Page 112 - Fundamentals of Light Microscopy and Electronic Imaging

OPTIMIZING THE MICROSCOPE IMAGE 95

field planes, and prepare a list of each set in order, beginning with the
lamp.
2. Adjust the condenser for bright-field mode. Image the diatom test plate
using green light (546 nm bandpass filter) and a 40 objective. Focusing
is difficult due to the extremely small size and transparent nature of the
specimen. First locate the diatoms under low power (10 ). Carefully
move the x and y coordinates of the stage until the specimen comes into
the field of view. Then swing in the 40 lens and refocus on the diatoms.
Compare the image of the diatoms with Figure 6-7 and note the indicated
spacings. Using bright-field optics and green light, open the condenser
aperture to the proper location using the telescope lens, and note the
species with the smallest spacing that it is possible to resolve with your
microscope. Indicate the species and note the distance between the striae
from the figure.
3. Calculate the theoretical resolution of the microscope under these condi-
tions. The NA is indicated on the barrel of the lens. Show your work. The
apparent and calculated resolution limits should roughly agree.
4. Now examine the diatom Pleurosigma with a dry 100 objective and
close down the field-stop diaphragm to illuminate just this one diatom and
no other. Make an accurate sketch of the hexagonal arrangement of the
pores. Close down the condenser diaphragm to its minimum size. Now
examine its diffraction pattern in the diffraction plane of the microscope
using the telescope lens. Make an accurate sketch of the diffraction pat-
tern.
5. How does the diffraction pattern relate to the spacing pattern of the
diatom pores in the image plane?
6. Examine the diatoms in red and blue light and with the condenser aperture
open or maximally closed. Which pair of conditions gives the lowest and
the best resolution of the striae?
7. Examine the diatom Pleurosigma or Diatoma with a 100 oil immersion
lens. Can the striae and pores be resolved? Examine the diffraction plane
under these conditions. Make sketches of both views. Now oil the con-
denser as well as the objective and repeat your observations. Make
sketches. Do you notice a difference in resolution?