The muscle-Q40-mRFP strain provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The neuronal-Q40-CFP was a kind gift of the Morimoto Lab. We acknowledge the DFG (KI-1988/5-1 to JK, NeuroCure PhD fellowship by the NeuroCure Cluster of Excellence to MLP), EMBO (Short term fellowship to MLP) and the Company of Biologists (travel grants to CG and MLP) for funding. We also acknowledge the Advanced Light Microscopy imaging facility at the Max Delbr&#252;ck Centre for Molecular Medicine, Berlin, for providing the setup to image the YFP constructs.

1. Synchronization of C. elegans
<ol>
	<li>Synchronize C. elegans either via alkaline hypochlorite solution treatment or via simple egg laying for 4 h at 20 &#176;C8.</li>
	<li>Grow and maintain nematodes at 20 &#176;C on nematode growth medium (NGM) plates seeded with OP50 E. coli according to standard procedures9. Age the nematodes until the desired developmental stage or day. 
	NOTE: In this protocol, young adults are imaged on day 4 and old nematodes are imaged on day 8 of life.</li>
</ol>
2. RNAi-mediated knockdown of chaperone machinery via feeding
NOTE: Perform knockdown of heat shock protein 1 (hsp-1) chaperone by feeding the corresponding RNAi vector to the nematodes10. The hsp-1 RNAi plasmid was obtained from the Ahringer library (clone ID: F26D10.3).
<ol>
	<li>Grow the HT115 (DE3) E. coli expressing the hsp-1 RNAi plasmid for 6 h to overnight in Luria Bertani (LB) medium containing 50 &#181;g/mL ampicillin.</li>
	<li>Prepare fresh NGM agar plates containing isopropyl &#946;-D-1-thiogalactopyranoside (IPTG; 1 mM) and ampicillin (25 &#181;g/mL) and seed with the hsp-1 RNAi bacteria. Leave plates to dry and induce at room temperature for 1-3 days.</li>
	<li>Place synchronized eggs on an siRNA plate and leave to hatch, or place gravid nematodes and allow to lay eggs for 4 h at 20 &#176;C before removing. Grow the nematodes until the desired age or stage. 
	NOTE: The second generation of nematodes might present a stronger phenotype of the knockdown. The siRNA protocol and conditions described here are general and adapted to hsp1. RNA interference via siRNA of a specific clone/gene needs to be established and optimized by the end user. It is important to note that not all siRNA have the same efficiency, and it is therefore recommended to test the efficacy of the knockdown by quantification by either quantitative reverse transcription polymerase chain reaction (RT-qPCR) or Western blot.</li>
</ol>
3. Preparation of microscopy slides
<ol>
	<li>On the day of imaging, start by preparing the imaging slides. Melt agarose in ddH2O at a concentration of 3% (w/v) and let cool slightly.</li>
	<li>Cut the tip of a 1 mL pipette tip and take roughly 200 &#181;L of 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 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. 
	NOTE: Each slide will be used to image between 5-10 nematodes. Slides can be prepared and stored for a few hours in a humified box to prevent the agarose from drying out.</li>
</ol>
4. Mounting nematodes onto microscopy slides
NOTE: FLIM requires the nematodes to be immobilized. Perform this step once the imaging setup (e.g., microscopes, lasers, detectors) is ready to use.
<ol>
	<li>Using a platinum wire pick, place nematodes of the desired age onto a fresh unseeded plate and let them crawl to remove the excess OP50 bacteria from their bodies.</li>
	<li>Prepare the anesthetic compound (sodium azide or levamisole) to immobilize the nematode. Keep a 500 mM NaN3 stock in the dark at 4 &#176;C and dilute in fresh ddH2O to a final concentration of 250 mM. If using levamisole, dilute a 20 mM stock to 2 mM working solution in ddH2O. 
	CAUTION: Sodium azide (NaN3) is highly toxic. Use gloves and protective eyewear and work under a ventilated hood.</li>
	<li>Working under a stereomicroscope, place a 10 &#181;L drop of anesthetic compound onto an agarose pad and gently transfer 5-10 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.</li>
	<li>Carefully overlay the nematodes with a coverslip. Take measurements within 1 h after mounting. 
	NOTE: Both anesthetics will eventually kill C. elegans. The nematodes must be completely immobile during imaging, because 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.</li>
</ol>
5. Acquisition of FLIM data
NOTE: 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 user. Lifetime measurements are achieved by detectors and electronic equipment installed alongside a conventional microscope. In this protocol, measurements are performed on three different laser scanning confocal microscopes with detectors and software provided by two different companies (Table of Materials) for acquisition of mRFP, CFP, and YFP lifetimes, respectively. Check that the correct filters of emission/excitation are in place and minimize any background or monitor backlight before starting. Before starting any experiment, establish the photostability of the chosen fluorophore. If the fluorophore bleaches within a short time within the nematode tissues, it is not suitable for FLIM measurements in C. elegans.
<ol>
	<li>Open the FLIM acquisition software. The FLIM software also allows control of the confocal microscope. Locate the tab/button to allow for the detector&#39;s outputs to be enabled and press Enable Outputs.</li>
	<li>Acquire the instrument response function (IRF), which describes the timing precision of the instrumental setup. 
	NOTE: This step should be performed preferably before mounting the nematodes.
	<ol>
		<li>If available, remove the excitation/emission filters.</li>
		<li>Place an empty coverslip above the objective and find its surface. Record the scatter signal obtained from the coverslip for a minimum of 30 s. 
		NOTE: For lifetimes of several nanoseconds, the acquisition software can automatically estimate the IRF shift. Acquiring an IRF is always recommended.</li>
	</ol>
	</li>
	<li>Place the slide with the mounted C. elegans on the stage. Using a 10x magnification lens in transmission mode and localize the position of the nematodes on the slide.</li>
	<li>Remove 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 localize the nematodes.</li>
	<li>Locate the Pinhole Manager on the acquisition software and open it to the Maximum. Start scanning the sample, select a region of interest (e.g., head, upper body), and focus on its maximum projection plane.</li>
	<li>Monitor the laser pulse rate and the three other values present on the interface of the software: The Constant Fraction Discriminator (CFD), the Time-to-Amplitude (TAC), and the Analogue-to-Digital Converter (ADC). 
	NOTE: The laser should have a maximum gate of 1 x 108 single photon counts. This number represents the maximum number of photons supplied by the laser. The CFD provides information on the receipt of the single photon pulse in reference to the laser pulse by the detector. This value should be roughly 1 x 105. The TAC discriminates between the time one photon was detected and the next laser pulse. Finally, the ADC converts the TAC voltage into a storable memory signal11. 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.</li>
	<li>On the interface of the FLIM software, preview the number of photons detected: the ADC value should be between 1 x 104 and 1 x 105. If necessary, shift the focus on a different plane or increase the laser power to collect more photons. 
	NOTE: In general, the number of recorded photons per second should not exceed 1% of the laser&#39;s repetition rate.</li>
	<li>In the menu bar, select the tab to set the acquisition parameters. Select scan sync in to allow for single photon detection.</li>
	<li>Set the acquisition to a fixed amount of time or a fixed number of photons. For example, acquire a lifetime decay curve for 2 min or until a single pixel reaches a photon count of 2,000 single events. Press Start to begin acquisition. 
	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, which will not interfere with the lifetime. These protocols use the following excitation/emission settings: YFP ex500/em520-50 nm, mRFP ex561/em580-620 nm. A pulsed two photon laser was employed for CFP measurements using ex800/em440 nm. The amount of time and photon count required for acquisition of a FLIM map will need to be empirically established for each setup and each experimental purpose.</li>
</ol>
6. Analysis of FLIM data using FLIMfit software
NOTE: Perform data analysis using the FLIMfit software tool developed at Imperial College London12 (see Figure 1).
<ol>
	<li>Open the software and import FLIM data files via File | Load FLIM Data. Load all samples from one condition, even if obtained in different sessions and from different biological repeats.</li>
	<li>If necessary, segment a single nematode from any FLIM picture via 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.</li>
	<li>Select a small region where the intensity-based image of a C. elegans appears (Figure 1, Arrows 1). The decay curve of that region will appear in the large decay window on the right side of the interface (Figure 1, Arrow 2). 
	NOTE: The decay can be displayed linearly or logarithmically.</li>
	<li>Set the correct parameters to extrapolate the lifetime via the software&#39;s algorithm as described in steps 6.5-6.8.</li>
	<li>On the Data tab (Figure 1, Arrow 3):
	<ol>
		<li>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.</li>
		<li>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. 
		NOTE: For example, for the analysis of mRFP, the events prior to 800 ps and after 4,000 ps were excluded. These values depend on the lifetime of the fluorophore and need to be determined by the end user.</li>
		<li>Do not change the preset Counts/Photon of 1.</li>
		<li>Input the Repetition Rate, in MHz, of the laser utilized during acquisition. 
		NOTE: For the current protocol, different lasers were utilized with various repetition rates. The two photon laser used for acquisition of CFP lifetimes possesses a repetition rate of 80 MHz, for YFP the laser repeats at 40 MHz, and for mRFP the value is 78.01 MHz. These values were inputted into FLIMfit according to the sample analyzed.</li>
		<li>Input a Gate Max value to exclude all saturated pixels. 
		NOTE: For lifetime measurements in C. elegans, this value is set to any large number (e.g., 1 x 108).</li>
	</ol>
	</li>
	<li>On the Lifetime tab, select a global fitting to be used (e.g., a pixel-wise fitting). See Figure 1, Arrow 4. 
	NOTE: 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.</li>
	<li>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. 
	NOTE: In the present protocol, this function was utilized to calculate the lifetimes of the biexponential CFP fluorophore.</li>
	<li>Upload 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 this is established, do not change any other parameters of this tab.</li>
	<li>Once all parameters are set, press Fit Dataset (Figure 1, Arrow 5). The algorithm will produce a fit for the decay curve and establish a lifetime value for each image. 
	NOTE: 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.</li>
	<li>Click the Parameters tab (Figure 1, Arrow 6), located within the top right menus of the software&#39;s interface, and select Statistic: w_mean (weighted mean) and check that the chi2 value is as close as possible to 1. 
	NOTE: A chi2 close to one ensures the accuracy of the fit. The lifetime value of the selected image is thus revealed as tau_1.</li>
	<li>Export any information of interest: File | Export Intensity Images/Fit Result Table/Images/Histograms. Save the data settings used to calculate the lifetime: File | Save Data Settings. 
	NOTE: The parameters employed will be saved for future analysis of the selected samples.</li>
</ol>
7. Graphical representations of FLIM data
NOTE: The lifetimes collected from different samples can be visually represented in various ways. Select to denote the lifetime values either in nanoseconds or picoseconds.
<ol>
	<li>Show the quality of the fit and the accuracy of the curve by exporting the decay curve directly from FLIMfit.</li>
	<li>Represent the distribution of the photons by plotting the frequency of the photon count versus the lifetime value in a histogram.</li>
	<li>Finally, 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.</li>
</ol>

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The authors have nothing to disclose.

The protocol presented here describes a microscopy-based technique to identify aggregated species in the C. elegans model system. FLIM can accurately characterize the presence of both aggregated and soluble species fused to a fluorophore via measurement of their 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 lowe...

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 their 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, as happens 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 that express human disease-causing proteins in vulnerable tissues have been created. Importantly, many of the strains containing aggregation-prone proteins recapitulate the hallmark of amyloid disorders, the formation of large inclusions. Thanks to C. elegans&#39; transparent body, these aggregates can be visualized in vivo, noninvasively and nondestructively2. Generating any protein of interest (POI) in fusion with a fluorophore allows to investigate its locations, trafficking, interaction network, and general fate.
We present a protocol to monitor the aggregation of disease-causing 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 (tau, &#964;) 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&#39;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&#39;s structure, binding, and environment3,4. 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, fluorescent intensity, and of the imaging methodology. However, within a biological system, it can 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 behavior of the fused protein. When a fluorophore is surrounded or encapsulated in a tightly bound environment, such as the antiparallel 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&#39;s lifetime will stay closer to its original, higher value. In contrast, when a protein starts to aggregate, its lifetime will inevitably shift to a lower value6,7. Therefore, it becomes possible to monitor the aggregation propensity of any amyloid-forming protein at different ages in living C. elegans.
Here we describe a protocol to analyze the aggregation of a fusion protein comprising different polyglutamine (CAG, Q) stretches (Q40, Q44, and Q85). 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, including 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.

<table><tbody><tr><td>Agar-Agar Kobe I</td><td>Carl Roth GmbH + Co. KG</td><td>5210.2</td><td>NGM component</td></tr><tr><td>Ahringer Library hsp-1 siRNA</td><td>Source BioScience UK Limited</td><td>F26D10.3</td><td></td></tr><tr><td>Ampicillin</td><td>Carl Roth GmbH + Co. KG</td><td>K029.3</td><td>Antibiotic</td></tr><tr><td>B&amp;amp;H DCS-120 SPC-150</td><td>Becker &amp;amp; Hickl GmbH</td><td></td><td>FLIM Aquisition software</td></tr><tr><td>B&amp;amp;H SPC830-SPC Image</td><td>Becker &amp;amp; Hickl GmbH</td><td></td><td>FLIM Aquisition software</td></tr><tr><td>BD Bacto Peptone</td><td>BD-Bionsciences</td><td>211677</td><td>NGM component</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; iQ44-YFP</td><td>CAENORHABDITIS GENETICS CENTER (CGC)</td><td>OG412</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; iQ85-YFP</td><td></td><td></td><td>Kind gift from Morimoto Lab</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; mQ40-RFP</td><td></td><td></td><td>Kind gift from Morimoto Lab</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; nQ40-CFP</td><td></td><td></td><td>Kind gift from Morimoto Lab</td></tr><tr><td>Deckgl&amp;auml;ser-18x18mm</td><td>Carl Roth GmbH + Co. KG</td><td>0657.2</td><td>Cover slips</td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranosid (IPTG)</td><td>Carl Roth GmbH + Co. KG</td><td>2316.4</td><td></td></tr><tr><td>Leica M165 FC</td><td>Leica Camera AG</td><td></td><td>Mounting Stereomicroscope</td></tr><tr><td>Leica TCS SP5</td><td>Leica Camera AG</td><td></td><td>Confocal Microscope</td></tr><tr><td>Levamisole Hydrochloride</td><td>AppliChem GmbH</td><td>A4341</td><td>Anesthetic</td></tr><tr><td>OP50 &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>CAENORHABDITIS GENETICS CENTER (CGC)</td><td>OP50</td><td></td></tr><tr><td>PicoQuant PicoHarp300</td><td>PicoQuant GmbH</td><td></td><td>FLIM Aquisition software</td></tr><tr><td>Sodium Azide</td><td>Carl Roth GmbH + Co. KG</td><td>K305.1</td><td>Anesthetic</td></tr><tr><td>Sodium Chloride</td><td>Carl Roth GmbH + Co. KG</td><td>3957.2</td><td>NGM component</td></tr><tr><td>Standard-Objekttr&amp;auml;ger</td><td>Carl Roth GmbH + Co. KG</td><td>0656.1</td><td>Glass slides</td></tr><tr><td>Universal Agarose</td><td>Bio &amp;amp; Sell GmbH</td><td>BS20.46.500</td><td></td></tr><tr><td>Zeiss AxioObserver.Z1</td><td>Carl Zeiss AG</td><td></td><td>Confocal Microscope</td></tr><tr><td>Zeiss LSM510-Meta NLO</td><td>Carl Zeiss AG</td><td></td><td>Confocal Microscope</td></tr></tbody></table>

characterization of amyloid structures in aging c. elegans using fluorescence lifetime imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;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 a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. 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 noninvasive manner and with the progression of aging 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 results 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 different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

The protocol shows how to accurately monitor the formation of aggregated species in living C. elegans, both during its natural aging and when subjected to stress. We selected four different strains of transgenic nematodes expressing polyglutamine proteins of either 40Q, 44Q, or 85Q repeats. These proteins are synthesized in different tissues and were fused to different fluorophores. The C. elegans strains either expressed Q40-mRFP in the body wall muscles (mQ40-RFP), Q40...

Watch this Scientific Journal Video about Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging at JoVE.com

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

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Research

JoVE Journal

Biochemistry

7.0K Views.  Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund Berlin. Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Video: Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Developmental Biology

DiI Dye-Filling as a Simple and Inexpensive Tool to Visualize Ciliated Sensory Neurons in C. elegans

C. elegans have long been used as a simple and accessible model to study neuronal structure and the many functions of the nervous system. Of the 302 neurons within the adult hermaphrodite nervous system, 60 are classified as ciliated sensory neurons. These neurons are central to a number of C. elegans behaviors, including but not limited to chemo-, mechano-, and osmosensing, male mating, and dauer formation. For several decades now, members of the C. elegans community have used the red fluorescent lipophilic dye DiI to visualize a subset of the ciliated sensory neurons that are directly exposed to the external environment. This dye enters the ciliated ends of the neurons and distributes in a relatively uniform pattern throughout the dendrites, cell bodies, and axons. This simple and powerful method makes an excellent first-pass tool to identify genetic mutants that impart structural or functional defects in ciliated sensory neurons. Here, we present a streamlined version of this staining method to visualize the eight pairs of amphid and two pairs of phasmid neurons that are environmentally exposed in C. elegans. We discuss tips for using this inexpensive method for imaging cellular dye-filling patterns in anesthetized animals.

DiI dye-filling is a method commonly used in C. elegans to visualize a subset of the ciliated sensory neurons, allowing for the identification of genetic mutations that alter sensory neuron structure or function.

C. elegans have long been used as a simple and accessible model to study neuronal structure and the many functions of the nervous system. Of the 302 ...

Genetics

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Neuroscience

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Biology

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Analyzing Mitochondrial Morphology in C. elegans

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

Analyzing Amyloid Structures in a Tissue Section Using Fluorescence Lifetime Imaging Microscopy

<a href='https://app.jove.com/v/56279/imaging-amyloid-tissues-stained-with-luminescent-conjugated'>Source: Nystr&#246;m, S., et al.,&#160;Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. J. Vis. Exp.&#160;(2017).</a>This video demonstrates analyzing amyloid structures with fluorescence lifetime imaging microscopy or FLIM, using hFTAA dye to reveal compact cores as blue/green and unstable peripheries as red/yellow in color-coded images, reflecting amyloid organization.

Source: Nystr&#246;m, S., et al.,&#160;Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy ...

JoVE Encyclopedia of Experiments

Neuroimaging

Neurodegeneration & Disease Monitoring

Fluorescence Lifetime Imaging of PolyQ Protein Aggregation in Caenorhabditis elegans Neurons

Source:&#160;Pigazzini, M. L.,&#160;et. al. Characterization of Amyloid Structures in Aging&#160;C. Elegans Using Fluorescence Lifetime Imaging.&#160;J. ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

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Przeglądaj więcej filmów

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

DiI Dye-Filling as a Simple and Inexpensive Tool to Visualize Ciliated Sensory Neurons in C. elegans

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Analyzing Amyloid Structures in a Tissue Section Using Fluorescence Lifetime Imaging Microscopy

Fluorescence Lifetime Imaging of PolyQ Protein Aggregation in Caenorhabditis elegans Neurons