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BIOL201 Study Guide - Final Guide: Secretion, Karyopherin, Nuclear Pore

Biology (Biological Sciences)
Course Code
Mike Harrington
Study Guide

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Bio 201 Midterm Study Notes 2015
Light Microscope Fundamentals:
Magnification of 1000X
Use Visible or UV light
Types of light microscopes:
Brightfield Microscope
Phase Contrast Microscope:
oTypically not stained
oTypically ALIVE!!!!
oUses more complex lenses to make darks darker, and make lights lighter.
oSame as PC MS but, causes image to have a slight bit of depth and is also gray
Widefield Fluorescence MS:
oFluorescent molecules added to sample and then short wavelength light is used to
observe. Areas that have fluorescent molecules expel high wavelength light, areas
that don't have fluorescent molecules shine the same wavelength of light
originally used.
Composite Images:
oCombines images from different microscopes to show unique aspects from all ms.
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Confocal Fluorescence Microscope:
oBasically reveals an area within specimen by shining light (laser) from different
oMore clear image than Widefield
oType of fluorescent ms that shows intensity of light coming off sample (concentration
of fluorescent molecules in area of specimen .
Specimen Prep:
Most specimen that use any kind of light ms have to be cut prior to being observed
oThis is done using a Microtome. ← basically cuts the cell up!
Large Molecules:
oSample is crushed using a slide and slide cover.
Probes and Fluorescence:
Regular stains show color
Fluorescent stains:
oONLY in fluorescent ms.
DNA Probes:
oHave a High Affinity for DNA
oHave Fluorescent molecules attached to them
oAttach to single stranded DNA (denatured) using base pairing, then are observed
through Fluorescent MS.
Fluorescent Antibodies: → Used to view Cells, Proteins, Tissue Section, Etc.
Typically use Widefield Fluorescence Microscope
Used to view specific molecules, Also Antibodies made from White Blood Cells
Direct Immunofluorescence:
oAntibodies made for YFP (specific antigen), with fluorescent molecules attached
Antibodies attach to antigens (specific molecules), so you can see different parts.
Indirect Immunofluorescence:
oHow it Works: YFP Antibodies are made (by taking blood from organism and
collecting antibodies from it 2 months after blood taken) (blood has to be isolated so
it’s just antibodies), These antibodies are added to the sample, then Fluorescent
antibodies that can attach to the YFP antibodies are added, they attach 2 PER
ANTIBODY and therefore glow brighter. The sample viewed through fluorescence
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Antibody Purification:
First total sample of Antibodies (non specific) are put into a column,
Column has YFP on ONE SIDE and nothing on the other, the antibodies affiliated to the YFP
go to that side, they are then put through harsh conditions so that they are separated from the
YFP protein and are then collected and used typically for Indirect Immunofluorescence.
Electron Microscopes Fundamentals:
Has 2 types, Scanning Electron MS (SEM) and Transmitting electron MS (TEM).
Are around 20-100X stronger than Light MS
Uses electrons to view sample, and focuses them using electromagnetic lenses
SEM: Observes sample in 3D (electron bounces off sample) Mag, of 20 000X
TEM: Observes sample typically in 2D (electrons go through sample) Mag of 100 000X
Preparation for SEM (20 000X)
Sample is coated with gold atoms and viewed in 3D, very detailed and clear image (usually).
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