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

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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?

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