CP BIO: Microscopes

Use light or electrons to magnify Enable us to see the shape and structure of very small objects

Magnification

Total magnification = ocular lens X objective lens

Real size Magnified 400 X

Elodea canadensis Cell walls cytoplasm

Pond weed

chloroplasts central vacuole

How two lenses magnifies

Resolution or Resolving Power

Resolution

= sharpness, clarity of focused image • “Ability to show two close points as separate” • Depends on shape and perfection of lenses • A light microscope can show objects as small as 1  m high resolution lens lower resolution lens

Depth of Field • Thickness of layer in focus • Higher magnification 

thinner

layer

Light Microscopes (LM) • Light passes through a thin specimen • Use lenses to focus light – 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

Advantages of light microscopes Can magnify up to 2000 times Shows shape and structure of cells and tiny organisms Specimens can be alive Disadvantages Specimens must be thin enough for light to pass through Image appears inverted and backwards Often need stain to see better (stain kills cells)

Cheek cells with stain Common stains: methylene blue, Lugol’s iodine “Vital stains” - stain without killing cells

Phase-Contrast Microscope Increases contrast without staining good for living organisms Cheek cells without stain 11

Compound Microscope

cheek cells – stained

Phase-Contrast

cheek cells –unstained

nucleus cytoplasm cell membrane nucleus cytoplasm cell membrane

Amoeba, one-celled organism preserved, stained alive, moving

Compound scope Phase-Contrast scope

Cell cycle, under phase contrast 14

Stereomicroscopy “Dissecting microscope” Has ocular lens and objective lens for each eye  stereoscopic vision, 3-D Image NOT inverted Magnifies 10-50X 15

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 17

Electron Microscope Uses electrons instead of light Magnets focus the electron beam Image on monitor 18

Electron Microscope • Invented 1930s • Very high magnification and resolution • Show cell details – internal anatomy, “ultrastructure”

Advantages of electron microscopy • Electron are much smaller than the wavelength of light – show things that light cannot show • Very high magnification – up to 500,000X • Very high resolution - up to 1 nanometer • DISADVANTAGE – specimen must be dead, dried, coated, in vacuum chamber

Scanning Electron Microscope SEM • Electron beam skims across specimen surface • Shows tiny surface details • 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 24

SEM of DNA Image made with special scanning “tunneling” microscope 25

Transmission Electron Microscope (TEM) shows

inside

cells • Electrons pass

through

thin specimen • Shows great detail of internal structure • Magnifies up to 500,000 times!!

Rough ER Mitochondria Nucleus

Comparing microscopes

Euglena

LM

Euglena

SEM

Euglena

TEM

Transmission Electron Microscope

Bacterium dividing Muscle fibers Phage virus Cilia and basal bodies Liver cells Chloroplast

28

Comparing microscopes

Euglena, LM Euglena, SEM Euglena, TEM

29

Which type of microscope produced these micrographs?

Amoeba

, preserved and stained

Amoeba

, alive and unstained Vacuole inside a cell 30

Which type of microscope made these micrographs?

Female and male fruit fly

Closterium -

Unicellular green alga 31

Name the microscope Leaf cross-section 400X chloroplast 5,000 X

Name the microscope Eye of a housefly Iridescent beetle 33

Which microscope?

34

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)

Confocal Microscopy

Specialized Cells in the Ear

E. Coli

bacteria