Microscopy Flashcards
Thinnest picture is by
Scanning Electron Microscopy
Light microscope kind
1- phase contrast
2- fluorescence microscope
Electron microscopes kind
1- transmission electrons
2- scanning electrons
Three elements needed for imaging
1- source of illumination
2- speciemen to be examined
3- system of lenses
Condenser lens
In front of light source
Objective lens
Closest lense to object
Ocular lens
Magnify image created by objective lense
Magnification
Size of the image vs object
Empty magnification
Maginified without resolution
Resolving power is
Resolution
Resolution allows object to
Be distinguished
Resolution
Minimum distance two points can be apart and remain apart
Magnification consider
1- refraction index of lens
1- medium that sample is immersed in
2- focal length of lens
Resolution considers
1- wavelength of illumination
2- refraction index
3- angular aperture
Longer wavelength
Larger object observed
Shorter wavelength
Smaller object obseved
Abbe equation
r=0.61landa/nsin(alpha)
Magnification in air and oil
1000 and 1400
Lighting
How light reflects off an object
Contrast
Difference in appearance between object and its background
Bright field microscopy
Light microscope
White light passed through specimen
Living sample are nearly invisible
Better image if stained:fixed
Bright field stain
Stain with dye absorb specific wavelength
Different dye for different biomolecules
Phase contrast
Light microscope
Use differences in refractive index and thickness
Without the need to section/stain
Turn Phase differences to brightness
Differential Interference Contrast
Shadow-casting effect
Cells dark on one side
Light on the other side
3D image
Fluorescence Microscopy
Detect fluorescent proteins
Use laser beam to illuminate a single plane of fluorescently
Excitation is ——— wavelength than emission
Smaller/shorter
Excitation filters
Transmit only light of particular wavelengths
Dichroic mirror
Reflect light below a certain wavelength
Emission filter
Prevent light from exiting microscope
Immunostaining uses ——— to locate specific molecules
Antibidies
Immunofluorescence microscopy advantage
-high specifity
-string signal
-identifies proteins in their native environment
Immunofluorescence microscopy disadvantage
Experiment on dead cell
Might not be any available antibody
GFP
Green Fluorescent Protein
GFP advantage
Cellular events in living cell
GFP disadvantaged
Introducing the gfp-tagged protein
Confocal fluorescence microscopy
Optical sectioning: produce in-focus images
3D reconstruction
Distinguish structures in the middle of the cell from top and bottom
Advantage of fixed cells
1- sectioning thick tissue
2- easier to manage samples
3- no need to recombinant protein
Advantage of live cell
1- movement of biomolecules within a cell
2- critical context to your observation
Electron microscope
Use of beam of electrons and electromagnets
Electron microscope types
1- scanning electron microscopes
2- transmission electron microscope
Scan electron microscopes
Surface is scanned
Detecting electrons from outer surfaces
Transmission electrons microscope
Electrons are transmitted through the specimen
Cryo-Et microscopy
Take multiple 2D images at different angles and crrate a 3D reconstruction
Fluorrescense recovery after photobleaching (FRAP)
Measure how fast a molecule moves
(Bleach an area and see how long takes for unbleached molecules to return)
Fluorescense Resonance Energy Transfer (FRET)
Measure if two molecules are touching
(If closer than 10nm one can excite other)
