Not all microscopes allow you to view
the specimen directly. In a scanning light microscope, a laser
beam is focused to a tiny spot on the specimen and scanned
rapidly back and forth, while a lens collects light leaving the
other side of the specimen. Each time the laser spot hits a dark
part of the specimen, little light leaves the specimen and a
computer records a dark spot. Each time the laser spots hits a
clear part of the specimen, considerable light leaves the
specimen and the computer records a bright spot. After studying
the light leaving a whole patch of illuminated spots, the
computer displays an image of the specimen's surface, formed from
these recorded spots.
1. To illuminate the smallest possible spot and thus achieve the highest spatial resolution, the microscope uses laser light rather than light from a bulb's hot filament. Why would light from a hot filament not focus as tightly as laser light?
Answer: Light from a hot filament consists of many different photons and they won't all focus at the same spot.
Why: Laser light is coherent and, from a properly designed laser, consists entirely of identical photons. Since they're all the same in wavelength and direction of travel, they focus at the same location.
2. The laser light is focused by a converging lens located about a centimeter from the specimen. This lens has two convex surfaces and is 5 millimeters in diameter. If you wanted to increase the distance between the lens and the specimen and still focus the laser light to a tiny spot, how should you change the size or shape of the converging lens?
Answer: Reduce the curvature of the lens.
Why: By reducing the curvature of the lens, you lessen the bending of the light and delay its coming to a focus.
3. A particular microscope allows you to choose between two different laser beams--one red and one blue. The focusing lens is a single piece of glass. If you shift from the red laser beam to the blue laser beam, you must move the specimen because the focus of the laser spot moves slightly. Why does the blue laser focus at a different distance from the lens than the red laser does and which way must you move the specimen to put it at the focus of the blue laser (toward the lens or away from it)?
Answer: Blue light travels more slowly in the lens than red light does, so blue light bends more and focuses closer to the lens. You would have to move the specimen toward the lens when you shift to blue light.
Why: Because dispersion in a clear material causes blue light to slow down more than red light, blue light bends more severely when it enters and exits glass or plastic and thus focuses more quickly after passing through a converging lens.
4. One of the beauties of this type of microscope is that it can look for fluorescence. If you insert a color filter into the light collecting system, it will no longer detect the laser light. Instead, it will detect other colors of light emitted by the specimen when exposed to the laser light. When a molecule in the specimen absorbs a photon of laser light and uses some of that photon's energy to emit a new photon with a different wavelength, the light collector detects this light and the computer records it. Why is the wavelength of the fluorescence light always longer than that of the laser light, never shorter than that of the laser light?
Answer: Each outgoing fluorescence photon can't have more energy than the incoming photon from which it was made. Since lower energy photons have longer wavelengths, fluorescence light is never shorter in wavelength than the light that initiates it.
Why: A fluorescent material obtains energy by absorbing a photon. The energy in this photon resides in the material for a short time and then some of it leaves as fluorescent light. Because of energy conservation, this outgoing photon of light can't have more energy than the incoming photon, but it can have less if some of the energy was lost as thermal energy in the material.
5. By carefully detecting the precise wavelengths of the fluorescence light as the laser spot scans across the specimen, you can identify the atoms or molecules that are doing the fluorescing and figure out where they are located in the speciman. Why do the wavelengths of the fluorescence light allow you to determine which atoms or molecules are present?
Answer: Each photon of fluorescent light is created by a radiative transition, in which an electron shifts from one atomic or molecular orbital to another. The wavelengths of the fluorescent photons are related to their energies and thus to the energy spacings between orbitals. Since these spacings are unique to each atom or molecule, so are the wavelengths of fluorescent light.
Why: The spectrum of light emitted by a material through fluorescence is a fingerprint of that material's atomic or molecular orbitals. Thus neon and helium have unique spectra because they have different arrangements of orbitals.
6. When a molecule absorbs a laser photon and emits a fluorescence photon, it can send that photon in almost any direction. To collect those fluorescence photons efficiently, the microscope has a large lens behind the specimen. This converging lens takes all the light that reaches it from that tiny illuminated spot and projects an image of this glowing spot onto a light sensor. What kind of image is this lens forming?
Answer: A real image.
Why: All the light striking the lens from one spot on the specimen is brought together on one spot on the light sensor. The pattern of light is hovering there in space on the surface of the light sensor--a real image.
7. Suppose that you had to move the collecting lens a little farther away from the sample. The image this lens projects onto the light sensor would also move. Which way--toward the lens or away from it?
Answer: Move the light sensor toward the lens.
Why: By moving the collecting lens away from the specimen, you decrease the divergence of the light striking the lens from the glowing spot on the specimen. The light is now easier for the lens to bend together and so the light focuses nearer to the collecting lens.
8. Suppose you increase the diameter (or area) of the light collecting lens without changing its curvature. How would that enlargement affect the brightness and depth of focus of the image it projects on the light sensor?
Answer: The brightness would increase but the depth of focus would decrease.
Why: As you enlarge the diameter of the lens, more of the fluorescence light will hit it and it will send more light onto the spot on the light sensor. But because light reaching the light sensor will now be arriving from a broader range of angles, the need for the light sensor to be at the exact focus becomes more critical. Just in front of or behind the focus, the rays of light will be more widely separated with the enlarged lens, to the out-of-focus nature of the light will be more obvious.