The overall goal of this experiment is to visualize complex formation of SNARE proteins in live cells. This method can help to answer key questions in the membrane trafficking fields, such as identifying the sets of SNARE proteins that catalyze the specific organella trafficking steps. The main advantage of this technique is that it allows direct visualization of cellular locations where SNAREs engage in complex formation.
In this experiment, 900, 000 HeLa cells divided between three Eppendorf tubes are used for transfection. Transfect the cells as described in the text protocol with the following constructs. Sample number one with the syntaxinin-4 mCitrine construct, sample number two with the syntaxinin-4 mCitrine and VAMP3-mCherry, and sample number three with syntaxinin-3 fused to both mCitrine and mCherry.
Culture the transfected cells in high-glucose DMEM supplied with 10%FCS and 1%antibiotic antimicotic at 37 degrees Celsius with 5%CO2 in a cell culture incubator overnight. On the following day, aspirate the medium and wash the cells once with live cell imaging medium for two minutes. After that, place the cells in fresh imaging medium.
To begin this procedure, place sample number two at 37 degrees Celsius, on a heated stage, under a time domain fluorescence lifetime imaging confocal microscope with a pulsed excitation source for the donor fluorophore and time resolved data acquisition. Prior to each fluorescence life time imaging microscopy or FLIM measurement, select a cell with visible expression of the mCitrine and mCherry labeled SNARE proteins and record a confocal image of 256 by 256 pixels with simultaneous excitation of both fluorophores. Record a FLIM image by clicking run FLIM with the FLIM image being recorded with the same dimensions and spacial resolution as the confocal image recorded earlier.
Record the image with at least 50, 000 photons for whole cell FLIM analysis or at least 400 photons per pixel. It is important to record the FLIM measurements at least one micrometer away from the surface of the glass to avoid a reflection view. Repeat the imaging from multiple cells in sample number two and for cells in sample number one and sample number three.
Next, place a clean glass cover slip on the microscope stage for recording an instrument response function or IRF. Tune the monochromator of the emission detector to the excitation wavelength and click the button run FLIM to record a FLIM image with the back scattering from the glass cover slip. The photo recordings will be converted to FLIM images with the PT32ICS conversion software.
Configure the software. Set output to ImageJ for generating FLIM images. Set the image size to 256 by 256 pixels and the channel to two.
Press convert and load one or more photon traces. Keep the control key pressed to select multiple photon traces. FLIM images should be generated in the same folder where the photon traces are saved.
Begin by opening the data analysis software program capable of fitting with deconvolution. Import the text file containing the histogram for each photon trace. Reorganize the table such that the IRF is in the second column of the table, next to the time values in column A.Determine the quality of the recorded photon traces by selecting all columns.
Do not analyze photon traces with a high reflection peak. An example is shown here. Use the control key to select all columns containing the quality controlled lifetime histograms that will be fitted, as well as the IRF in column B, and load the nonlinear fitting.
Load the deconvoluted fit function by adding this function to the analysis software. Select the fit function file. This function fits the fluorescence lifetime histograms with a mono exponential decay function deconvoluted with the IRF.
Perform X-axis scaling of the fitted curves. Fit the curves by pressing fit. This will convert the fit and generate a report sheet in the array with the table containing the fluorescence lifetimes offsets and amplitudes.
It will also generate a data sheet with the fitted curves and the residuals of the fit. Shown are representative confocal images of helocells that express only syntaxin four mCitrine, snytaxin four mCitrine with VAMP3 mCherry, or the syntaxin three mCitrine mCherry tandem construct. In these accompanying fluorescence lifetime images, the color indicates the average apparent fluorescent lifetime.
The images were then convoluted with the fluorescence intensities of the mCitrine donor fluorophore. Next are the same images with fitting with bi-exponential decay functions. The pixel colors indicate the estimated fractions of syntaxin four in complex with VAMP3.
The instrument response function of the setup was measured using the back-scattering on the glass water interface. Whole-cell lifetime histograms are shown for cells expressing only syntaxin four mCitrine, co-expressing syntaxin four mCitrine with VAMP3 mCherry, and expressing the syntaxin three mCitrine mCherry tandem construct. Panel E shows an overlay of the decay curves of panels B, C, and D.The inlays show the same graphs, but with logarithmic scaling of the Y-axis.
A fluorescent lifetime histogram recorded too close to the surface of the microscope cover slip resulted in a large reflection peak, depicted by the yellow shaded area. Lastly, a lifetime histogram with representative fit with bi-exponential decay function is shown. Once mastered, this technique can be done in two to four hours if it is performed properly.
While attempting this procedure, it's important to remember to record multiple cells while the expression levels of the donor and the interceptor SNARES can influence the lifetime. Following this procedure, the cells can be activated in order to answer cell type specific questions related to re-rooting of SNARE mediated membrane trafficking. After its development, this technique paved the way for researchers in the membrane trafficking field to explore the intercellular pathways of organella traffic in all eucaryotic cells.
After watching this video, you should have a good understanding of how to record and analyze the FLIM data. Don't forget that working with genetically modified organisms and high powered lasers can be extremely hazardous and precautions, such as gloves and eye protections, should always be taken while attempting this procedure.
