3 Flashcards
(1670s)
Anton Van Leeuwenhoek
The ________ enables us
to see the overall shape and
structure of a cell
light microscope
*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
TYPES OF LIGHT MICROSCOPY
- Bright-field microscopy
(simple light microscope) - 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
Fractionation of Cells
There are 2 types of sedimentations
- Velocity sedimentation
- Equilibrium sedimentation
Fractionation of Cells
- Restriction Enzyme Digestion and Electrophoresis
- SOUTHERN BLOTTING TECHNIQUE
- Determination of size and subunit of a protein by
SDS polyacrylamide-Gel Electrophoresis
