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Seeing skin deep: Special microscope gives live views of cells

Monday, January 10, 2000

By Byron Spice, Science Editor, Post-Gazette

The mole on my forearm measures perhaps a sixteenth of an inch. But Simon Watkins has pressed a flashlight-sized microscope against my arm, magnifying the mole so that just part of its image almost fills the screen of a computer monitor.

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Confocal applications


No magnifying glass could match the detail of this electronically processed image, in which individual skin cells can be seen. But the real wonder occurs when Watkins adjusts the controls and the image changes, revealing successive layers of living cells as the microscope peers deep beneath the surface of my skin. It's all painless and non-invasive; only laser light penetrates my skin.

It's as if Watkins is using X-ray Specs -- the novelty glasses that make it seem as if you can see the bones in your hand. The difference is that the device in Watkins' laboratory at the University of Pittsburgh sees into the skin, not through it.

And, unlike X-ray Specs, this thing actually works.

Called a confocal endomicroscope, the Australian invention gives researchers and doctors the ability to observe cells that not only are alive, but function and move within living beings.

"I am absolutely infatuated with this thing," said Watkins, who directs Pitt's Center for Biologic Imaging and who in November became the first U.S. microscopist to get his hands on one. "It's like getting a hand-held CAT scanner."

A microscope that allows doctors to see into the skin or other exposed surface potentially could replace surgical biopsies as a means of diagnosing skin cancer and could become a tool for monitoring treatments. It even might guide doctors to abnormal cells that can then be zapped with a laser.

Research applications are numerous. Rather than study cells grown in a petri dish, scientists can view cells as they move around or pump out substances in animals or patients. Lab animals need only be shaved and anesthetized, not sacrificed. When an experiment is over, Watkins said, "you pick him up, revive him and let him run back in the cage."

Confocal microscopes themselves are not new. Commercial versions have been available for more than a decade, but they typically fill a desk top and can only study small specimens that fit on the microscope stage beneath its objective lens.

Thanks to fiber optic technology, researchers at Optiscan Imaging Ltd., in Melbourne, Australia, were able to free the confocal microscope from its bulky laser light source, detectors and computers. They also reduced the size of its optics; the probe Watkins used to image my mole was a 7-inch-long stainless steel cylinder supported by a metal arm. A hand-held version the size of a cigar is scheduled to arrive soon at Pitt, the first U.S. research center to obtain the device.

"It's a pretty good job of miniaturization," said Alan Waggoner, director of the Center for Light Microscope Imaging and Biotechnology at Carnegie Mellon University. With the increase in applications that accompanies that miniaturization, he added, "I think it's going to become a much more important technology."

Conventional confocal microscopes cost about $300,000, but Optiscan expects to sell its endomicroscope for $50,000 to $70,000. General manager Roger Wallis said Optiscan hopes to bring the cost down further so it can be affordable for physician offices.

Confocal microscopes are valuable because they provide blur-free images and can differentiate between features at different depths within a specimen. This information can be combined by computer programs to produce three-dimensional images.

It was invented in 1955 by Marvin Minsky, a pioneer in artificial intelligence and robotics at the Massachusetts Institute of Technology's Media Lab.

Then a post-doctoral fellow at Harvard University, Minsky was interested in mapping the brain's circuitry. But that was impossible to do with regular microscopes, which produce images that include not only the spot in focus, but also blurry areas around it. That just wouldn't do for the study of interwoven, densely packed brain cells.

The ideal microscope, he reasoned, would gather light only from the point of focus. By scanning a specimen, gathering light from a succession of focal points, a blur-free image could be constructed electronically.

He accomplished this feat by combining two microscopes that were confocal -- sharing a common focus. The first microscope focuses intense light through a pinhole. This light passes into the main microscope and illuminates only the point of the specimen that is in focus. Light from the specimen then reflects back, retracing its path through the microscope optics.

At the beginning of its return flight, this light is contaminated by incidental light from areas outside the area of focus. But when the light again reaches the first pinhole, only the light from the focal plane passes through and the blurry light is stripped off.

Minsky won a patent and impressed visitors to his lab, but the confocal microscope was ahead of its time. Computers of the 1950s strained to process its images and the microscope required sources of intense light not widely available then.

The invention of the laser in 1960 solved the problem of light intensity and increases in computer power eventually solved the image-processing dilemma.

Also, the utility of confocal microscopes -- and light microscopes in general -- was boosted by the development of a variety of fluorescent dyes that release light when stimulated by certain wavelengths of laser light.

These dyes are being used to label any number of proteins or chemicals, which allows scientists not only to view parts of cells in detail but also to monitor their function.

Still, the combination of computers, lasers and complicated optics, including sophisticated mirror systems to rapidly scan the point of focus across and through a specimen, makes it "a pretty cumbersome device," Waggoner said.

The Australian researchers were able to streamline the instrument by substituting a long, flexible optical fiber for the pinhole. The laser, detectors and computers could thus be separated from the microscope lenses.

The fiber also is key to eliminating the scanning mirror system.

"Everything goes through the end of the fiber," Watkins explained, "and the fiber moves."

A small piezoelectric device in the microscope handpiece drives the fiber a thousand times a second, scanning the laser pulses across a specimen. The microscope gathers up to a million data points each second.

A limiting factor, however, is light penetration.

Blue-green laser light, which is used to trigger many fluorescent dyes, tends to be scattered by the skin. So the confocal endomicroscope can only see about 15-20 skin cells deep.

But Watkins and the Optiscan researchers plan to soup up the new scope, combining it with a technique called multi-photon fluorescence that uses longer wavelength, far-red laser light that penetrates farther.

If it works with the endomicroscope, he added, users could see into the skin to a depth of 100 cells -- down to the basal cells of the skin's epidermal layer.

Normal fluorescent dyes release a single photon of light for every photon of light they absorb.

The deep-penetrating, but low-energy light used in the two-photon method normally can't excite the dye molecules that way. But when that light is delivered with such intensity that two low-energy photons arrive at the dye molecule at the same time, the photons are absorbed and excite the dye, causing it to fluoresce.

Already, the new microscope is being used to monitor the effectiveness of gene therapy delivered via a device called a gene gun.

Researchers are trying to prompt the immune system to launch attacks on cancers by injecting genes into dendritic cells, a type of white blood cell.

The idea is that the dendritic cells, thus activated, will cause T-cells to fight cancer cells.

In experiments directed by Dr. Louis Falo, the genes -- bits of DNA -- are attached to millions of gold beads so tiny that they can enter cells without damaging them.

The gene gun uses pressurized helium to fire the beads into the skin.

The researchers want to be sure the beads are hitting dendritic cells within the skin

Falo said Watkins has used the endomicroscope to see if the beads are hitting dendritic cells in the skin of lab mice and if the genes are producing desired proteins in those cells.

"He is able to show us if we are hitting the right cells and if the DNA is being expressed," Falo said.

In just a few hours, the endomicroscope provides results similar to what Falo and Watkins previously were able to achieve only after months of processing samples of skin and lymph nodes that had been surgically removed.

The endomicroscope enables them to not only see where the dendritic cells go, but how they got there.

Watkins said the instrument's potential as a research tool remains to be tapped.

"We haven't realized all we can do with it," he added.

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