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Microscopy
I.
History
a. Hans and Zacharias Janssen, Late 1500s, Dutch Eyeglass Makers
A. Perfected Glass Production; Built compound microscopes
b. Robert Hooke, Late 1600s, English Scientist / Engineer
A. Improved upon early compound microscopes
B. Coined the word “cell”
c. Anton van Leeuwenhoek, Also Late 1600s, Dutch Scientist
A. Used simple “microscopes” but a premier lens maker
B. First to describe bacteria, protists a.k.a. “animucules”
II.
Basic Optics
a. Refraction
A. Light travels different speeds in different media
B. When light encounters the border between two media it bends
C. Lenses are designed to use this bending to direct light to a
specific place
b. Magnification
A. Microscope lenses are designed to enlarge the image created
through refraction
B. Amount of magnification called “Power” – designated like 10X
C. Compound microscopes combine the magnification of two lenses
1. Objective lens magnifies an image onto the focal plane of
the ocular lens (inverting the image in the process)
2. Total magnification is the product of the two separate
magnifications
c. Resolution
A. Resolving Power = the capacity to distinguish between two
adjacent objects
1. This power is limited by wavelength of visible light and the
aperture of the lens
2. Theoretical limit of resolving power is ~ 200 nm (0.2 um)
a. Macrophages ~= 15-20 um
b. E. coli ~= 2.0 um
c. Polio Virus ~= 0.02 um
d. Contrast
A. Eyes and Brain like to compare differences in either color or
intensity of light
B. Allows better detail to be “seen”; defines edges, borders, etc.
e. Aberrations
A. Lens are not perfect; chromatic, spherical aberrations occur
among others
B. The higher the quality of the lens, the less aberrations
III.
Brightfield Microscope Parts and Usage
a. Lenses
A. Objective – has rotating nose piece
1. Most ‘Scopes you will see have 4 choices; ours have 4X,
10X, 40X, and 100X
a. 100X is the largest lens and is called an “oil
immersion lens”
B. Ocular or eyepiece – can use prisms to split into any number of
eyepieces
1. ‘Scopes in our lab have 10X oculars
b. Light Source and Diaphragm
A. Need consistent source of light; Diaphragm regulates amount of
light – helps with contrast
B. Light source can also be filtered, typically to blue-violet, why?
c. Stage and Focusing Knobs
A. Knobs move stage, which in turn moves slide, which in turn
moves object – goal is to place object in focal plane of objective
lens
B. Do NOT use Coarse Focus Adjustment when using longer
(40X, 100X) objectives; coarsely focus first at low mag then use
fine with high mags
d. Base and Arm
A. Always transport a microscope with one hand on each the
arm and base
IV.
Procedures
a. Blood Slide
b. Wet Mount
A. Pros: Near to the natural state; Can view motility
B. Cons: Glass cover can damage larger cells; Can dry out quickly
c. Hanging Drop Slide
A. Can be a better alternative to wet mounts, but slightly more
complicated and slides slightly more expensive