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

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.