LESSON 1c: TOOLS USED IN MOLECULAR BIOLOGY

Question 1

-Dutch microscopist
-Made significant contributions to the field of microbiology
-Invented single-lens microscopes with high magnification
-Discovered bacteria, protozoa, sperm cells, blood cells, and other microscopic organisms
-His observations helped disprove the theory of spontaneous generation
-Known as the “Father of Microbiology

Answer 1

Anton Van Leeuwenhoek (1970s)

Question 2

First used the word “cell” to describe the microscopic cavities in cork.

Answer 2

Robert Hooke

Question 3

Evolution of Microscope: Circa late 1600s

Answer 3

Leeuwenhoek Microscope

Question 4

Evolution of Microscope: circa 1865

Answer 4

British Microscope

Question 5

Evolution of Microscope: circa early 1700s

Answer 5

Hand-held Microscope

Question 6

Evolution of Microscope: Circa 1927

Answer 6

Winkel-ZEISS Dissecting Microscope

Question 7

Evolution of Microscope: Circa 2010

Answer 7

ZEISS Primo Star

Question 8

*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 8

THE LIGHT MICROSCOPE

Question 9

The light microscope enables us
to see the overall ____ and ______ of a cell

Answer 9

shape and
structure

Question 10

TYPES OF LIGHT MICROSCOPY

Answer 10

-Bright-field microscopy
-Phase -Contrast Microscopy
-Differential Interference-Contrast Microscopy (DIC)
-Video-Enhanced Differential Interference-Contrast Microscopy
-Fluorescence Microscopy
-Confocal Microscopy
-Multi-Photon Excitation Microscopy
-Scanning electron microscope (SEM)
-Transmission electron
microscope (TEM)

Question 11

*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 11

Bright-field microscopy
(simple light microscope)

Question 12

*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 12

Phase -Contrast Microscopy

Question 13

*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 13

Differential Interference-Contrast Microscopy (DIC)

Question 14

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

Answer 14

Video-Enhanced Differential Interference-Contrast Microscopy

Question 15

*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 15

Fluorescence Microscopy

Question 16

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

Answer 16

Green Fluorescent Protein (gfp)

Question 17

*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 17

Confocal Microscopy

Question 18

*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 18

Multi-Photon Excitation Microscopy

Question 19

• 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 19

Scanning electron microscope (SEM)

Question 20

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

Answer 20

Transmission Electron Microscope (TEM)

Question 21

Manipulating Cells in the Culture

Answer 21

-Isolating Cells
-Growing cells in culture dishes

Question 22

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

Answer 22

Isolating cells

Question 23

Growing cells in culture dishes :

Answer 23

• Primary Cell Culture
  -Cell line

Question 24

Prepare directly from tissues
of an organism

Answer 24

Primary cell culture

Question 25

Immortalized, can grow
indefinitely

Answer 25

Cell line

Question 26

-Organelles and macromolecules can be
separated by ultracentrifugation
-There are 2 types of sedimentations
– Velocity sedimentation
– Equilibrium sedimentation

Answer 26

Fractionation of Cells

Question 27

Organelles and macromolecules can be
separated by _____________

Answer 27

ultracentrifugation

Question 28

2 types of sedimentations

Answer 28

– Velocity sedimentation
– Equilibrium sedimentation

Question 29

Used to detect specific DNA sequences in a sample.

Answer 29

Southern Blotting Technique

Question 30

Process of Southern Blotting Technique

Answer 30

-DNA is extracted from the sample and cut into smaller fragments using restriction enzymes.
-The fragments are separated by size using gel electrophoresis.
-The DNA is transferred from the gel to a membrane (usually nitrocellulose or nylon).
-A labeled probe, complementary to the target -DNA sequence, is added to the membrane and hybridizes to the target sequence.
-The membrane is washed to remove unbound probe.
-The labeled probe is detected, revealing the presence and location of the target DNA sequence.

Question 31

Determination of size and subunit of a protein by __________________________________

Answer 31

SDS polyacrylamide-Gel Electrophoresis