3

Question 1

(1670s)

Answer 1

Anton Van Leeuwenhoek

Question 2

The ________ enables us
to see the overall shape and
structure of a cell

Answer 2

light microscope

Question 3

*Basic tool of cell biologists
*Can magnify objects up to 1000x
*Most cells ( 1-100 µm in diameter) can be seen using light microscopy
*Also larger subcellular organelles like nuclei, chloroplasts, mitochondria
*Can not reveal details of cellular structure

Answer 3

THE LIGHT MICROSCOPE

Question 4

TYPES OF LIGHT MICROSCOPY

Answer 4

1. Bright-field microscopy
   (simple light microscope)
2. Phase -Contrast Microscopy
3. Differential Interference-Contrast Microscopy (DIC)
4. Video-Enhanced Differential Interference-Contrast Microscopy
5. Fluorescence Microscopy
6. Confocal Microscopy
7. Multi-Photon Excitation Microscopy
8. Scanning electron microscope (SEM)
9. Transmission electron
   microscope (TEM)

Question 5

*The most elementary form of
microscope illumination
*Used when there is enough contrast
in the specimen or when artificial
staining techniques are employed
* However, when an object of low
contrast to the background is being
viewed, such as protozoa, very little of
the specimen can be made out

Answer 5

Bright-field microscopy
(simple light microscope)

Question 6

*For live unstained cells
*Convert variations in density/thickness
between different parts of the cell to diff in
contrast that is seen in the final image
*Produces improved images of specimen

Answer 6

Phase -Contrast Microscopy

Question 7

*Same principle as phase contrast microscopy
*Converts phase differences to diff in contrast
*Differ from phase contrast in terms of the
optical basis upon which images are formed

Answer 7

Differential Interference-Contrast Microscopy (DIC)

Question 8

*Uses image-processing systems ( video
cameras and computers
*Allows visualization of small objects
through their movement
*Ex. Movement of organelles along
microtubules

Answer 8

Video-Enhanced Differential Interference-Contrast Microscopy

Question 9

*Studies intracellular distribution of
molecules
*Uses fluorescent dye to label
molecule of interest
*Ex. Labelling antibodies against a
specific antigen to determine its
distribution in the cell

Answer 9

Fluorescence Microscopy

Question 10

*From jellyfish
*Fused to protein of interest through recombinant DNA technology
*allows for detection of movement and localization of proteins within living cells

Answer 10

Green Fluorescent Protein (gfp)

Question 11

*Combines fluorescence microscopy with electronic
image analysis
*Fluorescent light emitted by specimen must pass
through a confocal aperture
*Series of images obtained at diff depths allows for
reconstruction of 3d image

Answer 11

Confocal Microscopy

Question 12

*Alternative to confocal microscopy that
can be applied to living cells
*2 or more Photons of light causes
excitation of fluorescent dye
*Highly localized excitation creates a 3d
image ( even w/o confocal aperture)
*Localization of excitation minimizes cell
damage ( living cells can be used)

Answer 12

Multi-Photon Excitation Microscopy

Question 13

• Electron microscopes
  were invented in the
  1950s
• The greater resolving
  power of electron
  microscopes
  – allows greater
  magnification
  – reveals cellular details
  -produces an image of
  the 3D structure of the
  surface of a specimen

Answer 13

Scanning electron microscope (SEM)

Question 14

EM tomography The structure
of 70Sribosome 3D structure
The resolution is 11.2
Angstrom.

Answer 14

Transmission electron
microscope (TEM)

Question 15

Manipulating cells in the culture

Answer 15

• Isolating cells
• Growing cells in culture dishes

Question 16

– Disrupt extracellular matrix with
proteolytic enzymes or EDTA
– Fluorescence-activated cell sorter

Answer 16

Isolating cells

Question 17

Growing cells in culture dishes

Answer 17

1. Primary cell culture
2. Cell line

Question 18

Prepare directly from tissues
of an organism

Answer 18

Primary cell culture

Question 19

Immortalized, can grow
indefinitely

Answer 19

Cell line

Question 20

Organelles and macromolecules can be separated by ultracentrifugation

Answer 20

Fractionation of Cells

Question 21

There are 2 types of sedimentations

Answer 21

1. Velocity sedimentation
2. Equilibrium sedimentation

Question 22

Fractionation of Cells

Answer 22

1. Restriction Enzyme Digestion and Electrophoresis
2. SOUTHERN BLOTTING TECHNIQUE
3. Determination of size and subunit of a protein by
   SDS polyacrylamide-Gel Electrophoresis