Flashcards in Microscopy Deck (13):
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.
– bright objects against a dark background. Can be used to examine small and thin cells, such as Treponema pallidum the etiologic agent of syphilis
Use smaller wavelengths to establish improved resolution (180 to 400 nanometers)
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.
–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.
Differential Interference Contrast (DIC)
– Same principle as phase contrast. Uses two beams of
light which pass through prisms. Image is in color and nearly 3D
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.
Electron Microscopy (Transmission E.M. or TEM and Scanning E.M or SEM) uses
a beam of electrons
In TEM the electron beam
moves through the specimen
In SEM the beam
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
The beam of electrons is extremely
weak and must move through a vacuum
other characteristics of electrons
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