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?
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?
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)?
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?
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?
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?
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?
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?