• Anton van Leeuwenhoek (compound microscope, bacteria)
• Galileo Galilei (compound microscope)
• Joseph Jackson Lister (achromatic lens)
• 1940 – Electron Microscope
What makes good microscope?
-adequate magnifying power (enlarge specimen)
-high resolving power (distinguish 2 points or objects from each other)
-provide good contrast (morphology, structure, between specimen and background)
-serve your purpose
LIGHT MICROSCOPES
• use light waves and mirrors
Simple
short focal length
only 1 lens
magnification: 300x
Compound or Complex
-2 sets of lenses
-magnification: 1000x
parts of light microscope
A) revolving nosepiece
B) eyepiece
C) objective lens
D) mechanical stage
E) coarse focus knob
F) stage
G) fine focus knob
H) diaphragm
I) condenser
J) illuminator
K) rheostat
L) x-y mechanical stage knobs
ELECTRON MICROSCOPES
• use electron beams as energy source
• has higher magnification and resolving power
• for objects smaller than 0.2 mm in diameter
• in vacuum
parts of electron microscope
A) electron source
B) sample port
C) viewing screen
D) evacuated chamber
Types of Light Microscopes
bright field
dark field
phase contrast
differential interference contrast
fluorescence
confocal
two-photon
BRIGHT FIELD
• objects under study are darker
• microscopic field is brightly lit
• gross morphology
Bacillus anthracis viewed in bright field
Treponema pallidum viewed in dark field
DARK FIELD
• microscopic field is dark
• objects under study are luminous
• for specimens that are:
• invisible in the ordinary light microscope
• cannot be stained by standard methods
• distorted by staining
capsule
PHASE CONTRAST
• principle is based on variations in the refractive indices (measure of bending or refracting of a beam of light on entering a denser medium)
• not necessary to fix or stain cells
• detailed examination of internal structure
Unstained Epithelial Cells in phasecontrast
DIFFERENTIAL INTERFERENCE CONTRAST
-principle is based on variations in the refractive indices
• advantage: no diffraction halo associated with phase contrast
• disadvantage: the three-dimensional appearance may not represent reality
Fonsecaea pedrosoi viewed in differential interference contrast
FLUORESCENCE
• makes use of fluorochromes
• visualizes specimens that fluoresce
• detection of immunological reactions
Neisseria gonorrhoeae viewed in fluorescence
Schistosoma mansoni viewed in fluorescence
CONFOCAL
-useful for thick specimens like biofilms
-used to visualize structures
Roof-dwelling Cyanobacterium Biofilm viewed in confocal
TWO-PHOTON
• useful for examining living cells within intact tissues
• currently limited to advanced clinical and research laboratories
Mouse Intestine Cells Stained With Fluorescent Dyes viewed in two-photon
Types of Electron Microscopes
TRANSMISSION ELECTRON MICROSCOPE (TEM)
SCANNING ELECTRON MICROSCOPE (SEM)
TRANSMISSION ELECTRON MICROSCOPE (TEM)
• ultrastructure in thin section of the cells
• can project images in a much higher resolution—up to the atomic level of thinner objects
• examine viruses
Cells in Biofilm viewed in Transmission Electron microscope
SCANNING ELECTRON MICROSCOPE (SEM)
• surface features of viruses and cells
• reveals a 3-D image
Staphylococcus aureus viewed in Scanning Electron Miscroscope (SEM)
Scanning Probe Microscope
-does not use light or electrons, but rather very sharp probes that are passed over the surface of the specimen and interact with it directly
Types of Scanning Probe Miscroscope:
scanning tunneling microscope (STM)
atomic force microscope (AFM)
scanning tunneling miscroscope
-uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen
atomic force microscope
-establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen.
Pure Gold Surface viewed under scanning tunneling microscope
Nanocellulose viewed under atomic force microscope
Living or Natural State
Advantages:
• observation of unaltered/undistorted characteristics
• cellular processes can be studied
• motility can be observed
• simple to prepare
Disadvantage:
• refractive index of cells almost similar to that of water