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Honors Biology Microscopes. Important tool for all biologists. Honors Bio: Microscopes. Use light or electrons to magnify Enable us to see the shape and structure of very small objects Cells and cell parts Tissues Molecules (only with electron microscopes) - PowerPoint PPT Presentation
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Honors BiologyMicroscopes
Important tool for all biologists
Honors Bio: Microscopes
Use light or electrons to magnify
Enable us to see the shape and structure of very small objects
• Cells and cell parts• Tissues• Molecules (only with electron microscopes)• Small and microscopic organisms
Value of Magnification
Real size Magnified 400 XElodea canadensis
Pond weed
cytoplasm
central vacuole
Cell walls
chloroplasts
MagnificationMagnification = object size ~ image size
Euglena, a one-celled organism 1000X
chloroplasts
flagellum
food vacuolenucleus
Total magnification = ocular lens X objective lens
Resolution or Resolving Power
• “Ability to show two close points as separate” • Depends on shape and perfection of lenses• Human eye can see objects as small as 0.2 mm• A light microscope can resolve objects as small as 0.2 m
high resolution lens lower resolution lens
Resolution = sharpness, clarity of focused image
Comparing ResolutionsOptical Instrument Resolving Power RP in Angstroms
Human eye 0.2 millimeters (mm) 2,000,000 A
Light microscope 0.20 micrometers (µm) 2000 A
Scanning electron microscope (SEM) 5-10 nanometers (nm) 50-100 A
(TEM) Transmission electron microscope 0.5 nanometers (nm) 5 A
Depth of Field
• Thickness or layer in focus
• Higher magnification thinner layer
Light Microscopes Send LIGHT through a thin specimen
binocular light microscope
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an early microscope
Light Microscopes (LM)
• Light waves pass through a thin specimen• Lenses bend light to magnify image
–Simple microscope – one lens–Compound microscope – two lenses
• Magnifies image twice
Leeuwenhoek’s Microscope• Anton von Leeuwenhoek, 1600s• First powerful scope with high resolution
– Single lens– Magnify ~ 300 X
Leeuwenhoek’s microscope
LE 4-1a
Eyepiece
Ocularlens
Objective lens
Specimen
Condenserlens
Lightsource
BINOCULAR MICROSCOPE – has ocular lens for each eye
How two lenses magnifies
Leaf cross-section (LM)
Epithelial cell
Photosynthetic cells
Chloroplast (dots inside cell)
Stoma (leaf opening)
Advantages of light microscopes- Can magnify up to 2000 times- Shows shape and structure of cells and
tiny organisms- Specimens can be alive
- Specimens must be thin enough for light to pass through
- Image appears inverted and backwards- Often need stain to see image
Disadvantages
Cheek cells with stain
Common stains: methylene blue, Lugol’s iodine
“Vital stains” - stain without killing cells
Light microscope LM “dark field”
Phase-Contrast Microscope“Differential Interference Microscope”
Increases contrast between tissue densities – don’t need stain; good for living organisms
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Cheek cells without stain
Compound Microscope
cheek cells – stained
nucleus
cytoplasm
cell membrane
Phase-Contrast Microscope
cheek cells –unstained
nucleus
cytoplasm
cell membrane
Amoeba, one-celled organism preserved, stained alive, movingCompound scope Phase-Contrast scope
Cell cycle, under phase contrast
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Phase-contrast micrograph of a roundworm 630X
Has ocular lens and objective lens for
each eye Stereoscopic vision,
3-D
Image NOT inverted
Magnifies 10-50X
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Stereomicroscope“Dissecting microscope”
Advantages of stereoscopes
• Image NOT inverted or backwards• Makes manipulation easy
• Specimens can be solid, living
• Disadvantage: magnifies up to ~50 X
Stereomicroscope – whole specimens
chick embryo soil worm
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Fluorescent Microscopy• Uses lasers on thin slices; confocal scope• Fluorescent dyes show different molecules
Cancer cells tagged with 3 fluorescent dyes shows cell microtubules (blue), microfilaments (yellow), DNA (green)
Fluorescent – shows different cell parts as different colors
• Details in a single layer
Fruit fly embryo – developmental layers
Green – microtubules in cytoplasm Red -DNA
http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html
Confocal Microscopy
E. Coli bacteria Specialized Cells in the Ear
Electron Microscope
• Uses electrons instead of light
• Magnets focus the beam
• Image shows on monitor
• Magnify up to 1 million times
• Show cell details, interior
- “ultrastructure
• Invented 1930’s
• Nobel for Ruska 1986
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Electron Microscope• How does it work?
– Specimen is coated with a metal film
– Electron beam hits metal, ejects electrons from metal atoms
– These electrons make the image
Advantages of electron microscopy
• Electron are much smaller than the wavelength of light – show things that light cannot show
• Very high magnification – up to 1,000,000X • Very high resolution - up to 1 nanometer
• DISADVANTAGE – specimen must be dead, dried, coated, in vacuum chamber
Scanning Electron MicroscopeSEM
• Electron beam skims across specimen surface
• Shows tiny surface structures in great detail
• Magnifies up to 50,000 times
• DISADVANTAGE: shows surface, but not interior
Compare LM and SEM
Blood cells (LM) Blood cells (SEM)
SEM micrographs
Euglena (protist) SEM
Ant head, SEM
Scanning Electron Microscope (SEM) shows surface details
Electrons scan across surface of specimen
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SEM of DNA
35Image made with special scanning “tunneling” microscope
Transmission Electron Microscope (TEM) shows inside cells
• Electrons pass through thin specimen
• Shows great detail of internal structure
• Magnifies up to 1,000,000 times!!
Rough ER
Nucleus
Mitochondria
Transmission Electron Microscope
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Phage virus
Bacterium dividing Muscle fibers
Cilia and basal bodiesLiver cells
Chloroplast
Comparing microscopes
Euglena LMEuglena SEM
Euglena TEM
Which type of microscope produced these micrographs?
Amoeba, preserved and stained
Vacuole inside a cell
39Amoeba, alive and unstained
Which type of microscope made these micrographs?
Female and male fruit fly
Closterium -Unicellular green alga
40
Name the microscope
Leaf cross-section 400X chloroplast 5,000 X
Name the microscope
Eye of a houseflyIridescent beetle
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Which microscope?
43