Characterisation of amyloid structures in aging C. elegans using fluorescence lifetime imaging

Abstract

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington’s and Alzheimer’s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are only a limited number of tools available to assess the aggregation process of amyloid proteins within an animal.

Here, we present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells such as neurons in a non-invasive manner with the progression of ageing and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which will result in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of e.g. different amyloid proteins, environmental stimuli or genetic backgrounds in vivo in a non-invasive manner.

Introduction

Protein aggregation occurs both in aging and disease. The pathways that lead to the formation and deposition of large amyloids or amorphous inclusions are difficult to follow and its kinetics are similarly challenging to unravel. Proteins can misfold due to intrinsic mutations within their coding sequences, as in the case of genetic diseases. Proteins also misfold because the proteostasis network (PN) that keeps them soluble and properly folded is impaired, which can occur during aging. The PN includes molecular chaperones and degradation machineries and is responsible for the biogenesis, folding, trafficking and degradation of proteins¹.

C. elegans has emerged as a model to study aging and disease, due to its short lifespan, isogenic nature and ease of genetic manipulation. Several C. elegans transgenic strains have been created that for instance express human disease-causing proteins in vulnerable tissues. Importantly, many of the strains containing aggregation-prone proteins recapitulate the hallmark of amyloid disorders: the formation of large inclusions. Due to C. elegans′ transparent body, these aggregates can be visualised in vivo, non-invasively and non-destructively². Generating any protein of interest (POI) in fusion with a fluorophore allows to investigate its locations, trafficking, interaction network and general fate.

We present here a protocol to monitor the aggregation of proteins in living and aging C. elegans via fluorescence lifetime imaging microscopy (FLIM). FLIM is a powerful technique based on the lifetime of a fluorophore, rather than its emission spectra. The lifetime τ is defined as the average time required by a photon to decay from its excited state back to its ground state. The lifetime of a given molecule is calculated with the time-domain technique of time-correlated single photon counting (TCSPC). In TCSPC-FLIM, the fluorescent decay function is obtained by exciting the fluorophore with short high-frequency laser pulses and measuring the emitted photon′s arrival times to a detector, in respect to the pulses. When scanning a sample, a three- dimensional data array is created for each pixel: the array includes information on the distribution of the photons in their x,y spatial coordinates, and their temporal decay curve. A given sample therefore becomes a map of lifetimes revealing information on the protein′s structure, binding and environment ^3,4.

Each fluorescent protein possesses an intrinsic and precisely defined lifetime, usually of a few nanoseconds (ns), dependent on its physiochemical properties. Importantly, the lifetime of a fluorophore is independent of its concentration and fluorescent intensity, and of the imaging methodology. Within a biological system, it can however be affected by environmental factors such as pH, temperature, ion concentrations, oxygen saturation and its interaction partners. Lifetimes are also sensitive to internal structural changes and orientation. Fusing a fluorophore to a POI results in a change in its lifetime and consequently information on the behaviour of the fused protein. When a fluorophore is surrounded or encapsulated in a tightly bound environment, such as the anti-parallel beta sheets of an amyloid structure, it loses energy non-radiatively, a process known as quenching⁵. Quenching of the fluorophore results in a shortening of its apparent lifetime. When soluble, a protein′s lifetime will stay closer to its original, ‘higher’ value. On the contrary, when a protein starts to aggregate, its lifetime will inevitably shift to a lower value ^6,7. It becomes therefore possible to monitor the aggregation propensity of a protein at different ages in living C. elegans.

Here we describe a protocol to analyse the aggregation of a fusion protein comprising different polyglutamine (CAG, Q) stretches (Q40 and Q44). We illustrate how the technique can be applied equally to different fluorophores, such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and monomeric red fluorescent protein (mRFP); and in all tissues of C. elegans, as for example, the neurons, muscles and the intestine. Moreover, in the context of proteostasis, FLIM is a very useful tool to observe changes upon depletion of molecular chaperones. Knocking down one of the key molecular chaperones, heat shock protein 1 (hsp-1), via RNA interference produces premature misfolding of proteins. The increase in aggregation load as a result of aging, disease, or deficient chaperones, is then measured as a decrease in fluorescence lifetime.

Protocol

1. Synchronisation of C. elegans

   • 1.1Synchronize C. elegans either via alkaline hypochlorite solution treatment or via simple egg laying for 4 hrs at 20 °C⁸.
   • 1.2Grow and maintain nematodes at 20 °C on nematode growth medium (NGM) plates seeded with OP50 E. coli according to standard procedures⁹.
   • 1.3Age the nematodes until the desired stage or day. In this protocol, young adults are imaged on day 4 and old nematodes are imaged on day 8 of life.

2. RNAi mediated knockdown of chaperone machinery via feeding

   Perform knockdown of heat shock protein 1 (hsp-1) chaperone via feeding the corresponding RNAi vector to the nematodes ¹⁰. The hsp-1 RNAi plasmid was obtained from the Ahringer library (clone ID: F26D10.3).

   • 2.1Grow the HT115 (DE3) E. coli expressing the hsp-1 RNAi plasmid for 6 hrs to overnight in Luria Bertani (LB) medium containing 50 μg / ml ampicillin.
   • 2.2Prepare fresh NGM agar plates containing IPTG (1 mM) and ampicillin (25 μg/ml) and seed with the hsp-1 RNAi bacteria. Leave plates to dry and induce at room temperature for 1-3 days.
   • 2.3Place synchronised eggs on plate and leave to hatch, or place gravid nematodes and allow to egg lay for 4 hrs at 20 °C before removing. NOTE: the second generation of nematodes might present a stronger phenotype of the knockdown.
   • 2.4Grow the nematodes until the desired age or stage.

3. Preparation of microscopy slides

   On the selected day of imaging, start by preparing the imaging slides.

   • 3.1Melt agarose in ddH₂O at a concentration of 3% (w/v). Let cool slightly.
   • 3.2Cut the tip of a 1 ml pipette tip and take roughly 200 μl of melted agarose.
   • 3.3Pipette the agarose onto a clean glass cover slip and immediately place a second glass slip on top, avoiding the formation of any bubbles. Leave to dry and gently remove the top cover slip. The result is a glass slide with an even agarose surface where the nematodes will be positioned.
   • 3.4Each slide will be used to image between 5-10 nematodes. NOTE: slides can be prepared and stored for a few hours in a humified box to prevent the agarose from drying out.

4. Mounting of nematodes onto microscopy slides

   FLIM imaging requires the nematodes to be immobilised. Perform this step once the imaging set-up (microscopes, lasers, detectors, etc) are ready to use.

   • 4.1Using a platinum wire pick, place nematodes of the desired age onto a fresh unseeded plate and let crawl. This removes the excess OP50 bacteria from the nematode′s body.
   • 4.2Prepare the anaesthetic compound to immobilise the nematode. Either sodium azide or Levamisole can be used. WARNING: sodium azide (NaN₃) is highly toxic. Use gloves and protective eye wear and work under a ventilated hood. Keep a 500 mM NaN₃ stock in the dark at 4°C and dilute in ddH₂O freshly to a final concentration of 250 mM. If using Levamisole, dilute a 20 mM stock to 2 mM in ddH₂O.
   • 4.3Place a 10 μl drop of anaesthetic compound onto an agarose pad and gently transfer 5-10 nematodes into it. Keep nematodes close but not touching; this will allow for easier localisation of the C. elegans during image acquisition.
   • 4.4Carefully overlay the nematodes with a cover slip. NOTE: both anaesthetics will eventually kill C. elegans. Measurements should be taken within one hour after mounting.

     IMPORTANT: the nematode must be completely immobile during imaging, as the map of the lifetime is recorded from each pixel. Any movement of the x,y parameters prevents the reading of the lifetime in the same excited pixel.

5. Acquisition of FLIM data

   In this protocol, the lifetime of the fluorophore is acquired via the time-domain TCSPC method. FLIM requires a pulse of light to be generated by the laser at a set and constant repetition rate. The repetition rate varies according to the laser type and needs to be known by the end-user. Lifetime measurements are achieved by detectors and electronic equipment installed alongside a conventional microscope. In this protocol, measurements are performed on a laser scanning confocal microscope.

   IMPORTANT: check that the correct filters of emission/excitation are in place and minimise any amount of background or monitor backlight before starting.

   IMPORTANT: before starting any experiment, establish the photostability of the chosen fluorophore. If, within the nematode tissues, the fluorophore bleaches within a short time, it is not suitable for FLIM measurements in C. elegans.

   Acquisition of the instrument response function (IRF), which describes the timing precision of the instrumental set-up.

   • 5.1.1Remove the emission/excitation filters.
   • 5.1.2Record the scatter signal obtained from a cover slip placed above the objective. First, find the cover slip surface.

     NOTE: for lifetimes of several nanoseconds, the acquisition software can automatically estimate the IRF shift. Acquiring an IRF is always recommended.

   • 5.2Using a 10x magnification lens in transmission mode, localise the position of the nematodes on the slide.
   • 5.3Remove the slide, switch the objective to a 63x magnification lens and apply the required immersion medium (e.g. oil). Replace the slide on the stage and localise the nematodes.
   • 5.4Open the pinhole to the maximum and start scanning the sample. Select a region of interest (e.g. head, upper body, etc) and focus on its maximum projection plane.
   • 5.5Switch to the FLIM acquisition mode and software.
   • 5.6Enable the outputs to allow the detection of emitted photons.

     NOTE: The laser should have a maximum gate of 1e+08 single photon counts. This number represents the maximum number of photons supplied by the laser. Monitor the three other values present on the interface of the software. The constant fraction discriminator (CFD) provides information on the receipt of the single photon pulse by the detector, in reference to the laser pulse. This value should be roughly 1e+05. The time-to-amplitude (TAC) discriminates between the time one detected photon and the next laser pulse. Finally, the analogue-to-digital converter (ADC) converts the TAC voltage into a storable memory signal ¹¹. The CFD, TAC and ADC should all have similar values, to ensure that photons emitted by the fluorophore are not lost. Correct evaluation of these parameters ensures that enough photons are being collected to create an accurate lifetime map.

   • 5.7Preview the number of photons detected: the ADC value should be between 1e+4-1e+5. If necessary, shift the focus on a different plane or increase the laser power to collect more photons. In general, the number of photons per pixel should not exceed 1% of the repetition rate.
   • 5.8Press Start to begin acquisition. Acquisition can be set to a fixed amount of time or a fixed number of photons. Acquire a lifetime decay curve for two minutes minimum or until a single pixel reaches a photon count of 2000 single events.

     NOTE: different fluorophores will require different excitation and emission lasers and filters. According to the brightness of the sample, the laser power can also be adjusted – this will not interfere with the lifetime. The excitation/emission settings utilised in these protocols are as follows: for YFP ex500/em520-50 nm, for mRFP ex561/em580-620 nm. A pulsed two photon laser was employed for CFP measurements using ex800/em440 nm.

6. Analysis of FLIM data using FLIMfit software

   Perform data analysis using the FLIMfit software tool developed at Imperial College London ¹².

   • 6.1Open the software and import FLIM data files: File>Load FLIM Data…. NOTE: load all samples from one condition, even if obtained in different sessions and from different biological repeats.
   • 6.2If necessary, segment a single nematode from any FLIM picture: Segmentation>Segmentation Manager…. Drag the cropping tool around the area of interest until it is highlighted. Once completed, press OK. NOTE: segmentation must be done for all images.
   • 6.3Select a small region where the intensity-based image of a C. elegans appears. The decay curve of that region will appear in the large decay window on the right side of the interface. The decay can be displayed linearly or logarithmically.
   • 6.4Set the correct parameters to extrapolate the lifetime via the software′s algorithm:
   • 6.5
     On the Data tab:

     • 6.5.1
       Set an arbitrary integrated minimum value to exclude any pixels that are too dim to produce a good fit. Depending on the C. elegans sample this value varies from 40-300. Input different values until a satisfactory preview is achieved.
     • 6.5.2
       Select a Time Min and a Time Max number to limit the FLIM signal to these values. All events that appear before and after this threshold will be excluded. Here, the events prior to 2000 ps and after 10000 ps, depending on the lifetime of the fluorophore, are excluded.
     • 6.5.3
       Do not change the pre-set Counts/Photon of 1.
     • 6.5.4
       Input the repetition rate, in MHz, of the laser utilised during acquisition.
     • 6.5.5
       Input a Gate Max value to exclude all saturated pixels. For lifetime measurements in C. elegans, this value is set to any high number, e.g. 1e+4.
     • 6.5.6
       On the Lifetime tab:
     • 6.5.7
       Select a global fitting to be used. A pixel-wise fitting will produce a decay fitted to each individual pixel. An image-wise fitting will produce a global fitting of each individual image and display a single lifetime value per image. A global-wise fitting will produce a single fitting across the whole dataset: a single lifetime value is provided for all images.
     • 6.5.8
       Do not change any other parameter except for the No. Exp selection if it is known that the chosen fluorescence decay is multiexponential and exhibits more than a single lifetime.
   • 6.6Upload the IRF via the IRF menu: IRF>Load IRF…. To estimate the IRF shift, select IRF>Estimate IRF Shift. A set of values will automatically appear on the IRF tab, once established, do not change any other parameters of this tab.
   • 6.7Once all parameters are set, press Fit Dataset. The algorithm will produce a fit for the decay curve and establish a lifetime value for each image. 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.
   • 6.8In the Parameters menu select Statistic: w_mean (weighted mean) and check that the chi2 value is as close as possible to 1. A chi2 close to one ensures the accuracy of the fit. The lifetime value of the selected image is thus revealed as tau_1.
   • 6.9Export any information of interest: File>Export Intensity Images/Fit Result Table/Images/Histograms…. It is also possible and advised to save the data settings used to calculate the lifetime: File>Save Data Settings…. The parameters employed will be saved for future analysis of the selected samples.

7. Graphical representations of FLIM data

   The lifetimes collected from different samples can be visually represented in various ways. Select to denote the lifetime values either in nanoseconds or picoseconds.

   • 7.1Show the quality of the fit and the accuracy of the curve by exporting the decay curve directly from FLIMfit.
   • 7.2Represent the distribution of the photons by plotting the frequency of the photon count versus the lifetime value in a histogram.
   • 7.3Finally, for statistical comparison, if comparing two or more samples, place lifetime values plus standard deviation of the mean in a scatter plot bar graph. Perform any desired statistical analysis.

Representative Results

The protocol shows how to accurately monitor the formation of aggregated species in living C. elegans, during its natural aging and when subjected to stress. We have selected four different strains of transgenic nematodes expressing polyglutamine proteins of either 40Q, 44Q or 85Q repeats. The proteins are synthesized in different tissues and fused to different fluorophores. The C. elegans strains either express Q40-mRFP in the body wall muscles (mQ40-RFP), Q40-CFP in the nervous system (nQ40) and either Q44-YFP or Q85-YFP in the intestine (iQ44-YFP and iQ85-YFP¹³). To illustrate how aging promotes aggregation, we have collected the lifetime of these polyQ strains in young nematodes, at day 4 of life, and old nematodes, at day 8. To show the effects of a deficiency in the PN, we have performed a knockdown of hsp-1 in the mQ40-RFP and the nQ40 strains.

Once the lifetime values have been extrapolated via the FLIMfit software, the obtained data point to a clear reduction of the lifetime of any of the polyQ constructs when aggregated due to either glutamine load, aging or stress. FLIM can distinguish between the soluble protein fraction and aggregated species, and their transition, by recording a shift in their lifetimes.

At day 4, mQ40-RFP displays an average fluorescence lifetime of 1.69 ns (Fig.1). Upon aging, more aggregated species arise, appearing as low lifetime foci in the microscopy images and shifting the histogram to reduced lifetimes (Fig.1 A). By plotting the mean fluorescence lifetime of every acquired image over the age of the nematodes a significant reduction of fluorescence lifetime and therefore accumulation of aggregated species, becomes visible (Fig.2 B). The protein folding capacity of the PN declines after day 4 of life in C. elegans ¹⁴ and aggregation prone proteins misfold and can form amyloid and amorphous aggregates. Apart from the PN, the intrinsic aggregation propensity of a certain protein plays an important role in the progression of aggregate formation. This was analysed by comparing the behaviour of iQ44-YFP and iQ85-YFP. The longer Q-stretch of the iQ85 is more prone to aggregation and exhibits a fluorescence lifetime shift in the histogram already at day 4 of life (Fig.2 A). At day 4 in fact foci formation was observed for iQ85, while those were absent in iQ44. Upon aging however, iQ44 also exhibits foci formation and a reduced fluorescence lifetime. Since iQ85 exhibits aggregates already in early adulthood the progression of aggregation upon aging is less pronounced, yet significant (Fig.2 B). We could not detect foci formation nor decreased fluorescence lifetime in the nQ40-CFP strain (Fig.3 A). For this strain, there were only subtle and not significant changes to the mean fluorescence lifetime upon aging due to aging (Fig.3 B), potentially due to the neurons being less susceptible for yet unknown reasons.

Figure 1. Fluorescence lifetimes of muscular Q40-RFP decrease with age.

A. Representative maps of C. elegans expressing muscular Q40-RFP on day 4 or day 8 of life generated by FLIMfit. Fluorescence lifetimes, fluorescence intensity and a merged image of both are provided. Scale bars are 25 μm. Histograms show a distribution of measured lifetimes for all analysed nematodes divided into 100 categories. B. Bar plots showing the weighted mean fluorescence lifetimes of all analysed animals on day 4 or day 8 of life, respectively.

Figure 2. Fluorescence lifetimes of intestinal Q44-YFP and intestinal Q85-YFP decrease with age.

A. Representative maps of C. elegans expressing intestinal Q44-YFP or intestinal Q85-YFP on day 4 or day 8 of life generated by FLIMfit. Fluorescence lifetimes, fluorescence intensity and a merged image of both are provided. Scale bars are 25 μm. Histograms show a distribution of measured lifetimes for all analysed nematodes divided into 100 categories. B. Bar plots showing the weighted mean fluorescence lifetimes of all analysed animals on day 4 or day 8 of life, respectively.

Figure 3. Fluorescence lifetimes of neuronal Q40-CFP do not change with age.

A. Representative maps of C. elegans expressing neuronal Q40-CFP on day 4 or day 8 of life generated by FLIMfit. Fluorescence lifetimes, fluorescence intensity and a merged image of both are provided (the second lifetime, τ2, is indicated in all samples). Scale bars are 25 μm. Histograms show a distribution of measured lifetimes for all analysed nematodes divided into 100 categories. B. Bar plots showing the weighted mean fluorescence lifetimes of all analysed animals on day 4 or day 8 of life, respectively.

Knocking down hsp-1 poses a challenge to the PN of mQ40 and nQ40 expressing nematodes. RNAi-mediated depletion of hsp-1 led to a significant increase in aggregation (Fig.4 and Fig 5). Q40 expressed in body wall muscles tends to form a small number of large foci surrounded by non-aggregated material. This results in two distinguishable peaks in the histograms (at around 1.7 ns and 1.4 ns) (Fig.4 A). The aged, RNAi treated nematodes show a strong increase in the low-lifetime peak ultimately decreasing the average fluorescence lifetime (Fig.4 B). Compared to this biphasic behaviour of Q40 in muscles, the neuronal Q40 displays a more diverse aggregation behaviour. We cannot directly correlate foci formation with aggregation as for the muscle expression (Fig.5 A). As FLIM offers an opportunity to assess the degree of aggregation, the histograms reveal that there is no distinct peak but a widespread distribution of fluorescence lifetimes. Thus, a complex composition of different oligomers and higher order aggregates can be concluded. Still, the overall degree of aggregation can be evaluated by plotting the mean fluorescence lifetime (Fig.5 B), showing that hsp-1 knockdown leads to a boost in aggregation.

Figure 4. Fluorescence lifetimes of muscular Q40-RFP decrease upon knockdown of hsp-1.

A. Representative maps of C. elegans expressing muscular Q40-RFP on day 4 or day 8 of life generated by FLIMfit. A merge of the fluorescence lifetime and intensity map is displayed. For both time points, nematodes are shown that have been grown on bacteria expressing an empty vector (control) or bacteria expressing the hsp-1 RNAi-construct. Scale bars are 25 μm. Histograms show a distribution of measured lifetimes for all analysed nematodes divided into 100 categories. B. Bar plots showing the weighted mean fluorescence lifetimes of all analysed animals on day 4 or day 8 of life, with control or hsp-1 RNAi, respectively.

Figure 5. Fluorescence lifetimes of neuronal Q40-CFP decrease upon knockdown of hsp-1.

A. Representative maps of C. elegans expressing neuronal Q40-CFP on day 4 of life generated by FLIMfit. A merge of the fluorescence lifetime and intensity map is displayed. Nematodes are shown that have been grown on bacteria expressing an empty vector (control) or bacteria expressing the hsp-1 RNAi-construct. Scale bars are 25 μm. Histograms show a distribution of measured lifetimes for all analysed nematodes divided into 100 categories. B. Bar plots showing the weighted mean fluorescence lifetimes of all analysed animals on day 4, with control RNAi or the hsp-1 RNAi.

It is important to note that the lifetime of the fluorophores, free from a fusion partner and outside of a biological system, is indeed higher. Since the lifetime is affected primarily by its environment, a slight reduction of the lifetime of YFP and RFP is already noticeable within C. elegans′ tissues. It is therefore important to obtain the lifetime of the soluble POI within the nematode as a suitable control. A comparison between the soluble fraction with a higher lifetime and aggregated fraction with a lower lifetime can then be made. Here, the decrease in lifetime correlates with the formation of visible foci within muscle and intestinal cells. Still, a fraction of foci exhibits no decrease of fluorescence lifetime (see Fig 1 and 2, white arrows). This feature highlights how only part of the fusion construct might be aggregated at a particular spatiotemporal point, and the presence and availability of unbound protein. A more complex scenario arises from investigation of the neuronal Q40-CFP strain. CFP intrinsically possesses two distinct fluorescence lifetimes. While CFP is an ideal fluorophore for Forster resonance energy transfer (FRET)¹⁵ measurements, in conjunction with YFP, it is not advisable to employ it to monitor formation of aggregates in C. elegans.

Discussion

The protocol presented here describes a microscopy-based technique to identify aggregated species in the C. elegans model system. FLIM can accurately characterise the presence of both aggregated and soluble species fused to a fluorophore via measuring fluorescence lifetime decays. When a fusion protein starts to aggregate its recorded average lifetime will shift from a higher to a lower value¹⁶. The propensity of aggregation can then be deduced by the drop in lifetime: the lower the lifetime, the higher the presence of aggregated protein species in the system. Thereby, it becomes possible to follow the proteotoxic effects of aging, disease or impairment of PN on the aggregation-propensity of any protein.

To highlight the versatility of the technique, our results clearly show that FLIM can identify the changes in structure of the various polyQ constructs, regardless of the tissue or fluorophore. Importantly, FLIM has been already successfully applied in the characterisation of other aggregation-prone proteins within C. elegans, such as α-synuclein¹⁷. Furthermore, it becomes possible to apply any stress factor to C. elegans and follow the unfolding and aggregation of any POI. Osmotic, metal-ion, redox or chemical stress can all promote toxic imbalances which can successfully be monitored in C. elegans employing FLIM. The reverse could also be possible: a delay in aggregation or even disaggregation due to the presence of beneficial compounds or boosting of the PN, can results in a rescue of the soluble protein and a consequent increase of lifetime.

FLIM has been widely employed to monitor changes in lifetime of any substance in a wide array of disciplines from chemistry to cancer biology¹⁸ to medical diagnostics, but some limitations remain. A main problem is the photostability of fluorophores. As FLIM requires the recording of a large number of photons, photobleaching reduces the number of photons collected and can change the resulting decay curve. Furthermore, especially within the nematode system, if the intensity of the fluorophore itself is not sufficiently high for enough photons to be collected, a higher excitation is required, leading to quicker photobleaching and an unreliable decay curve. Finally, a practical downside to the technique is that it requires sophisticated and costly equipment as add-ons to pre-existing microscopy systems, with one critical element being the sensitivity of the detectors¹⁹.

Conversely, one of the main advantages of calculating the lifetime of a fluorophore and its fusion protein, is that it provides information on different fractions of the same fluorophore-protein complex in diverse state of interactions within the environment, irrespective of its largely unknown concentration. FLIM can be used also to measure lifetime in any phase, gas, liquid or solid and in any medium or organism that can be normally imaged, from cells to organisms and organelles to immobilised, purified proteins²⁰. Microscopy techniques usually rely on the steady-state imaging of a fluorescently tagged protein. Unlike steady-state techniques, FLIM can resolve changes in binding, composition and conformation of a biological substrate. For investigating aggregation-prone protein, the presence of large fluorescent inclusions or foci can be imaged easily, but it represents only a static, intensity-based snapshot³. In the case of aggregated species, the visualised foci may also be misleading, as a high concentration of fluorophore does not necessarily result in its strong amyloid formation. Via FLIM it is instead possible to distinguish soluble from insoluble material. Finally, it becomes beneficial for any investigation to obtain both the intensity-based measurements and the parallel lifetime measurement. Within the complementarity of these microscopy techniques lies their strength.

SUMMARY.

Lifetime fluorescent imaging monitors, quantifies and distinguishes aggregation propensities of proteins in living, aging and stressed C. elegans models of diseases.