Tonight Amy prepared a brain sample for her two-photon microscope. A two-photon microscope works on a principle that violates the basic statement of Einstein’s work that won him the Nobel Prize.
Einstein was busy in 1905, often called his Annus Mirabilis (his “Miracle Year”). He produced his work on special relativity– but he never won a Nobel Prize for that. He produced the linchpin for an elementary explanation for thermodynamics–but never won a Nobel Prize for that either. Einstein’s Nobel-Prize winning work that year explained the most basic interaction of atoms with light, and forever changed our view of light.
Atoms are made of two charged particles, protons and electrons. Through those electric charges they interact with light. Modern physics has been studying these interactions for over 100 years and is the basis of everything from lasers to
making pornographic pictures in airports backscatter X-ray scanners at airports. Einstein’s Nobel Prize explained how only light above a certain energy will free an electron from atom: Giving a small amount of energy to an electron in an atom may wiggle its orbit a bit, but won’t break it free. Only with a large enough kick can an electron be separated from its atom. But physicists found that visible light, no matter how bright, would not knock electrons free from a material, while even a weak ultraviolet light could.
Einstein proposed a solution. He predicted that the energy of a beam of light was carried by individual particles of light, now called photons. He borrowed the idea from Max Planck, who had assumed such quantization to explain the color and brightness of hot objects. But now Einstein took the idea not just as a mathematical trick, but as a real description of the nature of a beam of light. So light would collide with atoms like two billiard balls. For most materials, visible light particles do not carry enough energy to knock electrons out of atoms. Only ultra-violet light has enough energyto knock out electrons. No matter how intense you make a the visible light, the story goes, you will never knock out electrons because the individual photons do not have enough energy. Because this represents one of the most basic interactions of light (the photons) with electrons is called the photoelectric effect.
During my own undergraduate days we demonstrated this effect in our student laboratory. We shone light onto a metal and looked for a charge buildup due to escaping electrons, just as it would if it were exposed to static electricity. But no matter how intense the visible light, no charge was built up. But a small ultraviolet lamp, such as you might use in a tanning bed, quickly charged the metal. Only ultraviolet photons have enough energy to knock out electrons, so there is a minimum for anything to happen. This is the same reason that rays damage your skin so easily, but visible light does not.
“Wait!” you might be thinking. What if the atom could absorb two particles of light, “two-photons”, at once? Couldn’t the combined energy of two low-energy, even infrared photons, be enough to kick out an electron? And you’d be both right and eighty years too late. The effect was predicted by Maria Goeppert-Mayer in her 1931 Ph.D thesis. She later went on to win a Nobel Prize of her own for completely different work, explaining the structure of the atomic nucleus.
The effect is very small, since two photons must combine with the atom at the same time. Most collisions of light with atoms are so small they are characterized by a small area. In my undergraduate lab we did not have anywhere close to the intensity for this effect to even begin. The effect predicted by Maria Goeppert-Mayer was so small that a new unit of measure, using the square of the small area, had to be invented. To this day, the unit is called the “GM” in her honor.
The effect would have remained just theory, without a light source intense enough to produce it experimentally. Charles Townes, the inventor of the laser, had such a device. His student, Isaac Abella (later one of my professors) was one of the first to demonstrate two-photon absorption experimentally, using infrared laser light to produce absorption at a factor of two less energy than Einstein’s model.
The two-photon technique has revolutionized the ability of scientists (and if I were Sheldon I might add “…and biologists”) to study thick biological and even living tissue. A two-photon microscope uses precisely this effect predicted by Goeppert-Mayer. Hence the discussion: Sheldon complained to Amy she was cutting the samples to thin. You can’t slice tissue too thin for an electron microscope, but thick samples is where a two-photon microscope comes into its own. Lucky for us, Newport Corporation lent us a two-photon microscope, the one you see on the show.
In a two-photon microscope, not enough energy is delivered to liberate the electron, but does excite the molecules of a flourescent dye with two photons. The resultant single photon fluorescence is a single photon of nearly twice the frequency of the incident red or infrared light, typically green and can be separated from intense incident light to form an image.
Another advantage is that the rate of exication now depends on the square of the illumination, since the technique needs two photons to be absorbed. By carefully focusing the incident light, only one small selected region of the sample glows, allowing a detailed picture without the extraneous light from everywhere else, as you would have in a conventional microscope.
And so we finally see scientists wearing lab coats on The Big Bang Theory. That’s what happens when you let in the biologists.