The method allow us to clearly distinguish integrated amyloid protein structures from the soluble and even accumulated counterparts. The technique is performed in vivo in a noninvasive manner with little or no toxic side effects. As it depends on the fluorescent properties of the fluorophore itself, it is highly robust and reproducible over time.
FLIM has been utilized in the field of cancer diagnosis. However, we employ it to study the basic mechanisms of protein aggregation which are indeed relevant to neurodegenerative diseases. The methodology can be used in the field of protein aggregation.
As long as the disease associated protein is tagged with a fluorescent probe, it can be tracked and monitored over time. Compounds and strategies that either promote or prevent aggregation can be successfully studied. Any biological system that allows to be visualized by light microscopy is suitable for FLIM either in vivo or ex vivo, for example in cell culture, in fixated or permeabilized samples and in any medium necessary.
Make sure to test the properties of your selected fluorophore within the nematode before obtaining experimental data and to test that the setup is fully functional before acquiring measurements. To begin, grow and maintain nematodes at 20 degrees Celsius on NGM plates. Age the nematodes until day four of life as the young adults and day eight of life as the old adults.
On the day of imaging, start by preparing the imaging slides. Place agarose and double distilled water at a concentration of 3%weight by volume. Transfer it into a microwave to melt and then let it cool slightly.
Cut the tip of a one milliliter pipette tip and take roughly 200 microliters of the melted agarose. Pipette the agarose onto a clean glass slide and immediately place a second one on top avoiding the formation of any bubbles. Leave the slides to dry and gently remove the top glass slide.
The result is a glass slide with an even agarose surface where the nematodes will be positioned. Once the imaging setup is ready to use, working under a stereo microscope, place a 10 microliter drop of anesthetic compound onto an agarose pad and gently transfer five to 10 nematodes into it. Use an eyelash tip to separate the nematodes.
Keep them close together but not touching to allow for easier localization of the nematodes during image acquisition. Carefully overlay the nematodes with a coverslip. Within one hour after mounting, take measurements.
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 10X magnification lens in transmission mode, localize the position of the nematodes on the slide. Remove the slide, switch the objective to a 63X 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 one times 10 to the fourth and one 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 to signal 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 multi-exponential 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 one. Export any information of interest through file, export intensity images, fit result table, images, histograms.
Representative maps of C.elegans expressing muscular Q40 RFP on day four or day eight of life are shown here. The longer Q stretch of the IQ85 was more prone to aggregation and exhibited a fluorescence lifetime shift in the histogram already at day four of life. In fact, at day four, foci formation was observed for IQ85 while still absent in IQ44.
Upon aging, however, IQ44 also exhibited foci formation and thus a reduced fluorescence lifetime. Finally, foci formation was not detected nor was decreased fluorescence lifetime in the NQ40CFP strain. For this strain, there were only subtle non-significant changes to the mean fluorescence lifetime upon aging.
Once the nematode model is established, the amount of photon collected is paramount to obtain a good lifetime fit. The method can be further conjoined with different techniques such as Forster resonance energy transfer, FRET, fluorescence recovery after photobleaching, FRAP, or measurement. All of these adaptations provide extra information on the properties of the protein studies and its aggregated form.
Within the field of protein aggregation and protein homeostasis, the technique allowed to investigate any perturbation imposed on the system, the distribution of different protein species, and the trafficking and in general the difference and significance of soluble and insoluble protein fractions within the living organism. The anesthetic sodium azide is toxic and should be handled with gloves and goggles and diluted under a fume hood. Because the technique is microscopy-based, it is important to follow all warnings related to work with lasers.
