MICROSCOPE
Antioni van Leeuwenhoek Delft, Holland, worked as a draper (a fabric
merchant); he is also known to have worked as a surveyor, a wine
assayer, and as a minor city official. Created a “simple” microscope
that could magnify to about 275x, and published drawings of
microorganisms in 1683. Discovered bacteria, free-living and parasitic
microscopic protists, sperm cells, blood cells, microscopic nematodes.
In 1673, Leeuwenhoek began writing letters to the Royal Society of
London - published in Philosophical Transactions of the Royal Society.
In 1680 he was elected a full member of the Royal Society.
Ernst Abbe together with Carl Zeiss published a paper in 1877 defining
the physical laws that determined resolving distance of an objective.
Known as Abbe’s Law“minimum resolving distance (d) is related to the
wavelength of light (lambda) divided by the Numeric Aperture, which is
proportional to the angle of the light cone (theta) formed by a point on
the object, to the objective”.
Abbe and Zeiss developed oil immersion systems by making oils that
matched the refractive index of glass. Thus they were able to make
the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light
microscopes to resolve two points distanced only 0.2 microns apart
(the theoretical maximum resolution of visible light microscopes).
Leitz was also making microscope at this time.
Microscopes
• Microscopes and microbiology linking advance
•The microscope is the microbiologist’s most basic tool .
• Microscopes use lenses to magnify object’s images.
• There are two types of microscopes:
◦ Light microscopes (4 types)
◦ Electron microscope (2 types)
Light microscope used to examine cells at relatively low magnifications and electron microscope
used to lock at cells and cell structure at very high magnification.
Some Principles of Light
Microscopy
oCompound light microscope uses visible light to illuminate cells
oMany different types of light microscopy:
oBright-field
oPhase-contrast
oDark-field
oFluorescence
Some Principles of Light Microscopy
Bright-field microscope
o Specimens are visualized because of differences in contrast (density) between specimen and
surroundings.
oContrast differences arise because cells absorb or scatter light to varying degrees.
oTwo sets of lenses form the image
o Objective lens and ocular lens (compound )
o Total magnification = objective magnification ocular magnification
o Maximum magnification is ~2,000
Some Principles of Light Microscopy
Magnification is not the limiting factor in the ability of seeing small things but also we need a
good resolution which is the ability to distinguish two adjacent objects .
Magnification can be increased with out limit but resolution cannot because it is the function
of physical properties of light.
Light microscopy limits resolution is about 0.2µm , electron microscope resolution is greater
than of light microscope .
Some Principles of Light
Microscopy
o Resolution: the ability to distinguish two adjacent objects as separate and distinct
◦ Resolution is determined by the wavelength of light used and numerical aperture of lens
Methods to Improve contrast to generate a better final image?
Staining
o Dyes are organic compounds that bind to specific cellular materials
o Examples of common stains are methylene blue, safranin, and crystal violet
Differential stains: the Gram stain
oDifferential stains separate bacteria into groups
Bacteria can be divided into two major groups: gram-positive and gram-negative
oGram-positive bacteria appear purple and gram-negative bacteria appear red after staining
Microscopic observation of gram-positive
(purple) and gram- negative (red) bacteria .
Phase-contrast and Dark-felid
Microscopy
Two form of light microscopy improve image with out using staining.
Phase –contrast microscopy: is based on the principle that cells differ in refractive index
from their surroundings .
Phase –contrast microscopy resulting a dark image on light background
The dark-fielded microscopy is a light microscope in which the light reaches the specimen
from the slides only . Thus the specimens appears light on a dark background.
The dark-fielded microscopy used in observed microbial motility .
Fluorescence Microscopy
Used to visualize specimens that fluoresce – emit light of one color following
absorption of light of another color .
Cells fluoresce either :
oContain naturally fluorescent substances such as chlorophyll
oBecause cells have satin with fluorescent dye .
DAPI (4`,6-diamidino-2-phenylindole) is widely used fluorescent dye staining cell`s
DNA .
Electron Microscopy
Electron microscopes use electrons instead of photons(visible light) to image cells and structures.
Electromagnets function as lenses in EM , whole system operates in a vacuum.
EM are fitted with cameras to allow a photograph to be taken
Two types of electron microscopes:
◦ Transmission electron microscopes (TEM)
◦ (need thin section),negative stain
◦ Scanning electron microscopes (SEM)
◦ Coat with heavy metal
Electron Microscopy
Transmission electron microscopy is used to examine cells and cell structure at very high
magnification and resolution , even enabling one to view structures at the molecular level .
This is because the wavelength of electrons is much shorter than the wavelength of visible light and
wavelength affects resolution .
Unlike visible light , electron beams can not penetrate very well. So, special techniques of thin
sectioning are needed to prepare specimens before observing them .
To obtain sufficient contrast, the preparation are treated with stains such as osmic acid ,
permanganate , uranium , lanthanum ; because these substances are composed of atoms of high
atomic weight , they scatter electrons well and thus improve contrast.
Figure 2.9
Electron
source
Evacuated
chamber
Sample
port
Viewing
screen
© 2012 Pearson Education, Inc.
Electron Microscopy
Scanning electron microscopy used to observed external features of an organisms or cell .
No need for thin sections
The specimen is coated with a thin film of a heavy metal such as gold .
An electron beam then scans back and forth across the specimens. Electrons scattered from
the metal coating are collected and activate a viewing screen to produce an image.
Cytoplasmic DNA
Septum Cell wall (nucleoid)
membrane
© 2012 Pearson Education, Inc.