Good Vibrations

Perspectives of CARS Microscopy in Life Science Research

April 13, 2011

The ability to see the cell’s molecular machinery at work has contributed immensely to our understanding of cellular functioning. In recent years, new molecular imaging techniques, such as coherent anti-Stokes Raman scattering microscopy (CARS), have been developed for rapid vibrational imaging of living cells. The intrinsic molecular vibrations leave specific fingerprints in the vibrational spectrum. Eric Potma, Ph.D., assistant professor at the University of California, Irvine, USA explains, how his team advance and apply this novel imaging technique for unveiling the molecular secrets of microscopic biological systems.

The first touches with CARS microscopy

When I was a graduate student in the Netherlands I did ultrafast spectroscopy. And my adviser suggested I apply those techniques to microscopy. This was a hot trend in the late ’90s, when the two-photon microscopy technique was becoming increasingly popular. We also saw a bit of second harmonic microscopy in those days, but not a lot. I was playing with a laser system that had two colors, and thought about doing a pump-probe type of microscopy.

In 1999, I saw Andreas Zumbusch of the University of Konstanz, Germany (then a postdoc in the laboratory of X. Sunney Xie) and Michiel Müller of the University of Amsterdam, the Netherlands, showing their work on CARS microscopy at the FOM conference. I was very impressed with the capabilities and the contrast of CARS microscopy. I immediately went back to my laboratory in the Netherlands and lined up the instrument to do CARS. The next day we got the first CARS signal. I have been doing CARS microscopy ever since.

Studying biological and material samples

The first sample we looked at was Dictyostelium discoideum, an amoeba cell. We used these cells as model systems to learn about water distribution in cells. We focused on the water band OH-stretching and did dynamic measurements on flushing water through the cells to examine water diffusion in living cells. I was in a physical chemistry department and we focused on biophysical applications in those days. We collaborated with biologists and cell biologists and those people brought us the samples.

It was great to work with biologists since they brought the real questions to the table, which made us tweak our instruments in such a way that we could see things that actually matter. There is a great collaborative spirit between biological researchers and those more involved in physics.

CARS became popular on the biology side, but you can probe anything else with this particular type of contrast. You can probe vibrational and electronic features as well. We are looking at carbon nanotubes, and trying to understand the propagation and coherent evolution of primary excitations. We also study the nonlinear optical properties of plasmonic structures on a microscopic level. CARS is a great way to perform these applications. So, it is finding its way into material science as well.

Fig. 1: Kidney tubules: CARS image at 2,845 cm–1 of the mouse kidney, showing the tubules and intracellular lipid droplets.
Fig. 3: Rayon anisotropy: CARS anisotropy image of rayon fibers. The anisotropy was determined at the 2,880 cm–1 methylene stretching mode of rayon. The brightness in the image corresponds to the degree of orientation of the methylene mode in the fiber.

Insight into fundamental cellular process

The real benefit of CARS microscopy is that you look at molecules just the way they are. There is no need to put labels on them, no need to dress them up in a certain way to make them fluorescent. That’s really where the technique is advantageous. You can look at all molecules that have a good Raman signature and so there are a couple of biomolecular candidates that can be easily visualized using the CARS microscope.

Any question dealing with lipid metabolism is where CARS can make a difference, as well as any question dealing with the mobility of water molecules, membrane dynamics, and variations in protein density distributions. And CARS holds great promise for following extrinsic agents like drug molecules or any molecular compound with a strong vibrational signature in tissue. This is great, since these molecules are typically hard to visualize otherwise, as they cannot always be labeled. Usually they are too small – if you label them you don’t get them into the cell or you change their functionalities.

With CARS we image such targets at a rate that is much faster than conventional vibrational imaging.We are talking about imaging in real-time, which is important for imaging all things biological, like living cells and tissues in vivo – these are the situations where CARS microscopy really helps.

Fig. 2: AortaPCA: This is a CARS image of an atherosclerotic lesion in a mouse aorta. The left panel shows a regular CARS image of the lesion at 2,845 cm–1, highlighting the lipophilic components. The right panel shows a spectral decomposition (principal component analysis) of the CARS signal in the 2,750–3,050 cm–1 range, showing that different areas in the image correspond to different lipophilic compounds. Each color in the image corresponds to a different CARS spectrum. Scale bar is 50 microns

CARS in life science research

There is a very important research direction that aims at visualizing endogenous molecules in living animals. The CARS microscope is great because of its speed, so you can monitor molecules in real-time. For instance, people have used CARS to look at myelin degradation – which is a way to investigate diseases like multiple sclerosis. There is no other way you could do this; visualizing myelin in real time in a living animal is really difficult. CARS is the only avenue for people to do this.

Another example is the use of CARS for skin imaging: attempts are under way to do this with a system that is currently optimized such that you can put your arm under a microscope and look at tissue morphology and abnormalities. This has direct implications for improving human health.

The future of CARS microscopy

I think we can anticipate that with CARS the same thing will happen as with confocal imaging. CARS is the latest edition of contrast methods. You want to inspect your samples, look at molecules, have depth-resolved imaging and do it with the least amount of perturbation to the sample – no harsh treatment or staining protocols – this is especially true for living systems.

So, having a system that adds this type of contrast to the microscope is an enormous step forward. It clearly widens the current landscape of scientific investigation. You can see more than was previously possible. It enables you to do more and to work on key applications. Just one breakthrough application is a tremendous success in itself.

For instance, people would have never foreseen the impact of being able to directly visualize lipid metabolism, an essential process in our bodies that was previously difficult to study without the use of perturbing labels.

By creating such new research avenues, CARS is a true asset to the label-free imaging approach. Other applications are waiting to be discovered. It is inevitable that CARS will continue to have a major impact in the biological and material sciences. It’s the way forward.

Fig. 4: Nanotubes: Electronic CARS signal from carbon nanotubes between Ti electrodes. The wider white lines correspond to the electrodes.
Fig. 5: Meibomian: Transversal cross section through a mouse meibomian gland. CARS signal was measured at 2,845 cm–1 to visualize the lipids in the gland. The center portion of the image corresponds to the duct of the gland, whereas the peripheral regions show the lipid rich meibocytes.
Fig. 6: Nanowires: Electronic CARS image of semiconducting CdSe nanowires using ps laser pulses. CdSe wires are 330 nm wide, 60 nm high and were lithographically fabricated on a glass coverslip.

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