One of the biggest problems in looking at biological tissues through a microscope is the lack of contrast. Basically, everything, with the exception of red blood cells, is transparent to visible light. The faint structures that are visible can be seen due to the tiny amount of light scattered from things like cell walls, the nucleus, and organelles.

To get around this problem, researchers usually label the structures they are interested in with a dye. When the sample is illuminated by a laser, the dye fluoresces like crazy, providing nice, bright, false-color images of the structure. Of course, you need to know what you want to label and how to label it, and you need to know that the label isn't changing what you are looking at. If you are interested in looking at the dynamics of living cells, you have to hope that the dye doesn't kill the cell as it breaks down. In short, using dyes is both a wonder and a curse.





Immunofluorescence is great if you

like your cells dead.

Some recent research, published in the Journal of the Optical Society of America B, shows that the visibility of some very specific features in label-free images can be significantly enhanced with a little bit of optical trickery. The imaging technique is called coherent anti-Stokes Raman Spectroscopy (CARS), and it's being touted as a label-free imaging technique that can provide resolution competitive with standard confocal microscopes at video frame rates.

CARS makes use of the vibrational modes of molecules to selectively image them. Two atoms in a molecule are held together through a chemical bond that is much like a spring. The mass of the atoms, and the strength of the spring provide a set of vibrational motions and rates that, when viewed globally, are unique to the molecule. Some of these characteristic vibrations can be indirectly detected through Raman scattering, where a relatively high-energy photon hits the molecule and excites a vibrational mode. In doing so, it loses energy and is shifted to a redder part of the spectrum, which is called a Stokes shift.

By measuring the change in wavelength, one can detect the presence of different vibrational modes. CARS modifies this by using both high-energy photons (called the pump beam), and a second beam of photons at the expected redder wavelength (called the Stokes beam). The pump beam provides the driving energy to set the molecules vibrating, while the Stokes beam stimulates the molecules to emit in that particular vibration mode. Since the pump beam is still present, the molecules get whacked a second time and, since they are already vibrating, the photons that come out are bluer than the pump light (called an anti-Stokes emission).

This anti-Stokes light is easier to detect for a number of reasons, the principle one being that this is a coherent process, so all the light travels in a single direction—if we just used the Stokes side, the light would be emitted in every direction. On the other hand, the CARS signal has two components: the resonant signal, which is what you really want to see, and some unwanted, nonresonant background. Getting rid of the background signal while not eliminating the resonant signal is one of the major challenges of CARS research today.

The work of Lu and coworkers, from the National University of Singapore, uses a clever trick to simultaneously filter the nonresonant background and amplify the resonant signal. The key is that the nonresonant background light has a slightly different polarization than the resonant signal. If one puts in a polarization filter at the right angle, the nonresonant background is eliminated, but so is most of the resonant signal that we want.

The researchers took the Stokes beam and divided it into two beams. One of the beams has its polarization rotated so that the two beams are 90 degrees from each other. At this point, the two Stokes beams can't interact with each other at all.

The resonant part of the two anti-Stokes beams can interact though and, due to the coherence of the process, this interaction depends on the relationship between the two Stokes beams. By shifting a mirror between two positions, the researchers obtained two images. One was basically the nonresonant background, and the second contained the resonant signal, the nonresonant background, and the two multiplied together—think of the nonresonant background as amplifying the resonant signal. Subtracting the two images provided an amplified version of the resonant signal.

They showed that, compared to filtering, they obtain a 20-fold increase in the signal-to-background ratio. Furthermore, the signal intensity, compared to the total CARS signal, is only a factor of two smaller, indicating that this is a pretty effective way of getting rid of the nonresonant background. There are a few pretty pictures of cells and polystyrene beads to back up their data—I have to say, I was impressed.

CARS microscopy is quite complicated in terms of equipment, setup, and alignment. This adds a little complexity to the system, but that addition is nothing compared to the complexity of the original set up. Although I like the CARS technique, and I know of places that are trying to set them up outside the research lab, I don't think we are going to be looking at widespread adoption anytime soon.

Journal of the Optical Society of America, 2008, DOI: 10.1364/JOSAB.25.001907