Microscope Techniques Microscopes

Microscopes
Most important tool for studying microorganisms Use viable light to observe objects Magnify images approximately 1,000x
Electron microscope, introduced in 1931, can magnify images in excess of 100,000x Scanning probe microscope, introduced in 1981, can view individual atoms

Principles of Light Microscopy

Light Microscopy
Light passes through specimen, then through series of magnifying lenses Most common and easiest to use is the brightfield microscope Important factors in light microscopy include
Magnification Resolution Contrast

Principles of Light Microscopy

Magnification
Microscope has two magnifying lenses
Called compound microscope Lens includes
Ocular lens and objective lens Most bright field scopes have four magnifications of objective lenses, 4x, 10x, 40x and 100x

Lenses combine to enlarge objects

Magnification is equal to the factor of the ocular x the objective
10x X 100x = 1,000x

Principles of Light Microscopy

Resolution
Usefulness of microscope depends on its ability to clearly resolve two objects that are very close together
Resolving power is defined as the minimum distance existing between two objects where those objects still appear as separate objects Resolving power determines how much detail can be seen

Principles of Light Microscopy

Resolution
Resolution depends on the quality of lenses and wavelength of illuminating light (blue filter gives the best resolution)
How much light is released from the lens

Maximum resolving power of most brightfield microscopes is 0.2 m (1 um =1x10-6 M)

This is sufficient to see most bacterial structures (>1um) Too low to see viruses (< 0.3 um)

Principles of Light Microscopy

Examples of light microscopes that increase contrast
Phase-Contrast Microscope Interference Microscope Dark-Field Microscope Fluorescence Microscope Confocal Scanning Laser Microscope

Principles of Light Microscopy

Phase-Contrast
Uses special lenses to amplify differences between refractive indexes of cells and surrounding medium Uses set of rings and diaphragms to achieve resolution

Principles of Light Microscopy

Dark-Field Microscope
Reverse image
Specimen appears bright on a dark background
Like a photographic negative

Achieves image through a modified condenser

Principles of Light Microscopy

Fluorescence Microscope
Used to observe organisms that are naturally fluorescent or are flagged with fluorescent dye
Fluorescent molecule absorbs ultraviolet light and emits visible light Image fluoresces on dark background

Principles of Light Microscopy

Electron Microscope
Uses electromagnetic lenses, electrons and fluorescent screen to produce image Resolution increased 1,000 fold over brightfield microscope
To about 0.3 nm (1 nm = 1x10-9 M)

Magnification increased to 100,000x Two types of electron microscopes

Transmission Scanning

Principles of Light Microscopy

Transmission Electron Microscope (TEM)
Used to observe fine detail Directs beam of electrons at specimen
Electrons pass through or scatter at surface
Shows dark and light areas Darker areas more dense

Specimen preparation through

Thin sectioning Freeze fracturing or freeze etching

Principles of Light Microscopy

Scanning Electron Microscope (SEM)
Used to observe surface detail Beam of electrons scan surface of specimen
Specimen coated with metal
Usually gold

Electrons are released and reflected into viewing chamber Some atomic microscopes capable of seeing single atoms