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 spec...

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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 ...

Stimulated Raman scattering microscopy is a chemical imaging technology that enables the rapid and quantitative detection of lipids and allows the tracking of lipids in live animals in a label-free manner. The most important advantage of SRS microscopy over traditional lipid detection techniques is that it is label-free and therefore, is not as sustainable to photo bleaching as other fluorescence and staining matters. The use of SRS microscopy in conjunction with the genetic and biochemical tools available for model organisms, particularly Caenorhabditis elegans provides a powerful framework for studying normal regulators of lipid biology and metabolism.To feed laser beams into the SRS microscope, first, set the periscope to lift the beam from a picosecond light source exit to the infrared laser input port on the scanner of the microscope and open the laser control software to start the lasers. Lower the pump laser power to 50 milliwatts and set the pump signal to 750 nanometers. Use a beam expander to adjust the beam diameter to fit the back aperture of the microscope objectives.Use two relay mirrors, M1 and M2, to guide the laser beam to the periscope and set the nods of the periscope at the center of the tuning range. Select the initial position and angle of each mirror such that the laser beam approximately hits the center of the mirror to launch the beam into the microscope and use an empty port on the objective turret of the microscope to perform the course alignment. Place the power meter probe at the objective port to measure the power of the transmitted light and set the scanner of the microscope at the highest zoom, 50X.Measure the power of the transmitted light with the focused laser beam spot and optimize the knobs of mirrors M1 and M2 iteratively to achieve the highest transmitted laser power. Use the alignment tool to perform the fine alignment of the transmitted light to make sure the light is passing through the center of the objective and place the fluorescence target alignment cap on the empty objective seat. Adjust the knobs of the first steering mirror, M1, to center the laser beam spot and introduce the extension tube with the alignment cap on the other end to an empty objective seat for monitoring the laser beam spot.Then, tune the knobs of the second steering mirror, M2, to center the laser beam spot again on the other end of the extension tube. To set up the SRS detection module, place a beam splitter cube after the condenser to direct the transmitted laser to the photo diode module. To set up the electronics connection, use a built-in electro optic modulator at 20 megahertz inside the picosecond tune-able laser to modulate the intensity of the Stokes beam and feed the output signal of the photo diode into the lock-in amplifier for demodulation of the stimulated Raman loss.Then, feed the lock-in amplifier output into the analog box of the microscope to convert the electrical analog signal to a digital signal. To optimize the imaging conditions, add five microliters of oleic acid to a mini microscope slide chamber and cover the sample with a cover slip. Place the sample onto the microscope stage and use the edge of the pad to locate and focus on the droplet.Adjust the condenser for Kohler illumination and use an infrared sensor card and infrared viewer to check whether the pump beam path is still correct. Confirm that the delay stage is set to zero femtoseconds and set the pump beam wavelength to 795.8 nanometers. Set the power of the pump beam to 50 milliwatts and set the Stoke beam to 100 milliwatts.Open the shutter for both the beams, as well as the main shutter of the laser. Scan the sample and check the image on the computer screen. Change the display mode to high low and adjust the range to see an approximately 50%saturation.Check whether the saturation is centered in the image, carefully adjusting the steering mirrors inside the picosecond tune-able laser system for optimized overlap of the pump and the Stokes to maximize the image intensity and to center the peak intensity as necessary. Before each imaging session, add 100 microliters of warm 2%agarose to a clean glass slide placed between support slides with two layers of laboratory tape and quickly place a second slide on top of the first slide. Gently pressed to create a thin, even, agarose pad and allow the agarose to cool.To mount the worms for imaging, place a four to five microliter drop of anesthetic agent per 10 to 20 worms onto a cover slip and use a dissection microscope to pick the worms to be imaged, placing the worms onto the droplet of anesthesia as they are collected. When all of the worms have been collected, cover the worms with the glass slide with the agarose pad. For imaging, mount the worm sample with the cover slip facing the objective lens and direct the bright field light source to the eyepiece to locate the worms.Bring the worms into focus and adjust the condenser position accordingly. Adjust the laser powers by setting the pump laser power to 200 milliwatts and the IR power to 400 milliwatts. Set the lock-in amplifier for demodulation of the worm SRS signal.Begin scanning the first worm at a fast scanning rate, adjusting the fine focus to find the area of interest. When the signal has been optimized, switch to a slower scanning rate and a higher pixel resolution to obtain the SRS image and save the image in a format that enables high resolution. When all of the samples have been imaged, place the laser source on standby and turn off all of the associated equipment.To analyze the images, open the files in ImageJ and select analyze and set measurements to select the properties to be analyzed. Use the polygon selection tool to outline the region of the worm intestine to be quantified and click analyze and measure again. The measurements will appear in the results window.When all of the worms have been outlined, copy and paste the measurements for each of the worms in a given genotype into a spreadsheet. For the background measurement value, select an area in the vicinity of the worm that does not have any SRS signal and subtract the background mean gray value from the measurement for each individual worm to calculate the average and standard deviation of the background subtracted mean gray values from all of the worms in a given genotype or test group. These values can then be normalized to the average of the control group.In this representative analysis, the intestinal lipid levels in age-synchronized wild type worms and daf-2 mutants lacking the insulin receptor were quantified during adulthood. As expected, a decline in lipid levels is observed in wild type worms, starting at day one and continuing until day nine of adulthood. The lipid levels in daf-2 mutants, however, increase until they are five days old, at which point they remain constant at about 2.5 to three fold higher than the wild type.Daf-16 inactivation suppresses the increase in lipid levels of daf-2 mutants. Expression of the daf-16a a or daf-16b isoform in the daf-2, daf-16 double mutants does not restore the lipid levels. The expression of the daf-16d/f/h isoform, however, is sufficient to restore the lipid levels in daf-2, daf-16 double mutants to the levels observed in daf-2 single mutants.These results indicate that the daf-16d/f/h isoform specifically modulates lipid metabolism in daf-2 mutant worms. It is important to use the same laser power for pump and Stokes beams throughout the imaging session and to include a control group in every six and four consistent quantification. In addition to quantifying lipid storage, SRS microscopy can be coupled with bioorthogonal tags, such as the deuterium, and implemented to visualize the dynamics of lipid incorporation, synthesis and degradation.SRS microscopy permits multiplexing and can be implemented to image multiple biomolecules in different subcellular compartments. So-called hyperspectral SRS microscopy has a great future in exploring the metabolic dynamics.

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

Research

JoVE Journal

Biology

4.0K vues.  Baylor College of Medicine. La microscopie à diffusion Raman stimulée est une technologie d’imagerie chimique qui permet la détection rapide et quantitative des lipides et permet le suivi des lipides chez les animaux vivants d’une manière sans étiquette. L’avantage le plus important de la microscopie SRS par rapport aux techniques traditionnelles de détection des lipides est qu’elle est sans étiquette et n’est donc pas aussi durable pour le photo blanchiment que d’autres questions de fluorescence et d...