Components of a compound microscope:
Ocular lens - magnifies the image.
Objective lens - forms the primary image.
Condenser lens, and light source
Magnification = mag. of ocular lens x mag. of objective lens.
Brightfield (unstained specimen):
Creating contrast with the light microscope
Passes light directly through specimen; unless cell is naturally pigmented or artificially stained, the image has little contrast.
Brightfield (stained specimen):
Creating contrast with the light microscope
Staining with various dyes enhances the contrast, but most staining procedures requires cells to be fixed (preserved).
one method: to label specific structures.
Phase constant:
Creating contrast with the light microscope
Enhances contrast of unstained cell by amplifying variations in refractive index within specimen; especially useful for examining living, unpigmented cells.
second method: uses prisms to bend the waves of light.
Refractive index:
The measure of how much the speed of light is reduced.
Differential interference contrast:
Creating contrast with the light microscope
Also uses optical modifications to exaggerate differences in refractive index.
Fluorescent molecules:
Are able to absorb ultraviolet light and emit visible light.
Fluorescence microscopy:
- Able to visualize the location of molecules (ex. Tagging proteins with a fluorescing molecule (GFP), Fluorescent dyes, Fluorescently tagged antibodies).
- Can see more than one molecule/structure at a time.
Confocal microscopy:
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Super resolution microscopy:
- A form of light microscopy
- A technique that allows the capture of images with a higher resolution than the diffraction limit
- Resolution of ~ 250nm
Light microscopy:
Use light to "illuminate" the sample.
� Can magnify structures up to 1,000X
� Can resolve structures as small as 0.2 �m (like eukaryotic organelles).
Transmission Electron Microscopy (TEM):
- Use electrons to "illuminate" the sample
- Can magnify objects up to 100,000X
- Can resolve structures smaller than 1 nm (i.e. molecules)
- 1-200X increase in resolution over light microscope
- Allows viewing of the detailed structure of organelles.
Electron tomography:
Technique for obtaining detailed 3D structures of sub-cellular macro-molecular objects, Rearrangements induced
16 hours after HCV infection
Scanning Electron Microscopy (SEM):
- Generates images with 3D quality
- Resolution 3-20 nm
- Reveals surface architecture
Study of cells in cell culture:
Cells can be removed from animals, treated with trypsin, and grown on glass coverslips or on plastic culture dishes for long periods of time.
First human cells to be grown in culture:
HeLa, 1951.
- Henrietta Lacks
- Cervical cancer cell line.
Primary cell culture
Cells that have been removed from an animal tissue and grown in culture.
Cell line
Cells that can be grown in cell culture and passaged multiple times - they have typically been modified to express genes expressed by cancer cells.
Passaging
Removal of cells growing in a dish filled with tissue culture media to another dish.
Tissue culture media
An aqueous solution of salts, growth factors and nutrients that will support cell growth.
What enzymes are typically used to create a single cell suspension?
Enzymes that digest proteins involved in cell-cell contacts and cell-extracellular matrix contacts
(i.e. trypsin)
Passaging cells in cell culture:
- If there's a confluent monolayer of cells, to keep cells alive, must passage.
- Trypsin is used to detach cells.
- Replate of fraction of cells.
Cells can be cultured as genetically _____________ populations or genetically _____________ populations:
Diverse or Identical
Clonal cell line:
All of the cells in the population are derived from a single parent cell.
Cell transfection
A method for modifying gene expression within cells by introduction of nucleic acids that encode a specific protein (plasmid).
Basic elements of a plasmid:
An inducible promoter, a gene that encodes a specific protein, a gene that confers antibiotic resistance for selection, and an origin of replication used to produce many copies of the plasmid.
Two common methods for cell transfection:
Lipid delivery agent and electroporation
Lipid delivery method:
Membrane enclosed vesicles containing substance X. Induced membrane fusion between vesicles and plasma membrane of target cell releases substance into the plasma.
Electroporation:
Cell is placed in substance X between two electrodes and subjected to a very short electrical shock. Transient pores made in the cell membrane allow substance to enter before the cell reseals.
The "substance" is typically..
A nucleic acid that has been created through molecular
biology techniques to express a "gene of interest.
Green fluorescent protein (GFP):
A protein isolated from the bioluminescent jellyfish Aequorea victoria that can be transfected into other cell types to label cells and proteins in live cells.
Using molecular biology GFP can be..
Added to other proteins to make a fusion protein.
Western blot
A biochemical method where proteins are extracted from a cell population and separated by size and the amount of protein in a cell population can be quantified.
Immunocytochemistry
A method where the location of a protein can be identified in single cells grown in cell culture.
Fluorescent immunocytochemistry labels..
Specific molecules such as microtubules and DNA.
Immunohistochemistry
A method where the location of a protein can be identified in cells within a thin section of tissue.
Antibodies (Immunoglobulin):
- Used by the immune system to identify foreign objects (antigens).
- Recognize sequence and shape of antigen (primary-quaternary structure of proteins).
- Important tool in cell biology to recognize proteins in cells.
Western Blot method:
- Also known as a "protein immunoblot".
- Proteins are transferred from an SDS-PAGE gel to a piece of paper (nitrocellulose).
- The nitrocellulose is incubated with a protein that will block non-specific binding of the antibody (typically powdered milk is
Detergents used in membrane biochemistry:
Triton X-100 = nonionic, uncharged detergent.
Nonionic detergents:
Can solubilize membrane components and remove integral
membrane proteins. This treatment leaves "holes" in the cell membrane.
Method for Immunocytochemistry:
- Cells are fixed with a chemical that crosslinks
the proteins together and kills the cell
- Triton X-100 is used to "poke holes" in the cell membranes.
- An antibody is incubated with the cells.
- A fluorescent tag on the antibody or other method is used
Method for Immunohistochemistry:
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