Name: label-free imaging of lipid storage dynamics in caenorhabditis elegans using stimulated raman scattering microscopy
Uploaded: 2021-05-28T14:00:00.000+00:00
Duration: 10 min 59 s
Description: Watch this Scientific Journal Video about Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy at JoVE.com

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&lt;br/&gt; 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

JoVE Journal

Biology

4.1K Views.  Baylor College of Medicine. Stimulated Raman scattering microscopie is een chemische beeldvormingstechnologie die de snelle en kwantitatieve detectie van lipiden mogelijk maakt en het mogelijk maakt lipiden bij levende dieren op een labelvrije manier te volgen. Het belangrijkste voordeel van SRS-microscopie ten opzichte van traditionele lipidendetectietechnieken is dat het labelvrij is en daarom niet zo duurzaam is voor het bleken van foto's als andere fluorescentie- en kleuringskwesties. Het gebruik van SRS-microscopie...

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

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

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Engineering

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 ...

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

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Bioengineering

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Medicine

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

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

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