KIN 155 Study Guide - Differential Interference Contrast Microscopy, Scanning Electron Microscope, Two-Dimensional Gel Electrophoresis

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5 Aug 2012
Department
Course
Professor
Biology
How Cells are Studied I
Optic techniques for cellular and subcellular architecture
The Light Microscopy
Limit of resolution
Scale of cell biology
 m, nm, and A
Compound microscopy
Types of light microscopy
Brightfield microscopy
basic form
inexpensive and easy
for color and fixed specimen and not for living species
Phase-contrast microscopy
phase plate
good for living, unstained specimen
Dark field microscopy
Fluorescence microscopy
fluorescent compounds
exciter filter
barrier filter
Differential -interference -contrast microscopy (DIC)
(Nomarski)
polarizer
analyzer
Wollaston prism
to produce 3-D image
Confocal microscopy
to produce 3-D image from a collection of optic sections
Sample preparation techniques in light microscopy
Fixation
Cryoprotection
Embedding and sectioning
Staining
Labeling
radioisotope
immunolabeling
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The Electron Microscopy
Use a beam of electron to produce an image
Two major types of electron microscopy
Transmission electron microscopy (TEM)
Vacuum system
Electron gun
Electromagnetic lenses and image formation
Photographic system
Sample preparation techniques in TEM microscopy
Fixation
Embedding, Sectioning, and poststaining
Electron microscopic autoradiography
Negative staining
Shadowing
Freeze-fracturing
Freeze-etching
Scanning electron microscopy (SEM): 3 D images
Second electrons
Sample preparation techniques in SEM microscopy
Fixation
Postfixation
Dehydration
Poststaining
Mounting
Coating
with a layer gold or a mixture of gold and palladium.
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How Cells are Studied II
Biochemical Techniques for Cellular and Subcelllular Functions
Isolation of cells
Source for the best yield
fetal or neonatal tissue
Disrupting the extracellular matrix and intercellular junctions
Proteolytic enzymes
Chelating agents
Approaches to separate cell types
Centrifugation
Cell sorter: fluorescence-activated cell sorter
What to do with a uniform population of cells
For biochemical analysis
For cell culture
Fractionation of organelles and macromolecules
Cell disruption: homogenate
Centrifugation
Separation by size
Separation by size and shape
Separation by buoyant density
Cell-free system
Isolation
Reconstitution
Chromatography
Partition chromatography
Column chromatography
Ion-exchange chromatography
Gel-filtration chromatography
Affinity chromatography
HPLC
Electrophoresis
Proteins usually have a net positive or negative charge that reflects the
mixture of charged amino acids they contain. If an electric field is
applied to a solution containing a protein molecules, the protein will
migrate at a rate that depends onits net charge and on its size and shape
SDS-PAGE
SDS
-mercaptoethanol
Coomassie blue
Silver stain
Western blotting
2-D gel electrophoresis
First dimension: isoelectrical focusing
Second dimension: SDS-PAGE
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Document Summary

Brightfield microscopy basic form inexpensive and easy for color and fixed specimen and not for living species. Phase-contrast microscopy phase plate good for living, unstained specimen. Differential -interference -contrast microscopy (dic) (nomarski) polarizer analyzer. Confocal microscopy to produce 3-d image from a collection of optic sections. Use a beam of electron to produce an image. Coating with a layer gold or a mixture of gold and palladium. Source for the best yield fetal or neonatal tissue. What to do with a uniform population of cells. Proteins usually have a net positive or negative charge that reflects the mixture of charged amino acids they contain. If an electric field is applied to a solution containing a protein molecules, the protein will migrate at a rate that depends onits net charge and on its size and shape. They are assembly of lipids and proteins held together by noncovalent interactions.

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