LESSON 1c: TOOLS USED IN MOLECULAR BIOLOGY Flashcards
-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
Anton Van Leeuwenhoek (1970s)
First used the word “cell” to describe the microscopic cavities in cork.
Robert Hooke
Evolution of Microscope: Circa late 1600s
Leeuwenhoek Microscope
Evolution of Microscope: circa 1865
British Microscope
Evolution of Microscope: circa early 1700s
Hand-held Microscope
Evolution of Microscope: Circa 1927
Winkel-ZEISS Dissecting Microscope
Evolution of Microscope: Circa 2010
ZEISS Primo Star
*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
THE LIGHT MICROSCOPE
The light microscope enables us
to see the overall ____ and ______ of a cell
shape and
structure
TYPES OF LIGHT MICROSCOPY
-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)
*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
Bright-field microscopy
(simple light microscope)
*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
Phase -Contrast Microscopy
*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
Differential Interference-Contrast Microscopy (DIC)
*Uses image-processing systems ( video
cameras and computers
*Allows visualization of small objects
through their movement
*Ex. Movement of organelles along
microtubules
Video-Enhanced Differential Interference-Contrast Microscopy
*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
Fluorescence Microscopy
*From jellyfish
*Fused to protein of interest through recombinant DNA technology
*allows for detection of movement and localization of proteins within living cells
Green Fluorescent Protein (gfp)
*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
Confocal Microscopy
*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)
Multi-Photon Excitation Microscopy
- 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
Scanning electron microscope (SEM)
-EM tomography The structure
of 70Sribosome 3D structure
The resolution is 11.2
Angstrom
Transmission Electron Microscope (TEM)
Manipulating Cells in the Culture
-Isolating Cells
-Growing cells in culture dishes
– Disrupt extracellular matrix with
proteolytic enzymes or EDTA
– Fluorescence-activated cell sorter
Isolating cells
Growing cells in culture dishes :
- Primary Cell Culture
-Cell line
Prepare directly from tissues
of an organism
Primary cell culture
Immortalized, can grow
indefinitely
Cell line
-Organelles and macromolecules can be
separated by ultracentrifugation
-There are 2 types of sedimentations
– Velocity sedimentation
– Equilibrium sedimentation
Fractionation of Cells
Organelles and macromolecules can be
separated by _____________
ultracentrifugation
2 types of sedimentations
– Velocity sedimentation
– Equilibrium sedimentation
Used to detect specific DNA sequences in a sample.
Southern Blotting Technique
Process of Southern Blotting Technique
-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.
Determination of size and subunit of a protein by __________________________________
SDS polyacrylamide-Gel Electrophoresis
