&Bullet; physics 14, 71

A new microscopy technique using micrometer-sized lasers can track impact-induced changes in the refractive index of a heart cell.

M. Schubert / University of St. Andrews

Micrometer-sized lasers in heart cells can track the beating of these dynamic systems.

There is no easy way to image individual cells in living animals. Marcel Schubert of the University of St. Andrews in the UK hopes that his newly demonstrated technique could change that. In a presentation at the Optical Society’s Biophotonics Conference last month, Schubert discussed how he and his colleagues tracked heart cells using micrometer and nanometer lasers placed directly in the cells. With the lasers, researchers could monitor the dynamics of cells deep in living tissues.

Medical imaging such as ultrasound and MRI can allow doctors to see inside the body, but the resolution of this in vivo Methods are usually too low to recognize individual cells. The only real way to look at cells in living organisms is by using fluorescence-based techniques, Schubert said. In these techniques, a camera records the light emitted by a fluorescent dye in the cells, which enables the characteristics of individual cells to be distinguished. Researchers can use fluorescence microscopy for imaging in vivo Brain cells, for example. The tool is difficult to use, however, and requires cutting out a piece of skull and replacing that bone with a porthole-like window for direct visual access to the tissue.

As well as the invasive nature of the tool for in vivo In applications, fluorescence microscopy has the problem that it can typically only image the few outer cell layers of a tissue. In the heart, for example, light only penetrates roughly


of tissue. “We really need a brighter light source,” said Schubert, what he and his colleagues have found out.

In their experiments, Schubert and his colleagues do not place dye molecules directly in the cell, but use a micrometer-sized plastic bead that is infused with dye molecules. It is known that this pearl works as a laser. Lasering works as follows: A pearl is illuminated with light, which causes its dye molecules to fluoresce. Due to the specific refractive properties of the bead, most of this fluorescent light is trapped inside the bead and circles around the outside of the bead.

For certain wavelengths this circling light disturbs constructively and builds up in intensity. The intensity also increases when the circling light hits a stimulated dye molecule, because – as with a normal laser – it gains additional photons through stimulated emission. Some of this circulating light exits and creates a laser signal that can be 1000 times brighter than the signal from traditional fluorescence microscopy techniques, making it much easier to see. It is this leaked light that the team examined in their experiments.

The team placed a bead next to an isolated mouse heart cell, which “swallowed” the bead. When they exposed the cell to light, they observed that the wavelengths of the laser light shifted up and down each time the cell contracted and expanded. At about 0.03% of each wavelength, those shifts were tiny, but they were clearly visible on the spectrometer, says Schubert.

M. Schubert / University of St. Andrews

The wavelength of the light emitted by a laser in a heart cell (left) shifts by a few hundredths of a percent every time the heart cell contracts (right middle graphic). Schubert and his colleagues believe that this shift is due to a change in the refractive index of the heart cell (graphic below right). The lasers used by the team emit five different wavelengths of light (graphic above right), each of which is tracked by the team.The wavelength of the light emitted by a laser in a heart cell (left) shifts by a few hundredths of a percent every time the heart cell contracts (right middle graphic). Schubert and his colleagues believe that this shift is due to a change in … show more

He and his colleagues explained these shifts as a result of local refractive index changes in proteins called myofibrils. These proteins, which look like long, thin rods, contract every time a heart cell beats. This contraction causes the local protein density in the cell to temporarily increase, which increases the cell’s refractive index. When the proteins relax and then lengthen again, the opposite happens. Using a confocal microscope, Schubert and his colleagues tracked changes in length in the myofibrils closest to the pearl and found that their shortening and elongation correlated perfectly with the change in wavelength.

The team also conducted experiments with live zebrafish hearts and with slices made from rat heart tissue. For the latter, they showed that they could recognize the striking signals of pearls through


of tissue. “Other people have tried unsuccessfully to monitor changes in the index of refraction deep in the tissue,” said Schubert. “Here we have shown that we can really do that with our laser beads.”

Schubert and his colleagues are also working on smaller lasers made from a semiconductor called indium gallium phosphide. Their first experiments show that these nanometer-sized disk lasers – like the pearl lasers – can be used to track changes in the refractive index of a cell. Due to their small size, ten of these lasers could be used in a single cell, which enables localized measurements within the cell, says Schubert.

Now that they know the technique works, the team plans to conduct experiments in 3D heart tissues grown in the laboratory. For example, researchers are considering placing their lasers in embryos to monitor the biomechanical properties of a heart as it develops.

– Katherine Wright

Katherine Wright is the assistant editor of physics.

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