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.
