MICROSCOPY
Introduction
•Microscopes produce magnified images of specimens
using a source of illumination and lenses
Typ e Light Electron
Illumination Light Electrons
Lenses Glass Electromagnetic
Magnification 1000X 100,000 – 2,000,000X
Resolution 200 nm 0.05 nm
Used to view Cells Cells, viruses, molecules
LIGHT MICROSCOPES
Parts and Functions
Microscope Part Function
Base Supports the microscope
Light source Source of light
Light switch (on/off) Turns the light on and off
Light rheostat knob Adjusts the light intensity
Condenser Focuses light on the specimen
Iris diaphragm lever Adjusts the light to provide contrast
Stage with stage clips Secures and holds the specimen in place
Stage adjustment knobs Moves the stage to view different parts of the specimen
Arm Supports the microscope
Objective lenses with revolving nosepiece
Magnify the specimen: 40x, 100x, 400X and 1000x (oil
immersion); use revolving nosepiece to change the
objectives
Parts and Functions
Magnification
•Objective lenses: 4X, 10X, 40X, 100X
•Ocular lens: 10X
•Total magnification = Objective lens x Ocular lens
•At 40X?
•Maximum magnification?
•Bacteria should always be evaluated at 1000X total
magnification
Resolution
•Ability to distinguish two objects
that are very close together
•Two close objects will appear as
one blurry object at low resolution
•Defined as the minimum distance
that 2 separate objects can be
distinguished (objects closer than
this distance will be blurred
together)
•Limited by wavelength of light
•Switch to electron microscope for
higher resolution
Resolution
•Oil immersion increases
resolution at 100X
objective
•Oil has similar refractive
index to glass
•Prevents refraction
(bending) and loss of light
rays
Contrast
•Difference in color intensity between object
and background
•Many microbes are thin & transparent, so do
not transmit enough light to the objective lens
and appear white against a white background
– low contrast
•Need to increase contrast in microscope
using the iris diaphragm lever or stains
•Why might staining not be desirable?
Because it kills cells
Types of light microscopes
•Bright-field
•Dark-field
•Fluorescence
Bright-field microscope
•Most commonly used is bright-
field, compound (uses 2 or more
lenses) light microscope
•Illuminates field evenly generating
a bright background
•Total mag. up to 1000X
•Resolution = 0.2 µm (human eye =
200 µm)
10X
40X
100X
Field
Bright-field microscope
•Light from bulb is focused on specimen using
condenser lens
•Specimen absorbs, reflects, and transmits
light, some of which enters objective lens
forming a magnified image
•Image is further magnified by ocular lens
•Field bright as light focused on it is
transmitted and enters objective lens
Light microscopes that increase contrast
•Dark-field
•A hollow cone of light is focused on field
& specimen, such that only the light
transmitted by the specimen enters the
objective lens to form an image, thus
specimen looks bright against a dark
field
Light microscopes that increase contrast
•Fluorescence
•Used to see specimens that are naturally
fluorescent or stained with fluorescent
dyes
•When UV light is focused on the
specimen, it emits visible light that forms
an image
Light microscopes that increase contrast
•Fluorescence
•A powerful way to detect specific microbes and subcellular
structures while avoiding signals from dust and other nonspecific
materials.
Green - E. coli sensitive to
tetracycline
Red - E. coli resistant to
tetracycline
2 different fluorophores used
Green V. cholerae in mouse
intestinal cells (blue) that are
secreting mucin protein in
mucus (red)
ELECTRON
MICROSCOPES
Electron microscopes
•A beam of electrons is focused on
specimen by electromagnetic lenses
to form an image
•Total mag. up to 100,000X
•Resolution = 0.3 nm
•Special preparation required to view
specimen
Electron microscopes
Typ e Tran smi ssi on Scanning
e- beam transmitted through
specimen
scans specimen surface
Specimen very thinly sliced sections
(100 nm)
thick (~cm)
Used to view internal structures surface details
Image 2-D 3-D
SEMTEM
Atomic force microscope
•Uses a laser probe that scans across a surface looking for
bumps/valleys to create a topographic map
•Better resolution than electron microscope
•Can see molecules
•No special preparation required Tetanus Toxin Antibodies
DNA
Cell membrane proteins
Comprehension check
Which type of microscope achieves contrast by
shooting ultraviolet light at the specimen?
1. Atomic force microscope
2. Dark-field microscope
3. Fluorescence microscope
4. Electron microscope
STAINING
rong
↳estamate
size
Eukaryot
-
of
cell
peokayol
bacteria
