UWM Research Profile Magazine - Understanding the physics of fundamental life

Understanding the physics of fundamental life.

UWM professor hopes to shed some (ultrashortpulse laser) light on the physics of how living cells operate and interact.

Laser microscopy using dyes shows molecules of nerve (green) and muscle (red) that are critical for developing functional connections. New developments in nonlinear optics provide the possibility that electrical activity of both nerves and muscles can be recorded in living animals without the use of dyes. (Image and caption courtesy David Heathcote)

by Peter Hansen

uch research has been done on the fundamental unit of life—the cell. With the help of powerful electron microscopes, scientists have learned the complex structures within the many different types of human cells. And by observing which nutrients are transferred from cell to cell, or measuring the changes in neurons’ electrical charges as signals are passed between them, scientists have a pretty good idea of what cells are doing.

But what exactly happens at the cell membrane to allow nutrients and signals through? And what happens inside a good cell to make it go bad? Answering these kinds of questions could help develop better treatments for or even prevent many different diseases. As strong as electron microscopes are, the static images they produce can’t tell the whole story; cells can’t survive in a vacuum, which the machine needs to do its work. So seeing what’s going on inside a living cell requires a different kind of instrument.

Deb Generotzky

Physicist Vladislav Yakovlev is teaming up with developmental biologist David Heathcote to try to understand the physics of human cells.

“You want to understand how molecules inside the cell interact with each other, how they transport, say iron or calcium from one place or another,” explains Vladislav Yakovlev, UWM associate professor of physics. “You have to somehow monitor the transport and to also try to make sense of it. You have to understand what are the mechanisms, because it all comes down to the physical laws. So we’re trying to study the physical laws on the level of cellular structures.”

The main tool Yakovlev uses is optical spectroscopy, a method used to identify and quantify matter by observing how it absorbs, emits, or scatters light. Using light as his tool offers the potential of non-invasive study of cellular activities.

“You could look at living tissue without inserting probes and things like that,” says David Heathcote, a professor of biological sciences collaborating with Yakovlev. “Just using light to look at changes in the structures of the membranes or different parts of the cell and getting an idea of what’s going on without putting in dyes or anything else.”

Yakovlev’s light source is a Cr4+:forsterite laser (a laser composed of forsterite doped with chromium). As he scans a cell with his laser, getting information about what’s inside, a sort of picture emerges. The smaller the size of his laser beam, the higher the resolution of the picture. So far, the best resolution is limited by the diffraction of light to about 200 nanometers, or billionths of a meter.

But even that tiny laser, with the intensity of a trillion watts per square centimeter, could easily destroy the thin cell membrane and the organelles inside, “without doing anything that is useful,” Yakovlev says dryly. The key is to keep the exposure time short. Ultrashort, to be exact.

The nerve cord of the worm contains 3 large, clear profiles of giant axons (bottom). They conduct electrical potentials that mediate the critical escape response of the worm. The large axons provide an ideal test system to try and record electrical activity in living cells using a laser microscope.

David Heathcote, a UWM developmental biologist and associate professor in the Biological Sciences Department, studies development and functioning of the nervous system. Through a collaboration with physics Professor Vladislav Yakovlev, Heathcote is hoping to get a clearer picture of just how neurons do what they do.
   In his teaching and his collabor ation with Yakovlev, Heathcote uses earthworms.
   “They have big neurons that you can see with a low-power microscope,” Heathcote explains. “You can also get them to fire action potentials very easily. We want to use them as a test system to see what kind of spatial and temporal resolution we can get. We know to stimulate those things and we can record their activity. At the same time, can we use the laser light to detect it and distinguish between the different cells in the earthworm: which ones are firing when?” Earthworms’ small size helps overcome the laser’s penetration limit of a few millimeters.
   Heathcote also hopes frogs, another type of animal in his lab, will shed light on his specific research interest —nerve-muscle junctions. Many such junctions in developing frogs are located just under the surface, he says.
   When the current optical limitations are overcome, Heathcote hopes, deeper images of things such as brain activity may be possible, “without doing anything nasty like jabbing electrodes in there.”
   “Those kinds of things are potentially very useful not only for understanding how things work normally, but for clinical purposes and diagnostic things as well.”
—Peter Hansen

The transparent nature of this tadpole makes it ideal to test the capabilities of laser microscopy in a living, functioning animal. (Images and captions courtesy David Heathcote)

Each ultrashort pulse with which he hits his target lasts just tens of femtoseconds, which are quadrillionths of seconds. Put another way, 100 femtoseconds equals .0000000000001 second. Or, as Yakovlev says it: “One femtosecond is related to one second as the nickel in your pocket is related to the federal budget deficit.”

How can he generate such short pulses? “In a typical laser an active material is somehow excited and is capable of emitting quanta of radiation within a certain range of a spectrum,” Yakovlev explains. This is known as stimulated emission radiation, which is the “ser” in the abbreviation laser. “By putting a set of mirrors around the active material, light can be cycled back and forth through the active material, resulting in light amplification,” (the “la” in laser).

“Typically, laser operates in many modes. Each mode is characterized by its individual wavelength within the gain spectrum of laser material. By introducing some external mechanism that is able to synchronize all these modes, one can generate ultrashort pulses, whose duration is limited only by the bandwidth of the laser material.”

Yakovlev then offers a somewhat less scientific explanation, making a comparison to Darwinian evolution. “You just have some kind of mechanism which says short pulses are better for survival,” he explains. “So you can just let this system develop itself and it will generate short pulses. So that’s the beauty of it: the system by itself selects shorter pulses as a way for survival.”

In addition to high resolution, images must be created at a high rate to capture the movement of signals or nutrients within cells. Yakovlev is able to create about 100 images per second, which is more than fast enough to capture activities such as cell reproduction. “But if you’re looking at transfer of signals from one neuron to another, it’s incredibly slow, because one pulse is formed within one millisecond, so you need kilohertz repetition rates; you need at least 1,000 frames per second in order to catch these events.” So far, no one has been able to achieve that rate, he says.

“It’s not only a problem of optics or biology. It’s basically getting your data fast enough, so it’s an engineering problem.”

Another tool Yakovlev uses is Raman spectroscopy, with which he can get information about vibrational properties of molecules.

“This way you get a fingerprint of your molecules,” he says. “That’s how you extract information. How do you know what the particular molecule is doing? You look for changes in the vibrational spectrum and, if you have a clever way to relate these changes to the variation of molecular structure, you can analyze molecular motions and structure changes associated with a particular biological process.

“But it’s a complicated task and nobody, so far, has been able to solve this problem, figuring out exactly what all the components of the cell are doing. That’s the nature of research: you start from something and try to get as much as possible.”

Deb Generotzky

Yakovlev is using Raman spectroscopy, with which he can get information about vibrational properties of molecules. Pictured are postdoctoral fellow Georgi Petrov (seated) and graduate student Vladislav Shcheslavskiy at the UWM Advanced Analysis Facility’s Raman spectroscope.

Collaboration has been central to Yakovlev’s research projects. In addition to two joint federal grants with Heathcote, Yakovlev has collaborated with materials engineering Professor Carolyn Aita, Chiu T. Law in electrical engineering, and investigators at the Medical College of Wisconsin.

Yakovlev also hopes his work will lead to better ways to diagnose and treat cancer. With a clearer picture of how cells operate, drug makers could design more effective treatments. “Something goes wrong with your cell—according to latest theories it’s most likely DNA—and it results in protein malfunction. We’re trying to find a better way to look at this,” Yakovlev says.

Or lasers could someday remove cancer cells from healthy tissue directly. “You basically kill the source of future disease. There are plenty of ways to use lasers.”

A current limitation with Yakovlev’s optical techniques is that his lasers can only penetrate tissue to depth of a few millimeters. “Someone has to come up with a very smart idea. I don’t have this idea yet.”

Still, Yakovlev is hopeful that his work and others’ will show results within 10 to 15 years.

“I don’t see the reason why a cure for cancer can’t be found within our generation.”