Lecture 9 - Microscopy Flashcards
what are the 3 elements always needed for imaging in a microscope?
- a source of illumination
- a specimen to be examined
- a system of lenses to focus the illumination on the sample and form an image
def: illuminates the sample
light source
def: placed in front of the light source to focus the light at the desired point on the specimen
condenser lens
def: forms the primary image of the specimen, its the lens closest to the object of interest
objective lens
def: magnifies the primary image produced by the objective lens
ocular lens
def: refers to the size of the image
magnification
the smaller the limit of resolution a microscope has, the greater its _________ _______
resolving power
def: the minimum distance two points can be apart and remain apart
resolution
what 2 things does magnification consider?
- the refraction index of the lens and the medium the sample is immersed in
- the focal length of the lens
what are the 3 things resolution considers?
- the wavelength of illumination
- the refraction index
- angular aperture
def: measure of the change in the velocity of light as it passes from one medium to another
refractive index
what does the Abbé equation give us?
resolution
what are the 6 kinds of light microscopy?
- brightfield(unstained)
- phase contrast
- fluorescence
- brightfieqld(stained)
- differential interference contrast
- confocal
how can one improve bright field microscopy to see live cell and tissues that lack compounds that absorb light and are invisible?
fix specimen or stain it
def: preserves cells, prevents degradation, formalin and formaldehyde
fixation
def: an intact object placed on a slide
whole mount
def: fixed and embedded tissue that is cut into thin pieces and placed on a slide
section
what is the key feature of phase-contrast microscopy?
takes advantage of differences in refractive index and thickness to image living cells without the need to section and stain
what is the most useful case to use phase contrast microscopy?
study dynamic events, like the movement of organelles within the cell
what is the key feature of differential interference contrast?
technique has a shadow-casting effect that makes cells appear dark on one side and light on the other as a result of differences in the optical path due to phase, resulting image has a 3D effect
what does fluorescence microscopy do?
detects fluorescent proteins or dyes to show locations of substances in the cell
what does confocal scanning do?
uses laser beam to illuminate a single plane of fluorescently labeled specimen
in fluorescence, the energy from an external source of light is ________ and almost immediately __________. Because energy must be conserved, the wavelength that comes out is ________ than what went in.
absorbed, re-emitted, longer
def: transmits only night of a particular wavelength
excitation filters
def: reflect light below a certain wavelength and transmits light above a certain wavelength
dichroic mirror
def: prevents light that does not match the emission wavelength from exiting the microscope
emission filter
what is fluorescent protein technology?
green fluorescent protein is fused to your favourite protein and then visualized in the cell
_______ uses antibodies to locate specific molecules
immunostaining
def: when the fluorescent dye is on the primary antibody
direct immunofluorescence
def: when the fluorescent dye is on a secondary antibody
indirect immunofluorescence
def: component of molecules which absorb light
chromophores
what are the advantages of immunofluorescence microscopy?
- high specificity
- strong signal
- identifies endogenous proteins in their native environment
what are the disadvantages immunofluorescence microscopy?
- experiments are done on dead cells (requires fixation)
- there might not be an available antibody
def: commonly used to visualize patterns of gene expression proteins in living cells and organisms
green fluorescent protein (GFP)
what is the advantage of GFP?
cellular events can be observed in living cells
what are the disadvantages of GFP?
- GFP is a protein that might negatively affect your target protein
- introducing the GFP tagged protein into cells
what are the key features of confocal fluorescence microscopy?
- produces in focus images of thick specimens (optical sectioning)
- excludes out of focus light
- reconstructed with computer in 3D
what does confocal fluorescence allow?
allows one to distinguish structures in the middle of a cell from those at the top or bottom
def: mathematical algorithm based process used to remove the contribution of to out-of-focus light in an image
deconvultion
what are the advantages of viewing fixed cells?
- organs can be viewed by sectioning thick tissue
- easier to manage samples
- don’t need to introduce a recombinant protein
what are the advantages of studying live cells?
- can observe the movement of biomolecules within a cell
- provides critical context to your observations
def: technique where you “bleach” an area of the cell and see how long it takes for unbleached molecules to return to the area (measure how fast a molecule moves)
fluorescence recovery after photobleaching (FRAP)
def: technique when fluorescent molecules are close together, one can be used to excited the other (measure if two molecules are touching)
fluorescence resonance energy transfer (FRET)
def: use a beam of electrons and electromagnets, rather than light and gas lenses
electron microscopes
def: electron microscope - the surface of a specimen is scanned, by detecting electrons deflected from the outer surface
scanning electron microscopy (SEM)
def: electron microscope - electrons are transmitted through the specimen
transmission electron microscopy (TEM)
what is immuno EM?
when antibodies are conjugated/linked to substances that are electron dense
def: you take multiple 2D images at different angles and using a computer, create a 3D reconstruction
cyro-electron tomography
def: used for high-resolution structure determination of biomolecules in solution
cry-electron microscopy
