Unit 1 Flashcards
What is FRAP and how does it work
Fluoresecent Recovery After Photobleaching.
Refers to a technique where we photobleach an area after halting protein synthesis such that the recovery of protein in that area show us that other fluorescent proteins have moved there.
Explain EM Tomography
This is an electron microscope technique where we take images from a bunch of different angles to reduce noise/blur and gain the capability to build a 3D image.
How do we enhance contrast in Light Microscopy
-Stains: Stains only let out light of a specific wavelength and differentially stain different parts of the cell
-DIC (Normarski): This technique takes advantage of changes in refractive index of dense and less dense areas and translates this into sharp edges
-the other one: This technique amplies the phase changes of light as it passes through the sample.
What is Deconvolution Microscopy
This is a blur-reducing technique in Light Microscopy. It involves using a computer with the ability to precisely manipulate the Z-axis such that it eliminates all light from above or below the axis of choice.
What is Confocal Microscopy
This is a blur reducing technique in Light Microscopy. It involves shining light through a pinhole that is confocal with the another pinhole infront of the detector. This ensure that only focused light reaches the detector.
What is Single Particle Reconstruction (SPR)
This is a technique in cryo-electron Microscopy. It involves getting a bunch of individual pure molecules crystalized in vitreous ice at various orientations and then using the different orientations to get a full 3D structure.
What is FRET and how does it work
Fluorescent …. It involves having two molecules you think might interact conjugated to 2 different fluorescent proteins. The emission spectrum of one should match the excitation spectrum of the other such that if they get REALLY close ( ) the second one will glow.
What is immunolabelling
This is when we use primary or secondary antibodies conjugated to a marker (Ex: fluorescent molecule or electron dense gold balls) in order to label a specific antigen.
what is immunofluorescence
This is when the antibodies are conjugated to a fluorescent molecule.
what is immunoelectron microscopy
This is when the antibodies are conjugated to gold balls of one (or various) size(s)
what is GFP? Where does it come from?
This is a B-barrel shaped green fluorescent molecule. It comes from the jellyfish aquoria victoria.
What do we use GFP for?
The following are examples of uses
-Conjugate to protien to see where it comes
-Replace coding region to monitor promoter activity
-another one
How can we study protein dynamics in living cells? List 2 methods
-fluorescent protein fusions
-FRAP
What are the basic principles of fluorescence Microscopy?
We shine light through a filter such that only excitation light gets in. It fluorescents the molecule and then the detector has a filter such that only emission light reaches it. Thus, we see glow on a dark background
Explain how “caged” molecules work
We don’t activate a fluorescence molecule until it is ready.
