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

Research

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Biology

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

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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)

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Basic Caenorhabditis elegans Methods: Synchronization and Observation

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Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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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

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 ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

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

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Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

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Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...