Module 06: Microscopy Flashcards
(39 cards)
What do you during when you fix cells?
Covalently crosslink macromolecules to each other
What is the purpose of dehydration when you fix cells?
Prevention of microbial growth and hydrolysis
What is the purpose of permeabilizing cells during fixation?
Degrades the membrane, allows access of antibodies and stains
What does a microtome do?
It cuts tissue into sections thin enough that light can transmit rough
What are the steps of preparing a thick specimen?
Fixing the tissue, dehydration, embedding in block of wax, microtoming
What classes of chemicals are used in chemical fixation?
Acids (acetic acid) and aldehydes (formaldehyde)
What processing is required to use phase contrast microscopy?
None; cells are unprocessed
How does phase contrast microscopy work?
Uses the different in phases between direct (unaltered) and diffracted (altered by specimen) light
What does DIC microscopy stand for?
Differential interference microscopy
What is another name for DIC microscopy?
Normanski microscopy
Describe the basics of fluorescence.
A photon causes an electron to move to an excited state, which then drops to a lower, more stable state. This energy is released as an emitted photon (of a longer wavelength)
What is it called when a photon causes an electron to move to a higher energy state?
Excitation
What is it called when an electron drops to a more stable energy state and releases a photon?
Emission
excitation (n.)
the act of a photon exciting an electron into a higher energy state
How does immunofluoresence work?
Fluorescently labeled antibodies bind directly to antigen
How does indirect immunofluoresence work?
An unlabeled primary antibody binds to the antigen; secondary antibodies labeled with a fluorescent dye binds to the primary antibodies
What does DAPI stain?
DNA
What does GFP stand for and where does it come from?
Green fluorescent protein, and it comes from jellyfish
What is special about GFP?
It allows us to image and record live cells
How is GFP added onto tubulin?
DNA coding for GFP is added on to the sequence that encodes for tubulin; the end result is a fusion protein of tubulin and GFP
What does TEM stand for?
Transmission electron microscopy
What are two heavy metals used in TEM?
OsO4 and gold
What does SEM stand for?
Scanning Electron Microscopy
What is the preparation process for SEM?
Fixation, dehydration, soaked in OsO4, then sprayed with a fine coat of gold
