Fluorescence Lifetime Imaging of PolyQ Protein Aggregation in Caenorhabditis elegans Neurons

Begin with anesthetized, age-matched control and chaperone-deficient Caenorhabditis elegans worms. 

Both groups express fluorophore-tagged polyQ proteins in their neurons that are prone to misfolding. 

In control worms, chaperone proteins limit misfolded polyQ aggregation, whereas, in chaperone-deficient worms, misfolded polyQ readily forms aggregates. 

Place the control slide on a confocal microscope stage for Fluorescence Lifetime Imaging Microscopy (FLIM). 

Illuminate the worms using a pulsed laser. 

The laser excites the fluorophore, which emits a photon as it returns to the ground state. 

FLIM measures fluorescence lifetime—the duration the fluorophore remains in its excited state.

In chaperone-deficient worms, polyQ aggregation clusters fluorophores, promoting energy transfer between adjacent fluorophores instead of photon emission, thereby reducing fluorescence lifetime.   

The FLIM software processes these lifetimes to generate color-coded maps, enabling aggregation visualization.

A reduced fluorescence lifetime in chaperone-deficient worms, compared to controls, indicates increased polyQ aggregation.

Make sure the nematodes are completely immobile. Open the FLIM acquisition software. Locate the Tab button and press Enable Outputs. Place the slide with the mounted C. elegans on the stage and, using a 10 times magnification lens in transmission mode, localize the position of the nematodes on the slide. Remove the slide, switch the objective to a 63 times magnification lens, and apply the required immersion medium.

Place the slide on the stage and localize the nematodes. Start scanning the sample. Select a region of interest and focus on its maximum projection plane. On the interface of the FLIM software, preview the number of photons detected. The ADC value should be between 1 times 10 to the fourth and 1 times 10 to the fifth. If necessary, shift the focus on a different plane or increase the laser power to collect more photons.

- Ensure that you avoid photon pileup, but collect a sufficient amount of photons to create a good lifetime fit and measurement.

- In the menu bar, select the tab to set the acquisition parameters. Select Scan Sync In to allow for single photon detection. Set the acquisition to a fixed amount of time or a fixed number of photons. Press Start to begin acquisition. Open the software and import FLIM data files via File, Load FLIM Data. Load all the samples from one condition, even if obtained in different sessions and from different biological repeats.

If necessary, segment a single nematode for many FLIM picture via Segmentation, Segmentation Manager. Drag the cropping tool around the area of interest until it is highlighted. Once completed, press OK. Select a small region where the intensity-based image of a C. elegans appears. The decay curve of that region appears in the large decay window on the right side of the interface.

To extrapolate the lifetime on the Data tab, set an arbitrary integrated minimum value between 40 to 300 to exclude any pixels that are too dim to produce a good fit. Select a time minimum and a time maximum number to limit the FLIM signal to these values. Do not change the preset counts photon of one. Input the repetition rate in megahertz of the laser utilized during acquisition.

Input a gate max value to exclude all saturated pixels. On the Lifetime tab, select a global fitting to be used. Do not change any other parameter except the number of exponential selection if it is known that the chosen fluorescence decay is multiexponential and exhibits more than a single lifetime.

Upload the IRF via the IRF menu. To estimate the IRF shift, select IRF, Estimate, IRF Shift. A set of values automatically appears on the IRF tab. Once this is established, do not change any other parameters of this tab. Once all parameters are set, press Fit Data Set. The resulting fit highlighted in a blue line should overlap with all the events. A good fit is obtained when all events are aligned along the fit.

Click the Parameters tab located within the top right menus of the software's interface, and select statistic, weighted mean, and check that the chi-square value is as close as possible to one. The lifetime value of the selected image is thus revealed as tau 1. Export any information of interest through File, Export Intensity Images, Fit Result Table, Images, Histograms.