History of Microscopy
• Born in Netherlands
• Fabric Merchant
• Wine Assayer
• Minor City Official.
• Learnt to grind lenses
and started building
simple microscopes by
ANTONIE VAN LEEUWENHOEK
Common terms in Microscopy
• Aberration Unwanted Artifacts
– Dust or Oil on Optical Surfaces
– Improper alignment
– Improper aperture settings
– Imperfections in lens system
• Spherical aberration
– Image in focus I n the centre and blurred at the periphery
• Chromatic aberration
– Colour fringes within image field
Depth of field
• Vertical distance between the nearest and
farthest objects in specimen that appear to be
in sharp focus.
• Depth of field increases with numerical
aperture of lens
Immersion fluid ( Oil )
• Any liquid that occupies space between the
object and microscope objective lens
• Required for objectives that have working
distance of 3mm or less
• Immersion fluid should have the same
refractive index as that of slide (1.515)
• Immersion fluid maximizes effective numerical
aperture of objective lens
• Minimizes light refraction
• Measure of light gathering capacity of Lens or
Objective Mirror Condense
Objective Eye Total
Low Concave Lowest Partially
10 10 100
45 10 450
100 10 1000
Special Dark field condenser
BRIGHT FIELD DARK FIELD
• BLOCKS CENTRAL PATH
OF LIGHT & DIRECTS
LIGHT AWAY FROM
OBJECTIVE AT AN
• Bacteria have a refractive index slightly
different from the surrounding.
• Light passing through the bacteria enters
• Light not passing through the bacteria does
not enter objective
• So , end result
– Bright organisms against dark background
Why Phase Contrast?
• Many unstained biological specimens are
virtually transparent under bright field
• Visibility can be improved in wet mount and
cell cultures by reducing the opening size of
substage condenser and iris diaphragm Loss
of resolution. Artifacts
• Phase contrast improves the contrast in these
specimens without loss in resolution
• The phase contrast microscope uses the fact that the
light passing trough a transparent part of the specimen
travels slower and, due to this is shifted compared to
the uninfluenced light.
• This difference in phase is not visible to the human eye.
• However, the change in phase can be increased to half
a wavelength by a transparent phase-plate in the
microscope and thereby causing a difference in
• This makes the transparent object shine out in contrast
to its surroundings.
• When dealing with transparent and colorless
components in a cell, dyeing is an alternative
but at the same time stops all processes in it.
The phase contrast microscope has made it
possible to study living cells, and cell division
is an example of a process that has been
examined in detail with it.
• The phase contrast microscope was awarded
with the Nobel Prize in Physics, 1953.