Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging
March 27th, 2020
Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.
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.
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Overview
This study utilizes fluorescence lifetime imaging (FLIM) to monitor and quantify protein aggregation in living C. elegans models. The technique allows for noninvasive observation of protein structures relevant to neurodegenerative diseases.
Key Study Components
Area of Science
- Neuroscience
- Biophysics
- Cell Biology
Background
- Protein aggregation is a key feature in neurodegenerative diseases.
- FLIM provides a method to distinguish between different protein states.
- The technique is performed in vivo with minimal toxicity.
- FLIM has applications beyond neuroscience, including cancer diagnosis.
Purpose of Study
- To investigate the mechanisms of protein aggregation.
- To distinguish between integrated amyloid structures and soluble proteins.
- To apply FLIM in the context of aging and stress in C. elegans models.
Methods Used
- Fluorescence lifetime imaging (FLIM)
- In vivo imaging of C. elegans
- Noninvasive observation techniques
- Analysis of protein aggregation dynamics
Main Results
- Successful differentiation of amyloid protein structures from soluble counterparts.
- Demonstrated robustness and reproducibility of FLIM over time.
- Highlighted the relevance of protein aggregation to neurodegenerative diseases.
- Showed minimal toxic side effects of the imaging technique.
Conclusions
- FLIM is a valuable tool for studying protein aggregation in living organisms.
- The method can enhance understanding of neurodegenerative disease mechanisms.
- Future applications may extend to other fields, including cancer research.
Frequently Asked Questions
Executive Industry Relevance
Fluorescence lifetime imaging (FLIM) enables noninvasive, quantitative monitoring of amyloid fibrilization in living models, supporting target validation and mechanistic de-risking in neurodegenerative disease programs. By distinguishing integrated amyloid structures from soluble species without staining, FLIM provides predictive confidence in early discovery workflows. The method’s robustness and reproducibility facilitate cross-functional collaboration and scalable assay development for protein homeostasis research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by distinguishing amyloid fibrils from soluble and accumulated protein species in vivo.
- Operational Value: Supports biological de-risking through quantitative, fluorescence-lifetime-based readouts that correlate directly with aggregation levels.
- Predictive Value: Facilitates portfolio triage by allowing comparison of fibrilization across genetic backgrounds, environmental stimuli, or amyloid variants.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream screening by enabling label-free, longitudinal tracking of protein aggregation in specific cells such as neurons.
- Operational Value: Ensures assay standardization and reproducibility through FLIM’s independence from fluorophore expression levels and resistance to photobleaching.
- Scalability: Enables reliable compound evaluation by detecting aggregation-promoting or -preventing strategies in any light-microscopy-compatible biological system.
Translational & Preclinical Research
- Translational Continuity: Connects discovery through preclinical validation by monitoring aggregation dynamics across aging and proteostasis network perturbations in disease-relevant models.
- Risk-Adjusted Decisions: Supports advancement decisions by quantifying soluble versus insoluble protein fractions in living organisms over time.
- Mechanistic De-risking: Focuses on predictive value when studying protein homeostasis, enabling comparison of trafficking and distribution of protein species without further staining.
Pipeline & Workflow Integration
FLIM integrates into the discovery continuum from target validation through lead identification to preclinical studies by providing quantitative, noninvasive readouts of protein aggregation in living systems.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling visualization of amyloid structures in specific cells such as neurons during aging.
- Screening: Delivers assay readiness and quantitative outputs through photon collection and lifetime fitting that exclude dim pixels and saturated signals for robust measurements.
- Analytics: Provides statistical outputs such as chi-square values near one and weighted mean lifetime (tau one) to compare conditions and assess fit quality.
- Translational Research: Connects to preclinical continuity by enabling observation of aggregation in aging models and upon proteostasis network perturbation, relevant to neurodegenerative disease mechanisms.
- Enterprise Reuse: Functions as a reusable platform for comparing fibrilization across different amyloid proteins, genetic backgrounds, or environmental stimuli in vivo.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation, reduction of mechanistic ambiguity in protein aggregation studies.
- Operational Value: Standardization, reproducibility, and scalability of amyloid detection in vivo without staining or bleaching.
- Strategic Value: Improved go/no-go decisions, capital efficiency, and reduced late-stage biological risk through early detection of aggregation propensity.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions based on quantitative, longitudinal aggregation data in disease-relevant models.
Implementation Considerations
- Requires expertise in fluorescence lifetime imaging and laser safety protocols.
- Needs FLIM-capable microscopy setup with appropriate objectives (e.g., 63X immersion) and photon detection capabilities.
- Demands cross-team standardization of acquisition parameters such as scan sync, repetition rate, gate max, and photon collection thresholds (10⁴–10⁵ ADC).
- Involves adaptation considerations across model systems, including agarose pad preparation, anesthetic use (e.g., sodium azide), and immobilization protocols.
- Includes practical limitations such as the need to pre-test fluorophore properties in the nematode model and avoid photon pileup during acquisition.
Biopharma FAQs
Chapters in this video
0:05
Introduction
1:25
Synchronization of C. elegans and Mounting Nematodes onto Microscopy Slides
2:58
Acquisition of FLIM (Fluorescence Lifetime Imaging Microscopy) Data
4:32
Analysis of FLIM Data Using FLIMfit Software
7:15
Results: Fluorescence Lifetimes
8:15
Conclusion
