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...

<ol>
	<li>Klaips, C. L., Jayaraj, G. G., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+cellular+proteostasis+in+aging+and+disease.">Pathways of cellular proteostasis in aging and disease.</a> Journal of Cell Biology. 217 (1), 51-63 (2018).</li><li>Kikis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+struggle+by+Caenorhabditis+elegans+to+maintain+proteostasis+during+aging+and+disease.">The struggle by Caenorhabditis elegans to maintain proteostasis during aging and disease.</a> Biology Direct. 11, 58 (2016).</li><li>Becker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+-+techniques+and+applications.">Fluorescence lifetime imaging - techniques and applications.</a> Journal of Microscopy. 247 (2), 119-136 (2012).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. , (2006).</li><li>Berezin, M. Y., Achilefu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+measurements+and+biological+imaging.">Fluorescence lifetime measurements and biological imaging.</a> Chemical Reviews. 110 (5), 2641-2684 (2010).</li><li>Kaminski Schierle, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET+sensor+for+non-invasive+imaging+of+amyloid+formation+in+vivo.">A FRET sensor for non-invasive imaging of amyloid formation in vivo.</a> ChemPhysChem. 12 (3), 673-680 (2011).</li><li>Sandhof, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+INS-IGF1+signaling+protects+against+non-cell+autonomous+vesicle+rupture+caused+by+SNCA+spreading.">Reducing INS-IGF1 signaling protects against non-cell autonomous vesicle rupture caused by SNCA spreading.</a> Autophagy. , 1-22 (2019).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+Synchronization+and+observation.">Basic Caenorhabditis elegans methods: Synchronization and observation.</a> Journal of Visualized Experiments. 64, e4019 (2012).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook the online review of C. elegans biology. 1999, 1-11 (2006).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome Biology. 2 (1), 1-10 (2001).</li><li>Becker, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Lifetime+Imaging+by+Time-Correlated+Single-Photon+Counting.">Fluorescence Lifetime Imaging by Time-Correlated Single-Photon Counting.</a> Microscopy Research and Technique. 63 (1), 58-66 (2004).</li><li>Warren, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+global+fitting+of+large+fluorescence+lifetime+imaging+microscopy+datasets.">Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.</a> PloS one. 8 (8), e70687 (2013).</li><li>Moronetti Mazzeo, L. E., Dersh, D., Boccitto, M., Kalb, R. G., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+aging+induce+distinct+polyQ+protein+aggregation+states.">Stress and aging induce distinct polyQ protein aggregation states.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (26), 10587-10592 (2012).</li><li>Ben-Zvi, A., Miller, E. A., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+of+proteostasis+represents+an+early+molecular+event+in+Caenorhabditis+elegans+aging.">Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (35), 14914-14919 (2009).</li><li>Wallrabe, H., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+molecules+using+FRET+and+FLIM+microscopy.">Imaging protein molecules using FRET and FLIM microscopy.</a> Current Opinion in Biotechnology. 16 (1), 19-27 (2005).</li><li>Chan, F. T. S., Pinotsi, D., Kaminski Schierle, G. S., Kaminski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-Specific+Intrinsic+Fluorescence+of+Protein+Amyloids+Used+to+Study+their+Kinetics+of+Aggregation.">Structure-Specific Intrinsic Fluorescence of Protein Amyloids Used to Study their Kinetics of Aggregation.</a> Bio-nanoimaging: Protein Misfolding and Aggregation. , 147-155 (2013).</li><li>Laine, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Fluorescence+Lifetime+Imaging+Reveals+the+Aggregation+Processes+of+&#945;-Synuclein+and+Polyglutamine+in+Aging+Caenorhabditis+elegans.">Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of &#945;-Synuclein and Polyglutamine in Aging Caenorhabditis elegans.</a> ACS Chemical Biology. 14 (7), 1628-1636 (2019).</li><li>Kelbauskas, L., Dietel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+of+Aggregated+Photosensitizers+by+Tumor+Cells:+Subcellular+Time-resolved+Fluorescence+Spectroscopy+on+Derivatives+of+Pyropheophorbide-a+Ethers+and+Chlorin+e6+under+Femtosecond+One-+and+Two-photon+Excitation.">Internalization of Aggregated Photosensitizers by Tumor Cells: Subcellular Time-resolved Fluorescence Spectroscopy on Derivatives of Pyropheophorbide-a Ethers and Chlorin e6 under Femtosecond One- and Two-photon Excitation.</a> Photochemistry and Photobiology. 76 (6), 686-694 (2002).</li><li>Becker, W., Su, B., Holub, O., Weisshart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIM+and+FCS+detection+in+laser-scanning+microscopes:+Increased+efficiency+by+GaAsP+hybrid+detectors.">FLIM and FCS detection in laser-scanning microscopes: Increased efficiency by GaAsP hybrid detectors.</a> Microscopy Research and Technique. 74 (9), 804-811 (2011).</li><li>Suhling, K., French, M. W., Phillips, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+fluorescence+microscopy.">Time-resolved fluorescence microscopy.</a> Photochemical and Photobiological Sciences. 4 (1), 13-22 (2005).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;H DCS-120 SPC-150</td>
			<td>Becker &#38; Hickl GmbH</td>
			<td></td>
			<td>FLIM Aquisition software</td>
		</tr>
		<tr>
			<td>B&#38;H SPC830-SPC Image</td>
			<td>Becker &#38; 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>C. elegans iQ44-YFP</td>
			<td>CAENORHABDITIS GENETICS CENTER (CGC)</td>
			<td>OG412</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans iQ85-YFP</td>
			<td></td>
			<td></td>
			<td>Kind gift from Morimoto Lab</td>
		</tr>
		<tr>
			<td>C. elegans mQ40-RFP</td>
			<td></td>
			<td></td>
			<td>Kind gift from Morimoto Lab</td>
		</tr>
		<tr>
			<td>C. elegans nQ40-CFP</td>
			<td></td>
			<td></td>
			<td>Kind gift from Morimoto Lab</td>
		</tr>
		<tr>
			<td>Deckgl&#228;ser-18x18mm</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>0657.2</td>
			<td>Cover slips</td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-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 Escherichia coli</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&#228;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 &#38; 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 ...

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.

characterization-amyloid-structures-aging-c-elegans-using

Research

JoVE Journal

Biochemistry

6.9K visualizações.  Leibniz Research Institute for Molecular Pharmacology im Forschungsverbund Berlin. O método permite distinguir claramente estruturas integradas de proteína amiloide das contrapartes solúveis e até acumuladas. A técnica é realizada in vivo de forma não invasiva, com pouco ou nenhum efeito colateral tóxico. Como depende das propriedades fluorescentes do próprio fluorohore, é altamente robusto e reprodutível ao longo do tempo.A FLIM tem sido utilizada no campo do diagnóstico de câncer. No entanto, nós o utilizamos para estudar os mecanismos básicos de agr...