Live Cell FRET

Note: this protocol has been superseded by two new FRET protocols.

Our microscope is capable of FRET measurements using conventional CFP/YFP pairs and their more modern derivatives. We recommend using mTurquoise2 and mVenus(L68V) due to their high quantum yield and good FRET overlap. While many FRET approaches can be used (e.g. acceptor photobleching), we suggest using the seFRET method of Kaminski et al, and their corresponding matlab scripts for analysis.

Required Controls and Samples

For seFRET each experiment requires cells expressing:

1. The donor fluorophore alone (e.g. mTurqoise2)
2. The acceptor fluorophore alone (e.g. mVenus)
3. A donor-acceptor fusion of known FRET efficiency (e.g. Vec50 - pmVenus-mTurquoise2, FRET efficiency of 42%)
4. The sample(s) expressing the reporter FRET pairs

Note: the donor and acceptors must be the same for all constructs - e.g. you cannot use CFP for controls and mTurquoise2 for the FRET pairs.

These samples should be prepared for each experiment, with the constructs expressed in the same cell line at a similar expression level. Additional fluoroscopes can be added, keeping in mind that their will be some spectral leakage of mVenus into the Cy3 channel, and that the DAPI/GFP channels are unavailable.

---

Microscope Configuration

All normal good microscopy practices should be followed, including proper sample preparation and labelling technique, selection of excitation energy and exposure time, etc. image the controls prior to imaging the samples to ensure all microscope settings are configured properly.

1. Exchange the normal excitation filter set for the FRET/ratio filter set (labelled Ratio-EX)
2. Startup the microscope as per usual
3. Switch to the EM-CCD camera
4. Configure other settings (Z-stacking, point visiting, time-laspe, etc) as needed

Donor-Only Sample

First image the donor-only sample. Two values are required from this sample - the intensity of the excited donor in the donor emission channel (I^DD), and the intensity of the excited donor in the acceptor emission channel (I^DA):

1. Configure the I^DD channel
   1. Set the ratio excitation wheel to 430 nm, emission wheel to 470/24
   2. Set the excitation intensity, EM-CCD gain, and exposure time to produce a reasonable image without saturation
2. Measure I^DA
   1. Do not change the ratio excitation wheel (keep at 430 nm)
   2. Keep the same excitation intensity, exposure time, and EM gain as for the I^DD channel
   3. Set the emission wheel to 535/50
3. Add additional wavelengths or DIC as required

Acquire at least 5 cells expressing the donor vector. Exposure time, excitation intensity and gain can be adjusted between images, but these settings must be kept the same between the I^DD and I^DA channels for each cell. Note that I^DD should always be brighter than I^DA, so the I^DD channel should be used to set excitation intensity, EM-gain and expsoure time for both the I^DD and I^DA channels.

Acceptor-Only Sample

Next, image the acceptor-only sample. Two values are required from this sample - the intensity of the excited acceptor in the acceptor emission channel (I^AA), and the intensity of the acceptor excited by the donor channel (I^DA):

1. Configure the I^AA channel
   1. Set the ratio excitation wheel to 490 nm, emission wheel to 535/50 nm
   2. Set the excitation intensity, EM-CCD gain, and exposure time to produce a reasonable image without saturation
2. Measure I^DA (note: this is not the same as the I^DA calculated with the donor-only sample)
   1. Do not change the ratio emission wheel (keep at 535/50 nm)
   2. Keep the same excitation intensity, exposure time, and EM gain as for the I^AA channel
   3. Set the ratio excitation wheel to 430 nm
3. Add additional wavelengths or DIC as required

Acquire at least 5 cells expressing the acceptor vector. Exposure time, excitation intensity and gain can be adjusted between images, but these settings must be kept the same between the I^AA and I^DA channels for each cell.

FRET Positive Control

The final required control is a positive FRET control with known FRET efficiency. Three channels must be collected - I^DD, I^DA and I^AA:

1. Configure the I^AA channel first, as this one is usually the brightest. Set the ratio excitation wheel to 490 nm, and the emission wheel to 535/50 nm
   1. Set the exposure time, EM-gain and exposure intensity to produce a reasonable image without saturation
2. Keeping the exposure time, EM-gain and exposure intensity the same, configure the I^DD and I^DA channels
   1. I^DD - Ex: 430 nm, Em: 470/24 nm
   2. I^DA - Ex: 430 nm, Em: 535/50 nm
3. Add additional wavelengths or DIC as required

Acquire at least 5 cells expressing the positive control vector. Exposure time, excitation intensity and gain can be adjusted between images, but these settings must be kept the same between the I^DD, I^DA and I^AA channels for each cell.

Sample Acquisition

1. Configure the I^DD, I^DA and I^AA channels as per the "FRET Positive Control" section, above
2. Configure additional fluorescent or DIC channels as needed
3. Configure additional acquisition parameters (time-lapse, point visiting, etc) as required
4. Image the sample, acquiring the I^DD, I^DA and I^AA channels for each cell at east time-point/z-position/etc

Shutdown

DO NOT FORGET TO RETURN THE RATIO FILTER WHEEL TO ITS HOLDER, AND THE DEFAULT WHEEL TO THE MICROSCOPE

1. Save all files, export to TIFFs as required
2. Close LAS-X and shutdown as per usual
3. Return the ratio filter wheel to its holder, and place the default excitation wheel back into the lightpath

DO NOT FORGET TO RETURN THE RATIO FILTER WHEEL TO ITS HOLDER, AND THE DEFAULT WHEEL TO THE MICROSCOPE

Analysis

Note: We are in the process of modifying the matlab code, so this section will change

1. Create separate folders for the donor alone, acceptor alone and positive control images
2. Export the controls as multi-channel 16-bit TIFFs to the corresponding folder
3. Export the FRET channels as multi-channel/multi-timepoint TIFFs to their own folder

<details to come>