Chapter 4 Visualizing & Culturing Cells Flashcards
Which has better resolution: electron or light microscopy?
Electron microscopy - but many light microscopy
techniques allow observation and manipulation of living cells.
What is a cell strain?
A lineage of cells originating from a primary culture taken from an organism. Cells are not transformed –> limited lifespan in culture.
What is a cell line?
Made of transformed cells and therefore these cells can divide indefinitely in culture –> immortal
How do you get a clone?
A single cell is cultured and gives rise to genetically identical progeny cells
When/why are chemical stains used?
- Required for visualizing cells and tissues with the basic light microscope because most cellular material does not absorb visible light and therefore cells are essentially invisible in a light microscope.
- May be used to absorb light and thereby generate a visible image usually bind to a certain class of molecules rather than a specific molecule within that class
- Certain stains may reveal where proteins are in a cell but not where a specific protein is located.
What advantage do fluorescent dyes and
fluorescence microscopy provide in comparison to the chemical dyes used to stain specimens for light microscopy?
- Overcomes limitation of chemical stain
- Fluorescent molecule may be either directly or indirectly attached to a molecule of interest which is then viewed by FL microscope
- Only light emitted by the sample will form an image, so the location of the fluorescence indicates the location of the molecule of interest
What advantages do confocal and deconvolution
microscopy provide in comparison to conventional
fluorescence microscopy?
- build on the ability of fluorescence microscopy
- using either optical (confocal scanning) or computational (deconvolution) techniques to remove out-of-focus fluorescence to produce much sharper images
- facilitate optical sectioning of thick specimens as opposed to physical sectioning and associated techniques that may alter the specimen
In certain electron microscopy methods, the specimen is not directly imaged. How do these methods provide information
about cellular structure, and what types of structures
do they visualize? What limitation applies to most forms of electron microscopy?
The metal coating acts as a replica of the specimen, and the replica rather than the specimen itself is viewed in the electron microscope. Methods that use this approach include metal shadowing, freeze fracturing, and freeze etching. Metal shadowing allows visualization of viruses, cytoskeletal fibers, and even individual proteins, while freeze fracturing and freeze etching allow visualization of membrane leaflets and internal cellular structures.
Explain why the process of cell fusion is necessary to produce monoclonal antibodies used for research.
Normal B lymphocyte cells can produce a single type of antibody molecule. However, such cells have a finite lifespan in culture. Researchers use cell fusion of B lymphocytes and immortalized myeloma cells to create immortalized, antibody-secreting cells. Such cells, called hybridoma cells, retain characteristics of both parent cells, allowing for production of a single-type, or monoclonal,
antibody.
What techniques do scientists commonly
use to isolate cells and organelles from complex mixtures,
and how do these techniques work?
Specific types of cells in suspension may be isolated by a fluorescence-activated cell sorter (FACS) machine in which cells previously “tagged” with a fluorescent-labeled antibody are separated from cells not recognized by the antibody. The scientist selects an antibody specifi c for the cell type desired. Specific organelles are generally separated by centrifugation of
lysed cells. A series of centrifugations of successive supernatant fractions at increasingly higher speeds and corresponding higher forces serves to separate cellular organelles from one another on the basis of size and mass (larger, heavier cell components pellet at lower speeds). This is often combined with density-gradient separations to purify specific organelles on the basis of their buoyant density.
Hoechst 33258 is a chemical dye that binds specifically to
DNA in live cells, and when excited by UV light, it fluoresces in the visible spectrum. Name and describe one specific
method, employing Hoechst 33258, an investigator would
use to isolate fibroblasts in the G2 phase of the cell cycle
from those fibroblasts in interphase.
FACS (Figure 9-2), whereby labeled cells pass through a laser light beam and the fluorescent intensity of light emitted is measured, allowing the computer to
assign each cell with an electric charge proportional to the fluorescence. Fibroblasts having twice the amount of DNA (G2 phase) compared to the normal diploid cells will emit more fluorescence and therefore have a different electric charge, which allows them to be separated and collected.
Proteomic analysis
can identify all the protein components in a preparation of a purified organelle
What technique is particularly useful in purifying organelles and vesicles of similar sizes and
densities?
Immunological techniques - which use antibodies against
organelle-specific membrane proteins
bright-field light microscopy
the simplest microscopes view cells under bright-field optics (Figure 4-9b), and little detail can be seen (Figure 4-10).
clone
(1) A population of genetically identical cells, viruses, or
organisms descended from a common ancestor.
(2) Multiple identical copies of a gene or DNA fragment generated and maintained via DNA cloning.
confocal microscropy
- uses optical methods to obtain images from a
specific focal plane and exclude light from other planes. - collect emitted fluorescent light from just one small area of a focal plane at a time in such a way that out-of-focus light is excluded.
deconvolution microscopy
uses computational methods to remove fluorescence contributed by out-of-focus parts of the sample
cryoelectron microscopy
an aqueous suspension of a sample is applied to a grid in an extremely thin film, frozen in liquid nitrogen, and maintained in this state by means of a special mount. The frozen sample is then placed in the electron microscope.
DIC microscopy
DIC = differential interference contrast (aka Nomarski interference microscopy)
- based on splitting the light into two perpendicular components before passing them through the specimen and then recombining them to observe their interference pattern, is the method of choice for visualizing extremely small details and thick objects
- Contrast is generated by differences in the refractive index of the object and of its surrounding medium
FACS
(fluorescence-activated cell sorter)
- based on flow cytometry
- can both analyze the cells and select the few fluorescent cells from thousands of others and sort them into a separate culture dish
FRAP
FRAP = fluorescence recovery after photobleaching
- allows the dynamics of a population of molecules to be analyzed.
1) use a high-intensity light to permanently bleach the fluorochrome (e.g., GFP) in the patch, there will initially
be no fluorescence coming from it, and it will look dark in the fluorescence microscope
2) if the components in the patch are in dynamic equilibrium with unbleached molecules elsewhere in the cell, the bleached molecules will be replaced by unbleached ones, and the fluorescence will begin to come back
3) The rate of fluorescence recovery is a measure of the dynamics of the molecules.
FRET
FRET = Förster resonance energy transfer
- used to determine if two proteins interact in vivo
- uses a pair of fluorescent proteins in which the emission wavelength of the first is close to the excitation wavelength of the second
hybridoma
A clone of hybrid cells that are immortal and produce
monoclonal antibody; formed by fusion of a normal antibody- producing B cell with a myeloma cell. (Figure 4-6)
immunofluorescence
microscopy
- the most widely used method of detecting specific proteins
- uses an antibody to which a fluorescent dye has been covalently attached
indirect immunofluorescence
microscopy
- most commonly used variation of this technique
- antibody specific to the protein of interest is detected indirectly (with secondary antibody)
monoclonal antibody
Antibody produced by the progeny of a single B cell and thus a homogeneous protein that recognizes a single antigen (epitope). It can be produced experimentally by use of a hybridoma. (Figure 4-6)
phase-contrast microscopy
- generates an image in which the degree of darkness or brightness of a region of a specimen depends on the refractive index of that region.
- suitable for observing single cells or thin cell layers, but not thick tissues
- particularly useful for examining the location
and movement of larger organelles in live cells.
PALM
PALM = photo-activated localization microscopy
- relies on the ability of a variant of GFP to be photoactivated; that is, it can become fluorescent only after being activated by a specific wavelength of light, different from its excitation wavelength
polyclonal antibody
many different cells in the animal have contributed the antibodies, and the antibodies are likely to bind to many different epitopes on protein X
TIRF microscopy
TIRF = total internal reflection fluorescence
- allows fluorescent samples adjacent to a coverslip to be seen
with great clarity.
TEM
TEM = transmission electron microscopy
- cannot be used to study live cells, and the absence of water in samples causes macromolecules to become denatured and nonfunctional
- electrons are emitted from a filament and accelerated in an electric field
chimeric proteins
By using molecular genetic techniques to fuse a protein
of interest with a naturally fluorescent protein
Two common methods for imaging live cells and unstained tissues to generate contrast take advantage of differences in the refractive index and
thickness of cellular material
1) phase contrast microscopy
2) differential-interference-contrast (DIC) microscopy
