Methods, Part 2 February 9, 2012. Learning Outcomes Discriminate between different types of...
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Methods, Part 2 February 9, 2012. Learning Outcomes Discriminate between different types of microscopy, and justify their use for answering research questions
Learning Outcomes Discriminate between different types of
microscopy, and justify their use for answering research questions.
Differentiate between conventional and confocal fluorescence
microscopy. Describe in writing how genes from different organisms
can be modified, inserted into, and expressed in cells.
Slide 3
Microscopy Resolution: The minimum distance between two objects
that can be detected Defined by The Abbe Equation: distance =
_0.612 * _ NA Visible light= 380-760 nm Best resolution of visible
light microscope: 200 nm Electron microscopes use electron beam,
wavelength 100,000 X shorter
Slide 4
Microscopy Contrast: The ability to interfere with the
illumination source Bright field microscopy uses dyes (stains) to
generate contrast H&E is popular dye for medical diagnostics
Sample must be dead
Slide 5
Microscopy Contrast: The ability to interfere with the
illumination source Phase contrast microscopy uses differences in
diffraction to generate contrast Image has grey background,
black/white contrast Colored filters can increase contrast a bit No
dyes used, cells can be alive when viewed Movies of cells! Neuron
in cell culture
Slide 6
Fluorescence Microscopy By far the most frequently used
microscopy technique in cell biology research Contrast generated by
fluorescent (visible) light emitted by target; background is black
Excitation wavelength is always shorter then emission wavelength A
dichroic mirror blocks excitation light, allows only emission light
to reach observer Can be used to visualize specific molecules
Slide 7
Fundamentals of Fluorescence Microscopy A nice summary of
fluorescence microscopy An explanation of confocal fluorescence
microscopy An explanation of confocal fluorescence microscopy
Slide 8
Slide 9
Basics of recombinant DNA technology Fundamental concept:
Because DNA is structurally identical in all organisms, it is
possible to combine DNA sequences from different organisms, and
insert the combined DNA into any organism. Isolating DNA from cells
is easy.easy Cutting DNA into pieces, according to DNA sequence, is
easy.easy Pasting the pieces together is easy, using DNA ligase.DNA
ligase Putting the hybrid DNA into cells (formerly called
transfection) is easy but expensive.easy
Slide 10
Basics of recombinant DNA technology Most often, genomic DNA is
not combined together; instead, a DNA copy of a single gene, called
complementary DNA (cDNA) is used instead. cDNA is derived from
mRNA, so the introns have already been removed from the gene, and
the DNA is thus smaller and easier to use. cDNA is frequently
combined with a small circular piece of DNA, called a plasmid,
before inserting it into a cell. The plasmid contains DNA sequences
that help control when and where the cDNA gene will be
expressed.
Slide 11
Basics of recombinant DNA technology It is becoming quite
common to build hybrid genes, which contain coding sequences for
two entirely different proteins. The product of these genes is
called a fusion protein. One of the most popular classes of fusion
proteins is genes fused to the gene that encodes Green Fluorescence
Protein (aka GFP) or its derivatives. GFP fusion proteins have
revolutionized cell biology: you can use fluorescence microscopy to
track specific proteins in living cells/tissues/organs/animals.
Those who discovered this gene and developed it for research were
awarded the Nobel Prize in 2008.Nobel Prize