MICROSCOPY

BIO205
 MICROSCOPY
Microscopes are specialized optical
instruments designed to produce magnified
visual or photographic (including digital)
images of objects or specimens that are too
small to be seen with the naked eye.
Scale
 Discovery of Microorganisms
• Antony van
Leeuwenhoek
(1632-1723)
– first person to
observe and
describe
micro-
organisms
accurately
It is critically important to understand the relationship
between the microscope and the human eye.

The human eye as a visual

detector (in combination with the
brain) is the most efficient image-
processing system.

There are no man-made devices

that can match the abilities of the
human eye in regards to imaging
speed and resolution.

The principles of operation

underlying modern cameras,
however, are strongly related to
the structure and operation of the
eye
Total Magnification = Objective Magnification x

Eyepiece Magnification
 Lenses and the Bending of Light
• light is refracted (bent) when passing from
one medium to another
• refractive index
– a measure of how greatly a substance slows
the velocity of light
• direction and magnitude of bending is
determined by the refractive indexes of the
two media forming the interface
Lenses
• The location at which a mirror or a
lens focuses rays parallel to the
optic axis or from which such rays
appear to diverge.
• Distance between center of lens
and focal point is the focal
• length
• strength of lens related to focal
length
– short focal length more
magnification
13
Microscope Resolution
• ability of a lens to separate or
distinguish small objects that are close
together
• wavelength of light used is major
factor in resolution
shorter wavelength  greater resolution
Numerical aperture and The numerical aperture of a microscope is the
Resolution measure of its ability to gather light and to
resolve fine specimen detail while working at a
fixed object (or specimen) distance.

Image-forming light waves pass through the

specimen and enter the objective in an
inverted cone. The smaller the object, the
more pronounced the diffraction of incident
light rays will be –less resolution

In order to increase the effective aperture and

resolving power of the microscope, a
condenser is added to generate a ray cone on
the illumination side of the specimen. This
enables the objective to gather light rays that
are the result of larger diffraction angles,
increasing the resolution of the microscope
system.
The sum of the aperture angles of the objective and the condenser is
referred to as the working aperture. If the condenser aperture angle
matches the objective, maximum resolution is obtained.
 Oil Immersion and Refractive Index

One way of increasing

the optical resolving
power of the
microscope is to use
immersion liquids
between the front lens
of the objective and the
cover slip.
 Microscopy
• General Principles of Microscopy
– Contrast
• Differences in intensity between two objects, or
between an object and background
• Important in determining resolution
• Staining increases contrast
• Use of light that is in phase increases contrast
 MICROSCOPY
Microscopes are specialized optical
instruments designed to produce magnified
visual or photographic (including digital)
images of objects or specimens that are too
small to be seen with the naked eye.
Scale
 The Light Microscope
• many types
– bright-field microscope
– dark-field microscope
– phase-contrast microscope
– fluorescence microscopes
• compound microscopes
– image formed by action of 2 lenses
Four kinds of light microscopy

Pearson education
The Bright-Field Microscope
• produces a dark image against a
brighter background
• has several objective lenses
– parfocal microscopes remain in focus
when objectives are changed
• total magnification
– product of the magnifications of the
ocular lens and the objective lens
 Microscopy
• Light Microscopy
– Bright-field microscopes
• Simple
– Contain a single magnifying lens
– Similar to magnifying glass
– Leeuwenhoek used simple microscope to observe
microorganisms
 Microscopy
• Light Microscopy
– Bright-field microscopes
• Compound
– Series of lenses for magnification
– Light passes through specimen into objective lens
– Oil immersion lens increases resolution
– Have one or two ocular lenses
– Total magnification = magnification of objective lens
X magnification of ocular lens
– Most have condenser lens (direct light through
specimen)
A bright-field
The Dark-Field Microscope
• produces a bright image of the object
against a dark background
• used to observe living, unstained
preparations
 Microscopy

• Light Microscopy
– Dark-field microscopes
• Best for observing pale objects
• Only light rays scattered by specimen enter
objective lens
• Specimen appears light against dark background
• Increases contrast and enables observation of
more details.
The light path in a dark-field
microscope
Principles of phase microscopy
 Microscopy
• Light Microscopy
– Phase microscopes
• Used to examine living organisms or specimens
that would be damaged/altered by attaching them
to slides or staining
• Light rays in phase produce brighter image, while
light rays out of phase produce darker image
• Contrast is created because light waves are out of
phase
• Two types
– Phase-contrast microscope
– Differential interference contrast microscope
The Phase-Contrast Microscope

• enhances the contrast between

intracellular structures having slight
differences in refractive index
• excellent way to observe living cells
The Differential Interference Contrast
Microscope
• creates image by detecting differences
in refractive indices and thickness of
different parts of specimen
• excellent way to observe living cells