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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 “Stokes” beam increases (stimulated Raman gain, SRG) and the intensity of the transmitted “pump” 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 μsec 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 300–500 μm. 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 organisms^12,13,14,15. 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.

Protokół

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 700–990 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.

1. Feeding laser beams into the microscope
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. Repeat steps 1.1.4 and 1.1.5 until the beam centers the target at both positions.
2. Connecting detection and electronics
   1. 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.
   2. Set up the electronics connection (Figure 1). The intensity of the Stokes beam is modulated by a built-in EOM at 20 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 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 10 or 20 MHz.
   3. 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.
3. Optimize imaging conditions
   1. 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 μL 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 2–3 months.
   2. 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.
   3. Set the imaging parameters. Check whether the delay stage is on the right numbers. Set the pump beam wavelength to 795.8 nm. Using an IR sensor card and IR viewer, check whether the pump beam path is still correct.
   4. 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.
   5. Set the lock-in amplifier gain to maximum. Open the shutter for both the beams, as well as the main shutter of the laser.
   6. 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.
   7. Fine tune the delay stage and find the maximum SRS signal intensity from the dodecane sample. The system is then ready for use.

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.

1. Preparation of the imaging slides
   1. First, prepare 2% agarose solution in distilled H₂O, and make sure all the agarose melts before preparing the agarose pads.
   2. Add one drop of warm 2% agarose onto a blank slide (approximately 100 μL) 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.
   3. 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.
2. Mounting the worms
   1. 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.
   2. Place a drop of anaesthetizing agent to a coverslip (approximately 4–5 μL for 10–20 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.
   3. 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.
   4. Separate the glass slides (from step 2.1) without perturbing the agarose pad.
   5. 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.
   6. Finally, mark the location of the worms using a permanent marker on the glass slide.

3. Image acquisition and analysis

1. Acquiring the images using the SRS microscope system.
   1. Before imaging any sample, determine the imaging parameters and verify SRS signals (as explained in Protocol 1).
   2. 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.
   3. Bring the worms into focus and adjust the condenser position accordingly.
   4. 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.
   5. Switch to a slower scanning rate (e.g., 2 μs/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.
   6. 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.
   7. 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.
   8. Remove the slide before placing the next sample slide. Repeat steps 3.1.2–3.1.7.
   9. 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.
2. Analysis of images using ImageJ
   1. 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).
   2. Select the properties to be analyzed (Analyze > Set Measurements). For total lipid storage quantitation, select Area, Min and Max Gray Value, and Mean Gray value.
   3. 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 > Measure. A new window with the measurement results will pop-up. Repeat this step for all the open images.
   4. Select the measurements for all the worms in a given genotype and copy/paste the results to a new spreadsheet. Repeat the steps 3.2.1–3.2.4 for all the genotypes in the same imaging session.
   5. 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.
   6. 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.
   7. Finally, perform the appropriate statistical analysis using the mean gray value as a measure for lipid levels in each worm. Typically, use Student’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 > Adjust > Brightness and Contrast).

Wyniki

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, the worm lipid storage tissue²¹

Dyskusje

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...

Materiały

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