Microscopy

Question 1

brightfield

Answer 1

know total magnification calculation, working distance, depth of field, resolution.
Resolution or resolving power is the ability to distinguish two points a specified distance apart.
Our microscopes have a resolution distance of 0.2 micrometers. Resolution capability is more
important than magnification, as this allows us to distinguish fine detail and structure.

Question 2

Darkfield

Answer 2

– bright objects against a dark background. Can be used to examine small and thin cells, such as Treponema pallidum the etiologic agent of syphilis

Question 3

Ultra-Violet (U.V.)

Answer 3

Use smaller wavelengths to establish improved resolution (180 to 400 nanometers)

Question 4

Fluorescence Microscopy

Answer 4

uses U.V. light and fluorescent dyes. Fluorescent dyed
specimens are best viewed with U.V. microscopes. In immunofluorescent microscopy, antibodies are complexed with fluorescent dyes and viewed with U.V. light.

Question 5

Phase

Answer 5

–Contrast – light is slowed down as it passes objects of increased density. Makes visible internal cell structures. Thinner areas light travels faster and structures are brighter. Thicker areas light travels more slowly and structures are darker.

Question 6

Differential Interference Contrast (DIC)

Answer 6

– Same principle as phase contrast. Uses two beams of

light which pass through prisms. Image is in color and nearly 3D

Question 7

Confocal

Answer 7

uses highly refined laser light. Successive planes and regions are illuminated until the entire specimen has been scanned. Computers construct image which can be rotated and viewed in any orientation.

Question 8

Electron Microscopy (Transmission E.M. or TEM and Scanning E.M or SEM) uses

Answer 8

a beam of electrons

Question 9

In TEM the electron beam

Answer 9

moves through the specimen

Question 10

In SEM the beam

Answer 10

of electrons is bounced off the specimen and is collected by a detector. Computers can then create the
image. The wavelength of the electron beam is 0.005 nm. The resolution is considerably improved
when compared to that of the light microscope. Magnifications of 100,000X and even better can be
achieved.

Question 11

The beam of electrons is extremely

Answer 11

weak and must move through a vacuum

Question 12

other characteristics of electrons

Answer 12

Furthermore, electrons have very little penetrating power and specimens (TEM) must be extremely thin. Thicknesses are in the order of 0.02 micrometers. Specimens can never be alive as they must be extremely thin and in a
vacuum

Question 13

“artifacts of preparation”

Answer 13

Since the slides are so highly processed prior to viewing, “artifacts of preparation” are sometimes created. These are artificially created structures due to operator error. In
addition, electron beams cannot be seen by the human eye. The images must be displayed on fluorescent screens, TV screens or on photographic plates