Lecture 1 Flashcards
What does the cell theory state?
All organisms consist of one or more cells, the cell is the basic unit of structure, and all cells come from preexisting cells.
What are the two groups of cells?
Prokaryotic and Eukaryotic.
What is the difference between prokaryotic and eukaryotic cells?
Prokaryotic cells have no nucleus or membrane organelles, but eukaryotic cells do.
What kind of microscopy is required to view plant cells, animal cells, and bacteria?
Light microscopy
What kind of microscopy is required to view ribosomes and microfilaments?
Electron microscopy
What is the mathematical formula for resolution?
R = 0.61(wavelength)/(numerical aperture)
Why is the limit of resolution for light microscopes 200nm?
Because the visible spectrum of light ends at 200nm and cannot be used to prove structural details smaller than its own wavelength
Describe phase contrast microscopy
The refractive index of the specimen determines how bright it appears
What is phase contrast microscopy good for?
It is good for viewing both intra and extracellular edge details.
Describe differential contrast microscopy
Light is split into closely spaced beams which generate contrast between the object and its surrounding medium due to differences in the index of refraction.
What is differential interference contrast microscopy used for?
To view surfaces of objects
What is fluorescence microscopy?
It can be used to visualize subcellular structures and individual proteins by a flour absorbing a specific wavelength of light and then emitting a wavelength in the visible spectrum.
What are the two major kinds of fluorescence microscopy?
Immunofluorescence and Green Fluorescent Protein (GFP)
Describe immunofluorescence
Antibodies (primary) detect and attach to a specific antigen, then a second antibody bound with a fluor (secondary) attaches to the primary antibody
What is immunofluorescence used for?
To detect and localize proteins in a cell.
What is an advantage and disadvantage of immunofluorescence?
Advantage: It is very specific and provides good resolution
Disadvantage: it requires fixed specimens
Describe GFP
A jellyfish gene was cloned and can be added to normal proteins to make them glow.
Name advantages and disadvantages of GFP
Advantage: allows for real-time localization and time-lapses, can use different colours, and can localize known proteins
Disadvantages: requires complicated genetic engineering
What is confocal microscopy?
It uses a pinhole laser beam to excite only a small plane of specimen with fluorescent light and gives very crisp images of this section
Name the advantages and disadvantages of confocal microscopy
Advantage: produces high-resolution images with distinct focal planes
Disadvantage: Takes a long time to scan an entire specimen plane by plane
Name the two kinds of electron microscopy
Scanning and Transmission electron microscopy
What is the difference between light and electron microscopy?
Electron microscopy uses a beam of electrons, not light, so it is more focused and gives a higher resolution but requires fixed cells.
How does SEM work?
It measures the amount of electron scatter of the surface of a heavy-metal coated object. Good for viewing surfaces.
How does TEM work?
It measures how the electrons scatter as they pass through the specimen. Can use antibody staining. Shows internal structures
What is centrifugation?
The separation of components in a solution by spinning them. Lower density moves up, higher density moves down.
What is electrophosphoresis?
The separation of molecules based on size (and sometimes charge).
How does SDS-PAGE gel separate proteins?
Only by size.
How does isoelectric point focusing gel separate proteins?
Based on size AND native charge.
In gel electrophosphoresis, where do larger proteins get stopped?
Closer to the top as it is difficult for them to navigate through the matrix.
What are the steps of a western analysis?
Blot the gel, expose the filter paper to primary antibody, incubate with secondary antibody, and expose to x-ray film to detect the secondary.
What is an example of a western blot?
The HIV test which uses a western blot to detect in HIV antibodies are present.
What is a western blot used for?
It detects specific proteins in a gel.
