The overall goal of this procedure is to demonstrate how to implement time gated fluorescence lifetime imaging microscopy, or FLIM, in an automated multi well plate work flow using opensource software and basic instrumentation. This methodology can be applied to FLIM based readouts in a series of fixed or live cells. And it's particularly useful for reading out cell signal processing or protein interactions.
The main advantages of this technique are that FLIM provides robust quantitative readouts and can provide interaction population fraction in a single measurement. Applying global analysis techniques to thousands of cells over hundreds of fields of view, lets us utilize foster resin energy transfer, or FRET readouts. And taking advantage of, for instance, lifetime changes down to about 20 picoseconds.
Demonstrating the procedure with Frederik Gorlitz will be Sunil Kumar, our research associate and Ian Munro, our software developer. Begin by starting the micromanager and open the openFLIM-HCA plugin. Under the FLIM control tab, select delay box calibration file, and open the calibration files.
Select the calibration file for the specific combination of the delay generator unit and laser repetition rate of interest. Under the file menu, select set base folder and select the folder where the data will be saved. After confirming that all the appropriate parameters have been set, manually set a gate width of four nanoseconds at the gated optical intensifier control box for the whole experiment.
To acquire instrument response function image data, manually place a beam block in the filter cube cassette before the objective to prevent the escape of any laser light and angle the beam block to minimize any reflection back into the microscope frame. Under the light path control tab, set the excitation, dichroic and emission filters in the spinning disk unit, such that the fraction of excitation light scattered from the spinning Nipkow disk can be imaged without saturating the system for a minimum of 200 milliseconds camera integration time. Under the FLIM control tab, use the find max point function over the full range of the delays covered by the programmable fast delay box.
Then check the displayed output trace. And set the course delay times, so that the full instrument response function profile, is within the scan range of the fast delay box. Adjust the neutral density and exposure time parameters.
As well as the frame accumulation, so that the camera is not saturated in the brightness time gate and the effective integration time is not less than 200 milliseconds. Under the FLIM control menu, select fast delay box and set 25 picoseconds delay steps over the full delay range. Next, select populate delays and click snap FLIM image to acquire an instrument response function image.
And wait until the acquisition is completed. Under the light path control menu, select neutral density and click blocked to block the laser. Open the FLIM control menu and select fast delay box to reduce the number of delay steps by increasing the value in the increment box.
Then click snap FLIM image to acquire a background camera image. To acquire reference die data, go to the XYZ control tab and enter the H4 well in the go to well dialogue box. Next, go to the light path control tab and select the appropriate excitation, dichroic and emission filters for imaging the M turquoise fluorescent protein using the find max point function over the full range of the fast delay box.
Then set the appropriate parameters for acquiring the reference die data and a corresponding background image as just demonstrated. To acquire the FLIM data from a multi well plate sample, go to the setup HCA sequenced acquisition menu. Select the XYZ positions tab and set which wells should be imaged and the number of fields of view to be acquired per well.
Select a representative field of view containing the sample to be imaged and set the optimal neutral density filter in the integration time of the camera to reach 75%of the dynamic range of the readout camera. Under the XYZ control tab, select return focus control in the auto focus dialogue box and manually focus the microscope on the cells or structures of interest. Set the auto focus search range to 2000 micrometers and press enter.
Then click AF now to initiate the auto focus procedure. An offset value will appear on the focus offset auto focus field. Enter this offset value into the AF offset window, and click AF now twice to repeat the auto focus procedure.
Offset values should now be set to zero, indicating a correct functioning of the auto focus system. Next, under the FLIM control tab, select the autogating function to ensure a logarithmic sampling of the delay points. Set accumulate frames to a value that yields the desired total data acquisition time.
Then, in the FLIM acquisition dialogue box, click the start HCA sequence button to run the FLIM multi well plate acquisition and acquire a background camera image as just demonstrated. Here a representative FLIM fret assay using the model fret system expressed in cocells and a multi well plate is shown, with examples of the automatically acquired fluorescence lifetime images of typical fields of view apparent in each well. In this experiment the negative control wells presented the longest lifetimes, and the donor lifetimes exhibited the lowest fret constructs with the shortest linker lengths.
Overall, the mean lifetime averaged over all the fields of view in each well varied across the multi well plates. Averaging the donor fluorescence intensity decay profile over all the cells imaged in well A1 together with the fit to a mono exponential decay model and the instrument response function, illustrates that the fitting model is valid. Further analysis of the average fluorescence lifetime for each column of the multi well plate, obtained from a pixel wise mono exponential fit, demonstrates the lifetime decreases in the fret constructs with shorter linker lengths, simulating an experiment with different fret efficiencies.
In this experiment, the FLIM data of a fret assay of the action of different inhibitors on protein oligomerization was fitted with a single exponential decay model. The fluorescence lifetime plate map illustrates the mean lifetime per well averaged across eight fields of view. One small set, imaging four field of views per well, while our FLIM assay can be completed in approximately two and a half hours, including the time to acquire the instrument response function, the reference die data and to run the analysis in FLIM fit.
When performing a FLIM assay it's important to remember to acquire the instrument response function and reference die data together with their background images so that you've got all the information needed to analyze the FLIM data. Using our opensource software, FLIM fit, it's also possible to fit this data into more complex decay models. For example, allowing the determination of the fret population fraction.
Our FLIM HCA technology is helping researchers in the drug discovery community make robust measurements of fret. In the future we hope this technology will enable dissociation constants to be measured in cell based assays. We hope that this video paper also the information in our Wiki will help other researchers build their own FLIM HCA instrumentation and apply it to fret and to other assays.
Don't forget that working with lasers can be hazardous and that precautions such as enclosing all the laser beams should always be taken when using such instrumentation.
