lec 3- light and electron microscopy methods part 2 Flashcards
(26 cards)
if you don’t have a confocal microscope, how can you get a confocal quality image using a regular fluorescent microscope?
deconvolve it
what is deconvolution?
it is when a software that is calibrated to the microscope system mathematically moves out of focus light back to focus
how is the control for deconvolution?
fluorescent beads are imaged as a control, because the size of the beads are known, the mathematical elements can be solved, making it easy to get the out of focused light back in focus
what is FRAP (fluorescence recovery after photo bleaching)?
it is when fluorescently tagged proteins are hit for a prolonged period of time with a laser until the fluorescence is no longer remaining, after time that area recovers and the time tells us the turnover rate of the proteins
what is FRET (fluorescent resonance energy transfer)?
it is when one fluorophore is excited, causing it to emit a light that can excite another fluorophore, letting us know if they are closely related when collecting the light of the second
what type of microscopy helps us go past the Abbe limit of 0.2 nm?
super-resolution microscopy
what are the 5 types of super-resolution microscopy?
-structured illumination (SIM)
-stimulated emission depletion (STED)
-photo-activated localization microscopy (PALM)
-stochastic optical reconstruction microscopy (STORM)
-Lattice light-sheet microscopy
how is SIM done?
-a grid is placed between the light path and the camera
-3 pictures are taken with the grid in 3 different places
-a sharp image with little out of focus light
-can get 100 micro m
-very slow cause need three pictures for every plane
how is STED done?
-works like the point scanning confocal
-has a ring (AKA a depletion beam) around the laser point making the excited area much smaller
-takes all points and puts them together
-resolution 30 micro m
-problem is that it is slow
how is PALM done?
-thousand of images are collected each with only a few molecules of a photo-activated fluorescent protein excited
-these molecules are black to start, then turn fluorescent when activated
-the center of the excited photon gives the location of the GFP molecule itself
-10 nm resolution
-problem is that it is very very slow
-dark –> light
how is STORM done?
-nearly identical to PALM, really slow
-20 nm when invented, now down to 5 nm
-instead of GFP, it uses lasers to excite a photoswitchable fluorochrome to a dark state
-the center position of each molecular image is plotted based off of the photons emitted
-light –> dark
how is lattice light shield microscopy done?
-uses a sheet of light to illuminate 1 entire plane of the sample being imaged in the same plane
-all planes are imaged very quickly (up to 1,000 images/second)
-resolution of 150 nm
-enables live super-resolution imaging
-uses transparent samples organisms
how do they use lattice sheet microscopy when the sample is too thick?
they do tissue clearing
what does tissue clearing provide?
a 3D arrangement of whatever your looking at within a huge volume
does image quality get worse with thicker samples? what type of light do thick samples use?
-yes
-they use infrared light
why is tissue clearing better than thick samples?
-much of the matter that will generate out of focused light is removed, allowing for the usage of light in the visible spectrum
how is tissue clearing done?
hydrogel imbedding:
-the sample is embedded into hydrogel
-lipids are removed in 8% SDS
-the sample is immersed into a clearing solution
-end up with a clear sample
-can take hours or months depending on the technique
-tissue may shrink or swell
-can easily be used for immunolocalization as there is no membrane hampering the permeability of the process being used
how is electron microscopy done?
-electron microscopy uses a high velocity beam of electrons to shoot at the sample
-all under an ultra high vacuum because air can absorb electrons
-electrons are contained within an electric field
-resolution of 0.0005 micro meter (size of an atom)
-it is 40,000 times better than a light microscope
-0.1 nm better for biological samples
-300x better than super-resolution
-problem is it cant do live imaging
what are the 3 types of electron microscopy?
- transmission EM (and immuno EM)
- scanning EM
- 3D cryoEM tomography
how is TEM done?
-lets you see structures within cells
-samples are fixed by cross-linking the proteins into positions using fixatives (paraformaldehyde/glutaraldehyde)
-embedded in resin, sectioned into 70 nm parts, stained with heavy metals
why and how is immuno EM done?
-it allows us to detect antigens on structures at the ultrastructural level
-gold particles are used to identify their location because they are electron dense
-dual labeling is also possible using gold particles of different diameters
why and how is SEM done?
-lets you see the surface of samples
-samples are coated with metals
-resolution of 10 nm
how is cryoEM done?
-no fixation or staining needed
-samples are frozen with liquid nitrogen
-so samples are viewed in their native state
-hundreds of images get arranged to generate the final image
-the sample is rotated on a platform so that larger samples can be imaged from many different angles
-image is reconstructed by computer for 3D organization
-this technique got the nobel prize in 2017
what is the problem with cryoEM?
-cant correlate something seen on a fluorescent microscope with a cryo electron microscope from the same sample
-need an antibody
