This work was supported by NIH grants R01AG045183 (M.C.W.), R01AT009050 (M.C.W.), R01AG062257 (M.C.W.), DP1DK113644 (M.C.W.), March of Dimes Foundation (M.C.W.), Welch Foundation (M.C.W.), and by HHMI investigator (M.C.W.). We thank the Caenorhabditis Genetics Center (CGC) for C. elegans strains.

1. Instrumental setup for Stimulated Raman Scattering Microscopy
NOTE: The SRS microscopy system is built upon a picosecond laser with pump integrated optical parametric oscillator and a confocal laser scanning microscope. The oscillator provides two picosecond pulse trains, including a Stokes beam at 1,064 nm and a pump beam tunable between 700&#8211;990 nm. Temporal and spatial overlapping of the two beams are achieved inside the laser. A built-in electro-optic modulator (EOM) is designed specifically for SRS microscopy. This protocol will focus on coupling the laser with the microscope, and the daily operation of this system (Figure 1). The configuration of an SRS microscope system is evolving in the last decade. It should be noted that the following description only represents one of the several configurations.
<ol>
	<li>Feeding laser beams into the microscope
	<ol>
		<li>Set up the periscope to lift the beam from picosecond light source exit to IR laser input port on the scanner of the microscope. 
		CAUTION: Read the laser system manual before operation and take all required precautions because near infrared radiation generated by this Class IV laser system can cause severe harm to the eyes and skin. Complete the trainings that are required by the institutional environmental safety departments. Wear protective goggles and lab coat with cuffed sleeves.</li>
		<li>First, set the pump beam at 750 nm and lower down the laser power to 50 mW, which can be visually inspected, affiliating the alignment. Then, use a beam expander (Figure 1, L1 and L2) to adjust the beam diameter to fit the back aperture of microscope objectives. Use a two relay mirrors (Figure 1, M1 and M2) to guide the laser beam to the periscope (Figure 1, P1 and P2). The periscope will lift the beam to the height of the scanner opening and serve as the steering mirror for alignment as well.</li>
		<li>Set the knobs on the periscope at the center of the tuning range and choose the initial position and angle of each mirror such that the laser beam hits the center of the mirror, approximately.</li>
		<li>Perform the coarse alignment using an empty port on the objective turret and place the power meter probe to measure the power of the transmitted light. Set the scanner of the microscope at highest zoom (50x), and then measure the power of the transmitted light with the focused laser beam spot. Optimize the knobs on each mirror, M1 and M2, iteratively to achieve the highest transmitted laser power.</li>
		<li>Perform the fine alignment with the alignment tool. Put the fluorescence target alignment cap on the empty objective seat and adjust the knobs of M1 to center the spot. Then, introduce the extension tube for the target and tune the knobs of M2 to center the spot.</li>
		<li>Repeat steps 1.1.4 and 1.1.5 until the beam centers the target at both positions.</li>
	</ol>
	</li>
	<li>Connecting detection and electronics
	<ol>
		<li>Set up the SRS detection module. Place a beam splitter cuber after the condenser to direct the transmitted laser to the photodiode module. The module includes a telescope to relay and change the beam size to fit the active area of the photodiode, as well as an optical filter to remove the modulated Stokes beam and let the pump beam go through for demodulation.</li>
		<li>Set up the electronics connection (Figure 1). The intensity of the Stokes beam is modulated by a built-in EOM at 20&#160;MHz inside the picosecond tunable laser. The modulation frequency output from the laser is input into the lock-in amplifier as the reference frequency for demodulation. Feed the output signal of the photodiode into the lock-in amplifier for demodulating the SRL. 
		NOTE: The one-box laser systems can be upgraded and thus their specifications can vary due to different production date. The previous version of picosecond tunable laser system, used in this protocol, has a Stokes beam of 1,064 nm, but the recent version has a Stokes beam of 1,031&#160;nm. The modulation frequency of EOM used in this protocol is customized to 8 MHz, but the default modulation frequency for the recent system can be&#160;10 or 20 MHz.</li>
		<li>Finally, feed the lock-in amplifier output into the analog box of the microscope to convert electrical analog signal to digital signal. The system should be ready to process the SRS signal.</li>
	</ol>
	</li>
	<li>Optimize imaging conditions
	<ol>
		<li>Make a chemical sample for system alignment. For example, use dodecane to optimize the microscope, because dodecane has a very strong SRS signal from the C-H bonds. Use a secure seal to make a mini chamber; put 5 &#956;L of dodecane and cover it with a coverslip. 
		NOTE: Replace the dodecane sample once it looks unclear or presents debris, with a new sample which could be used for alignment for 2&#8211;3 months.</li>
		<li>Place the sample on the microscope stage. Properly focus on the liquid droplet. It can be done faster by looking for the edge of the dodecane pad. Adjust the condenser for Kohler illumination.</li>
		<li>Set the imaging parameters. Check whether the delay stage is on the right numbers. Set the pump beam wavelength to 795.8&#160;nm. Using an IR sensor card and IR viewer, check whether the pump beam path is still correct.</li>
		<li>Set the power of the pump beam to 200 mW and the Stokes beam to 400 mW on the picosecond system control panel. 
		NOTE: The laser throughputs from laser exit to post objective of the two beams are approximately 25% for pump and 14% for Stokes for this system setup. Therefore, a post objective power of pump beam is approximately 50 mW and for Stokes beam it is around 56 mW. The pulse width of the recent picosecond laser system has changed from 6 ps to 2 ps, therefore, the applied laser power should be even lower.</li>
		<li>Set the lock-in amplifier gain to maximum. Open the shutter for both the beams, as well as the main shutter of the laser.</li>
		<li>Scan the sample and check the image on the computer screen. Change the display mode into Hi-Lo in the imaging software. Adjust the range to see an ~50% saturation. Check whether the saturation is centered in the image. If not, carefully adjust the steering mirrors, M1 and M2, to maximize the image intensity and to center the peak intensity.</li>
		<li>Fine tune the delay stage and find the maximum SRS signal intensity from the dodecane sample. The system is then ready for use.</li>
	</ol>
	</li>
</ol>
2. Preparation of C. elegans samples for SRS microscopy imaging
NOTE: As model organisms, Caenorhabditis elegans have been proven to be very useful for multiple imaging techniques. They are whole-body transparent, therefore various tissues can be imaged in the intact animal without the need for dissection. In C. elegans, neutral lipids are stored in lipid droplets located in the intestine, which consists of 20 epithelial cells located bisymmetrically around the intestinal lumen16. Hypodermal cells and oocytes also store lipids. The main form of storage lipids in C. elegans lipid droplets are the triacylglycerols (TAGs)17, which generate strong SRS signals due to the abundance of CH2 bonds present in their fatty acid chains. This section will focus on how to prepare C. elegans samples for SRS microscopy imaging and quantification of their total intestinal lipid storage levels.
<ol>
	<li>Preparation of the imaging slides
	<ol>
		<li>First, prepare 2% agarose solution in distilled H2O, and make sure all the agarose melts before preparing the agarose pads.</li>
		<li>Add one drop of warm 2% agarose onto a blank slide (approximately 100 &#956;L) that is placed between support slides with two layers of laboratory tape and quickly place a second slide on top of the first slide. Gently press to create a thin, even agarose pad.</li>
		<li>Set these slides aside in closed position and do not separate them until worms are ready to be mounted. Prepare fresh slides before each imaging session, as the pads will dry after 1 day. 
		NOTE: Use multi-well imaging chambers, if imaging several worms in high-throughput genetic screens18. Use live imaging setups to image spatio-temporal dynamics of lipid metabolism through development and aging19.</li>
	</ol>
	</li>
	<li>Mounting the worms
	<ol>
		<li>Prepare the anaesthetizing agent by adding sodium azide or levamisole in M9 buffer to the final concentration of 100 mM or 1 mM, respectively. 
		NOTE: Use levamisole if the worms need to be recovered after imaging for genotyping after genetic screens. 
		CAUTION: Several anaesthetizing agents, including sodium azide and levamisole, are harmful if inhaled. Wear protective gloves and clothing, as well as use a chemical fume hood when preparing those working solutions.</li>
		<li>Place a drop of anaesthetizing agent to a coverslip (approximately 4&#8211;5 &#956;L for 10&#8211;20 worms). 
		NOTE: Adjust the amount of the anaesthetizing agent according to the worm number to keep the worms as close to each other as possible. Using too much liquid for few worms may cause the worms to spread on the agarose pad.</li>
		<li>Pick the worms to be imaged under the dissection microscopy and transfer them to the anaesthetizing agent droplet. After all the worms are added to the droplet, move them around using the worm picker and make sure they do not overlap with each other.</li>
		<li>Separate the glass slides (from step 2.1) without perturbing the agarose pad.</li>
		<li>Cover the worm droplet with the glass slide that has the agarose pad. Make sure the agarose pad is facing toward the worms. Alternatively, the anesthetizing aging droplet can also be added onto the agarose pad and worms can be covered with the coverslip.</li>
		<li>Finally, mark the location of the worms using a permanent marker on the glass slide.</li>
	</ol>
	</li>
</ol>
3. Image acquisition and analysis
<ol>
	<li>Acquiring the images using the SRS microscope system.
	<ol>
		<li>Before imaging any sample, determine the imaging parameters and verify SRS signals (as explained in Protocol&#160;1).</li>
		<li>Mount the slide with the coverslip facing the objective lens. Turn on the bright field light source and direct it to the eyepiece to locate the worms. 
		NOTE: Use a 20x objective lens to image the lipid storage in the whole intestine. Consider using a 60x objective lens to image hypodermal fat storage or to quantify lipid droplet size/number.</li>
		<li>Bring the worms into focus and adjust the condenser position accordingly.</li>
		<li>Turn off the bright field light source and switch to the laser scanning unit. Begin scanning the first worm at a fast scanning rate (e.g., 512 x 512 pixels), and adjust the fine focus to find the area of interest. For the first sample to be imaged, adjust the laser powers to the level where SRS signals are not saturated.</li>
		<li>Switch to a slower scanning rate (e.g., 2 &#956;s/pixel) and higher resolution (e.g., 1024 x 1024 pixels) to obtain the SRS image. 
		NOTE: Keep the laser powers constant for every sample to be imaged during the imaging session.</li>
		<li>Save the SRS image in the desired format that enables high resolution (such as a .tiff file). Depending on the imaging software and setup used, save the location of the first worm before moving to the next.</li>
		<li>Complete imaging of all worms that were mounted on the slide. Turn off the shutter to block the laser beams. Do not turn off the laser source until all samples are imaged.</li>
		<li>Remove the slide before placing the next sample slide. Repeat steps 3.1.2&#8211;3.1.7.</li>
		<li>Once all the samples have been imaged, put the laser source on standby and turn off the associated equipment including the amplifier, the detector, the microscope and the computer.</li>
	</ol>
	</li>
	<li>Analysis of images using ImageJ
	<ol>
		<li>Open the image files (usually .tiff) to be quantified in ImageJ, by selecting the files and dragging and dropping them to the ImageJ window. Download the appropriate ImageJ plug-ins, if using specific Olympus microscope formats (such as .oib files).</li>
		<li>Select the properties to be analyzed (Analyze &#62; Set Measurements). For total lipid storage quantitation, select Area, Min and Max Gray Value, and Mean Gray value.</li>
		<li>Use the polygon selection tool and outline the region of the worm intestine to be quantified. To measure the parameters selected in step 3.2.2, click on Analyze &#62; Measure. A new window with the measurement results will pop-up. Repeat this step for all the open images.</li>
		<li>Select the measurements for all the worms in a given genotype and copy/paste the results to a new spreadsheet. Repeat the steps 3.2.1&#8211;3.2.4 for all the genotypes in the same imaging session.</li>
		<li>Select an area in the vicinity of the worm that does not have any SRS signal, to quantify as background. Make sure the area selected for background measurement does not contain any bacteria or debris that could also give an SRS signal.</li>
		<li>Subtract the background mean gray value from the measurements of each individual worm. Calculate the average and standard deviation of the background subtracted mean gray values from all of the worms in a given genotype/test group. Normalize this value to the average of the control group.</li>
		<li>Finally, perform the appropriate statistical analysis using the mean gray value as a measure for lipid levels in each worm. Typically, use Student&#8217;s t-test for two groups and use one-way ANOVA with an appropriate multiple comparison test for more than two groups. 
		NOTE: When selecting images for figure presentation, ensure that those selected images have the same pixel intensity distribution. Set the brightness and contrast to the same values for all the images in the same figure (Image &#62; Adjust &#62; Brightness and Contrast).</li>
	</ol>
	</li>
</ol>

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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+13C+isotope+labeling+strategy+reveals+the+influence+of+insulin+signaling+on+lipogenesis+in+C.+elegans.">A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.</a> Cell Metabolism. 8 (3), 266-274 (2008).</li><li>Elle, I. C., Rodkaer, S. V., Fredens, J., Faergeman, 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=A+method+for+measuring+fatty+acid+oxidation+in+C.+elegans.">A method for measuring fatty acid oxidation in C. elegans.</a> Worm. 1 (1), 26-30 (2012).</li><li>Ezcurra, M., 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=elegans+eats+its+own+intestine+to+make+yolk+leading+to+multiple+senescent+pathologies.">elegans eats its own intestine to make yolk leading to multiple senescent pathologies.</a> Current Biology. 28 (16), 2544-2556 (2018).</li><li>Lu, F. K., 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=Label-free+DNA+imaging+in+vivo+with+stimulated+Raman+scattering+microscopy.">Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (37), 11624-11629 (2015).</li><li>Long, R., 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=Two-color+vibrational+imaging+of+glucose+metabolism+using+stimulated+Raman+scattering.">Two-color vibrational imaging of glucose metabolism using stimulated Raman scattering.</a> Chemical Communications. 54 (2), 152-155 (2018).</li><li>Zhao, Z., Shen, Y., Hu, F., Min, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+vibrational+tags+in+biological+imaging+by+Raman+microscopy.">Applications of vibrational tags in biological imaging by Raman microscopy.</a> Analyst. 142 (21), 4018-4029 (2017).</li><li>Liao, C. 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=In+vivo+and+in+situ+spectroscopic+imaging+by+a+handheld+Stimulated+Raman+Scattering+Microscope.">In vivo and in situ spectroscopic imaging by a handheld Stimulated Raman Scattering Microscope.</a> ACS Photonics. 5 (3), 947-954 (2018).</li><li>Lee, H. J., 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=Assessing+cholesterol+storage+in+live+cells+and+C.+elegans+by+stimulated+Raman+scattering+imaging+of+phenyl-Diyne+cholesterol.">Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-Diyne cholesterol.</a> Scientific Reports. 5, 7930 (2015).</li><li>Wang, P., 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=Imaging+lipid+metabolism+in+live+Caenorhabditis+elegans+using+fingerprint+vibrations.">Imaging lipid metabolism in live Caenorhabditis elegans using fingerprint vibrations.</a> Angewandte Chemie (International Edition in English). 53 (44), 11787-11792 (2014).</li><li>Chen, W. 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=Spectroscopic+coherent+Raman+imaging+of+Caenorhabditis+elegans+reveals+lipid+particle+diversity.">Spectroscopic coherent Raman imaging of Caenorhabditis elegans reveals lipid particle diversity.</a> Nature Chemical Biology. 16 (10), 1087-1095 (2020).</li><li>Freudiger, C. 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=Stimulated+Raman+Scattering+microscopy+with+a+robust+fibre+laser+source.">Stimulated Raman Scattering microscopy with a robust fibre laser source.</a> Nature Photonics. 8 (2), 153-159 (2014).</li><li>Brinkmann, M., 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=Portable+all-fiber+dual-output+widely+tunable+light+source+for+coherent+Raman+imaging.">Portable all-fiber dual-output widely tunable light source for coherent Raman imaging.</a> Biomedical Optics Express. 10 (9), 4437-4449 (2019).</li></ol>

The authors have nothing to disclose.

In defense against obesity and its associated metabolic disorders, important research efforts have been implemented to better understand the regulatory mechanisms of lipid homeostasis. For quantitative detection of lipid molecules in biological samples, label-free imaging by SRS microscopy has been proven to be a reliable alternative to biochemical assays and other staining methods. Our group and others have revealed novel biological mechanisms in lipid metabolic regulation by combining the use of C. elegans and...

Obesity has become a global health problem threatening one third of the population around the world, and it presents a serious medical concern, given its association with poor mental health1 and deadly diseases including diabetes2, cardiovascular diseases3 and some types of cancer4. Study of lipid metabolism is essential to better understand the biological problems behind obesity. Rapid and specific quantification of lipid storage entails the detection of fatty acids and their derivatives, as well as sterol-containing metabolites, with high sensitivity and preferably with spatial information. Lipids are challenging targets to image because they lack intrinsic fluorescence and cannot be easily fluorescently tagged. The fluorescent tags are often larger than the lipid molecules and, therefore, can be chemically invasive and impractical for in vivo applications. Label-free or minimal labeling strategy is necessary to preserve the hydrophobic structure of the lipid molecules5. Recent developments in imaging technologies have created exciting opportunities for label-free imaging of lipids in living cells, tissues, and organisms.
Traditional approaches for lipid storage analyses in biological samples include biochemical assays and staining protocols with lipophilic dyes. Biochemical quantification assays involving mass spectrometry (MS) are unmatched in their molecular resolvability, but they require very large sample amounts and sample preparation usually takes several hours, limiting their application for real-time imaging of living systems5. Another major limitation of these assays is the lack of spatial information. On the other hand, lipophilic dyes such as Oil Red O and Sudan Black provide tissue and cellular distribution of lipid storage organelles and compared to MS techniques, these staining methods are also low in cost and easy to perform. However, these staining protocols require fixation, which can impact the hydrophobic nature of the lipid droplets, generate artificial changes in their structure and result in inconsistencies between experiments6. The technical difficulties associated with biochemical and staining techniques have led to the search for label-free methods to image lipid molecules and to the rapid increase in the usage of coherent Raman scattering (CRS) microscopy in lipid imaging.
The Raman effect was first recognized by Raman and Krishnan, where they reported that upon interacting with a photon, a molecule can generate scattered light with no change in wavelength (called Rayleigh scattering) or rarely with an altered wavelength (called Raman scattering) and this change in wavelength is characteristic of the functional chemical groups within the molecule7. When the chemical bonds within a molecule get excited to a higher vibrational energy level by an incident photon, called pump photon, the energy of the scattered photon, called the Stokes photon, become lower. Otherwise, the chemical bonds can reach a lower vibrational energy level if they are originally at a higher level, and the scattered photon gain energy to be the anti-Stokes photon. The frequency difference between the incident and scattered photons is known as the Raman shift. Each chemical bond within a molecule has a characteristic and quantifiable Raman shift. For example, the CH2 bond has a Raman shift of 2,845 cm-1, which is abundant in fatty acid chains8. This spontaneous Raman signal is generally very weak, which has vastly limited the imaging speed in conventional spontaneous Raman microscopy. Over the years, various approaches have been developed to increase the imaging speed and sensitivity of spontaneous Raman microscopy. Coherent Raman scattering microscopy, including Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy, is the most recent progress. CARS and SRS have slightly different working principles, but both are label-free techniques that have live imaging capability, can yield spatial and temporal information on lipid storage dynamics, and only require small sample size. CARS microscopy suffers from non-resonant background, which comes from various non-linear processes, and CARS signals also have non-linear relationship with the molecule concentration, which together complicate the quantification process9. Unlike CARS microscopy, SRS microscopy does not generate non-resonant background signals and offers linear dependence on the concentration of the molecule of interest. Thus, currently SRS microscopy is more widely used for lipid imaging.
In SRS microscopy, the weak spontaneous Raman signals can be amplified when excited by two synchronized laser beams with their frequency difference matching the chemical bond vibrational frequency. The molecule will experience an enhanced transition to an excited state due to coherent excitation. As a result, the rate of Stokes photon generation is boosted. Consequently, the intensity of the transmitted &#8220;Stokes&#8221; beam increases (stimulated Raman gain, SRG) and the intensity of the transmitted &#8220;pump&#8221; beam decreases (stimulated Raman loss, SRL). Detection of SRG or SRL signals underlies the basis for Stimulated Raman Scattering (SRS) microscopy imaging of molecules with specific chemical bonds10. If the frequency difference between the two laser beams does not match the vibrational frequency of the chemical bond within a molecule of interest, neither SRG nor SRL signals will be generated. The imaging speed of SRS microscopy is around 2 &#956;sec per pixel or 1 second per frame, which is much faster than spontaneous Raman microscopy11. Typical lateral resolution for SRS microscopy is diffraction limited and around 300 nm. In addition, the two-photon optical processes of SRS microscopy allow for volumetric 3D imaging of relative thick tissue samples and the imaging depth could reach 300&#8211;500 &#956;m. Overall, SRS microscopy presents an efficient, label-free imaging technique to detect specific biomolecules, especially lipids.
Lipid droplets are single-membrane organelles, which are the main cellular storage site for neutral lipids, including the triacylglycerols (TAGs) and cholesterol esters (CEs). CH2 bonds in the fatty acid chains of these lipid molecules generate strong SRS signals at 2,845 cm-1 when excited8, thus enabling the detection and quantification of storage lipid levels in intact cells, tissue sections and even whole organisms12,13,14,15. In particular, C. elegans are useful for lipid imaging studies owing to their transparency. Like mammals, C. elegans also store lipids in lipid droplets and the synthesis and degradation pathways of lipid molecules are highly conserved16. In this protocol, we will provide the working principle of SRS microscopy, its fundamental setup and describe the methods for its use in lipid imaging in C. elegans.

<table>
	<tbody>
		<tr>
			<td>A/D converter</td>
			<td>Olympus</td>
			<td>Analog Unit</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>GeneMate</td>
			<td>3119</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Alignment tool - adapter</td>
			<td>Thorlabs</td>
			<td>SM1A4</td>
			<td>For mounting the tool on scope</td>
		</tr>
		<tr>
			<td>Alignment tool - target</td>
			<td>Thorlabs</td>
			<td>VRC2SM1</td>
			<td>For viewing IR laser</td>
		</tr>
		<tr>
			<td>Alignment tool - tube</td>
			<td>Thorlabs</td>
			<td>SM1L40</td>
			<td>Length can vary</td>
		</tr>
		<tr>
			<td>Autocorrelator (Optional)</td>
			<td>APE</td>
			<td>pulseCheck</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter</td>
			<td>minicircuits</td>
			<td>BBP-21.4+ if modulated at 20MHz or KR Electronics 2724 if modulated at 8 MHz</td>
			<td>For signal with modulation frequency filtering</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>For handling and picking worms for imaging</td>
		</tr>
		<tr>
			<td>Dodecane</td>
			<td>Sigma-Aldrich</td>
			<td>44010</td>
			<td>Used for calibration of the SRS signal</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Chroma Technology</td>
			<td>890/220 CARS</td>
			<td>For removing Stokes beam</td>
		</tr>
		<tr>
			<td>General purpose laboratory labeling tape</td>
			<td>VWR</td>
			<td>89097</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>VWR</td>
			<td>48393-106</td>
			<td>For covering worms for imaging</td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Laser scanning microscope</td>
			<td>Olympus</td>
			<td>FV3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens</td>
			<td>Thorlabs</td>
			<td>L1: AC254-050-B 
			L2: AC254-075-B</td>
			<td>For beam expander</td>
		</tr>
		<tr>
			<td>Lock-in amplifier</td>
			<td>Zurich</td>
			<td>HF2LI</td>
			<td></td>
		</tr>
		<tr>
			<td>Lowpass filter</td>
			<td>minicircuits</td>
			<td>BLP-1.9+</td>
			<td>For power supply noise suppression</td>
		</tr>
		<tr>
			<td>Mirrors</td>
			<td>Thorlabs</td>
			<td>BB1-E03</td>
			<td>For relay and periscope</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UPlanSAPO 20x 0.75, UPlanSAPO 60XW 1.20</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>FDS1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Picosecond laser source</td>
			<td>APE</td>
			<td>picoEmerald</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>TEKPOWER</td>
			<td>TP1342U</td>
			<td>For photodiode, reversed 50V voltage</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>For anaesthesizing the worms</td>
		</tr>
		<tr>
			<td>Worm picker</td>
			<td>WormStuff</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
	</tbody>
</table>

label-free imaging of lipid storage dynamics in caenorhabditis elegans using stimulated raman scattering microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Insulin signaling is an important endocrine pathway that impacts development, reproduction, lifespan, and metabolism. In worms, insulin signaling consists of about 40 insulin-like peptide ligands, insulin-like growth factor receptor ortholog DAF-2, downstream PI3K/AKT kinase cascade, and the FoxO transcription factor ortholog, DAF-1620. daf-2 mutants, that lack the insulin receptor, have more lipid droplets in their intestine,&#160;the worm lipid storage tissue21<s...

Watch this Scientific Journal Video about Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy at JoVE.com

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

label-free-imaging-lipid-storage-dynamics-caenorhabditis-elegans

Research

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Biology

4.0K Views.  Baylor College of Medicine. Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Video: Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...