their spectra can be seen here.

There are many applications of FRET. One could study protein folding for example. If a FRET pair is tagged to the two ends of a protein, when it folds, the FRET pair gets closer to each other and exhibit stronger FRET signals. 

There are multiple ways to detect FRET. The following are some of them:

  1. Sensitized Emission: The 'Donor' is excited. The 'Donor' transfers energy to the 'Acceptor'. Acceptor fluorescence increases. 'Acceptor' fluorescence is measured.
  2. Acceptor Photobleaching: In a region of the sample, the Acceptor molecules are photobleached. This prevents the non-radiative transfer of energy from the Donor to the Acceptor. This results in an increase in 'Donor' fluorescence emission as more of the 'Donor' molecules in the region decays to the ground state radiatively. 'Donor' fluorescence before and after 'Acceptor' photobleaching is measured.
  3. Polarization Anisotropy: This technique is more useful for Homo-FRET ('Donor' and 'Acceptor' are the same species) measurements. Polarization anisotropy decreases with increasing FRET. Polarization anisotropy is measured.
  4. FRET by FLIM: This is probably the most sensitive FRET measurement technique. 'Donor' fluorescence lifetime decreases with increase in FRET. Donor fluorescence lifetime is measured most commonly using a Time-Correlated Single Photon Counting (TCSPC) based system.

For more details on the technique refer to the link

The idea of 'Sensitised Emission' would seem straightforward: Excite the 'Donor' and measure the fluorescence of the 'Acceptor'. However, there are few things we need to keep in mind. The very fact that we need the Donor's emission spectra to overlap with the absorption spectra of the Acceptor also means that there is inevitably some amount of:

  1. Cross Excitation: Some 'Acceptor' molecules will get excited directly by the 'Donor' excitation wavelength.
  2. Bleed Through: Some amount of 'Donor' emission will bleed into the 'Acceptor' channel.

This means that not all 'Acceptor' fluorescence is a result of FRET.  There are few other factors as well that can bias the results.

To have a more unbiased data from 'Sensitized Emission' based FRET experiments one needs to have a measure of these biasing factor. The following is the basic protocol:

a. Take three sets of images with the following sample sets:

  1. Sample with 'Donor' molecules only
  2. Sample with 'Acceptor' molecules only
  3. Sample with both 'Donor' and 'Acceptor' fluorophores

It is also possible to use only one sample if one can find regions within the sample with 1) only 'Donor',  2) only 'Acceptor' and 3) both 'Donor' and 'Acceptor'.

b. For each of these three sets take images using the following channels:

  1. Donor Channel: Excitation with 'Donor' excitation wavelength and collection in the 'Donor' emission spectrum.
  2. Acceptor Channel: Excitation with 'Acceptor' excitation wavelength and collection in the 'Acceptor' emission spectrum.
  3. FRET Channel: Excitation with 'Donor' excitation wavelength and collection in the 'Acceptor' emission spectrum.

In this write-up, I will describe how to use the Carl Zeiss LSM 780 confocal microscope to set up a 'Sensitized Emission' based FRET experiment using the 'ZEN' software.

Step 1: Setup the tracks and channels 

Setup three tracks with the following channels for sequential imaging with the following channels:

  1. Donor Channel
  2. Acceptor Channel
  3. FRET Channel

This is shown in Figure 1907/1.

Figure 1907/1: Setting up the three tracks 1) Donor Channel, 2) Acceptor Channel and 3) FRET channel

I used a fixed thin section of convallaria as the sample. While this is definitely not a good sample to demonstrate FRET, it is certainly enough for us to learn the software features. Convallaria is autofluorescent for excitations with most visible laser excitation sources. I used 488nm and 561nm excitation sources and spectral detector settings commonly used for Alexa 488 dye and Alexa 568 dye for the 'Donor' as well as 'Acceptor' and 'FRET' channels respectively. 

The channel configurations for the three tracks are shown in Figure 1907/2.

Figure 1907/2: Channel configurations for the three tracks- Left) Donor Channel, Middle) Acceptor Channel and Right) FRET channel

The following need to be kept in mind when configuring the channels:

  1. Use the same primary dichroic mirror for all channels
  2. Adjust the spectral detection bands to minimize bleed-through in the Donor and Acceptor channels.
  3. Use exactly the same spectral detection bands for both Acceptor and FRET channels

In addition, make sure that the Laser Power and PMT gain for each channel are set appropriately so as to avoid saturation of images. This is very important in any intensity quantification experiments. Saturation can be checked using the 'Range Indicator' feature in the software. It is also good to have the sequence mode as 'Change track every Frame' rather than 'Change track every Line'

Step 2: Acquire Images

Acquire three sets of images. Each set is imaged with one of the three samples using all the three tracks ( 1.only 'Donor',  2. only 'Acceptor' and 3. both 'Donor' and 'Acceptor'). This results in three image sets with 3 channels each (one each track).

In my case, I used a single sample and identified two regions 1) that had only 'Donor' and 2)only 'Acceptor' fluorescence. The whole field of view was used as the one with both 'Donor' and 'Acceptor'. For this, I first took an overview image with the track configuration described above. Figure 1907/3 shows the overview image.

Figure 1907/3: Overview image- Green: Donor Channel, Red: Acceptor Channel, and Blue: FRET channel

From the overview image, I identified regions with only green or red fluorescence. Using the 'Regions' feature the required region with only 'Donor' was marked for imaging. The 'Regions' setting is shown in Figure 1907/4.

Figure 1907/4: Regions setting

Make sure to uncheck bleach and check acquisition.

Check all the tracks and click 'Snap'. This will acquire an image of the ROI with only 'Donor' fluorophore in all the three channels. Save this image as 'Donor Only'

Similarly, use the 'Regions' feature to select a region with only 'Acceptor' fluorophores. Snap the image with all the three tracks and save the image as 'Acceptor Only'. 

The ROI images acquired using all the three tracks are shown in Figure 1907/5.

Figure 1907/5: ROI image with Donor Only (Left) and Acceptor Only (Right)

Now snap an image using all the three tracks of an ROI that has both 'Donor' and'Acceptor' fluorophores. I used the whole field of view. Call this image 'Donor and Acceptor'. In this case, the image will be same as the overview image I took. Now we have acquired three images each with three channels (one in each track). This is shown in Figure 1907/6.

Figure 1907/6: The three acquired images

Step 3: Set Donor and Acceptor parameters

Select the on the 'Donor Only' image and click on the 'FRET' tab on the side panel. This is shown in Figure 1907/7.

Figure 1907/7: FRET tab on the Visualization side panel

Note that the 'FRET' tab appears automatically under the following circumstances:

  1. Acquisition using 3 tracks (For 'Sensitized Emission')
  2. Acquisition using 2 tracks with one having a bleaching with time series (for 'Acceptor Photobleaching')

In the FRET GUI click on 'Parameters'. This is shown in Figure 1907/8.

Figure 1907/8: Setting Donor parameters

Do the following: 

  1. Select the Donor, Acceptor and FRET channels from the track assignments
  2. Click 'Donor' tab. This will calculate the Donor parameters Fd/Dd and Ad/Fd
  3. Select 'Donor' from the bottom drop down menu and click save. Save the parameters as 'Donor Parameters'

Similarly, select the 'Acceptor Only' image and click on the 'FRET' tab on the side panel. In the FRET GUI click on 'Parameters'. This is shown in Figure 1907/9.

Figure 1907/9: Setting Acceptor parameters

Do the following: 

  1. Select the Donor, Acceptor and FRET channels from the Track assignments
  2. Click 'Acceptor' tab. This will calculate the Acceptor parameters Fa/Aa and Da/Aa and Da/Fa.
  3. Select 'Acceptor' from the bottom drop down menu and click save. Save the parameters as 'Acceptor Parameters'

Kindly note that for the Convallaria sample I used has broad autofluorescence. The 'Donor' only and 'Acceptor' only regions may not exactly be that way. I selected them based on how they appeared in the overview image.

You can adjust the thresholds in the 'Thresholds' tab manually or select a background ROI in the image which will help the software calculate the thresholds. It would be advisable to define a background ROI (described below). The 'Thresholds' tab is shown in Figure 1907/10.

Figure 1907/10: 'Thresholds' Tab

For most applications set the 'Settings' tab to as shown in Figure 1907/11.

Figure 1907/11: 'Settings' Tab

Step 3: Analyze

Select the 'Donor and Acceptor' image and click on the 'Parameters' tab on the FRET GUI. Select 'Donor' from the drop-down menu. Load the 'Donor Parameters' file which was saved earlier. Then select 'Acceptor' from the drop-down menu and load 'Acceptor Parameters' file.

Subsequently, select the 'FRET' tab in the FRET GUI.  This is shown in Figure 1907/12. 

Figure 1907/12: 'FRET' tab

Select the Analysis method from the dropdown menu. There are three options available:

  1. Fc (Youvan)-"This method assumes that the signal recorded in the FRET channel is the sum of real FRET signal overlaid by donor crosstalk and acceptor signal induced by direct (donor) excitation. There is no correction for donor and acceptor concentration levels and as a result, the FRET values tend to be higher for areas with higher intensities". (Source: Zeiss LSM 880 ZEN 2 Manual)
  2. FRETN (Gordon) -"This method calculates a corrected FRET value and divides by concentration values for donor and acceptor. This method attempts to compensate for variances in fluorochrome concentrations but overdoes it. As a result cells with higher molecular concentrations report lower FRET values."(Source: Zeiss LSM 880 ZEN 2 Manual)
  3. N-FRET (Xia)-"This method is similar to the Gordon method with the difference that for concentration compensation the square root of donor and acceptor concentration is used. The resulting image is properly corrected for variances in the fluorochrome concentration." (Source: Zeiss LSM 880 ZEN 2 Manual)

Here I have used the N-FRET (Xia) method. 

Once a method is selected use the ROI tools in the FRET GUI to select a background ROI (a region with no signal). Check the 'Background' and 'Enabled' checkboxes for the background ROI. This helps the software to calculate the thresholds. 

Click the 'Analyze' button. This sted processes and analyzes the 'Donor and Acceptor' image to generate the FRET image. The FRET image will be displayed as shown in Figure 1907/13. 

Figure 1907/13: FRET image and FRET analysis results

Use the ROI tools in the FRET GUI to select an ROI on the image. The FRET analysis data for this ROI will be displayed below the image.

Kindly note again that I have used a highly autofluorescent sample which displays strong cross-excitation and bleedthrough. For the same reason, the calculated parameters might not be accurate enough as well. So please do not read too much into the FRET image and the results that are shown here. 

Posted in Fluorescence Microscopy, FRET, System Configuration and Troubleshooting.

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