Microscopy Flashcards
briefly describe optics and condenser lenses
-condenser lenses collect and refract light so it converges at a focal point (this is where specimen sits)
-the light passes thru the specimen and creates a larger upside down image
-the microscope converts this via an additional lenses to make a right side up image
magnification vs resolution
magnification = an image is made larger
resolution = the extent to which two objects that are close together can be distinguished
can an object be high resolution but low mag? what about the other way around?
yes high res and low mag
not really no low res and high mag as wont have usable images
bright field light microscopy (5)
- most common
- known as compound microscope
- mag = 1000x resolution = 0.2 um
- may need staining to see
- specimens appear darker/colored against a bright background
dark field light microscopy (5)
- light does not enter the object directly
- only light reflected by the specimen is transmitted thru the microscope
- specimen appears light against a dark background
- can be observed without staining
- can see live cells/motility
state the 3 types of stains and what must happen to them before the stain
- cells are fixed before staining (usually kills them) to ensure they don’t move around the slide
- simple
- negative
- differential
simple stain
cells or structures are stained by a single dye
negative stain
cells or structures repel a single dye, staining the background
differential stain
cell or structures can be distinguished based on their reaction to the stain
describe florescence microscopy
specimens are seen because they emit light (at a higher/longer wavelength)
~uses fluorochromes/phores
- they excite and omit at 2 DIFFERENT wavelengths
what does the microscope have to do in florescence microscopy
needs to produce light at the excitation wavelength and detect light at the emission wavelength
epiflourescence microscopy
light is emitted by the specimen NOT transmitted through it
natural autofluorescence
some cells fluoresce naturally
fluorescent dyes
some cells or structures fluoresce when the cell is stained
fluorescent tags & 2 examples
attached to cell components
ex.
-antibodies help to bind (immunofluorescence)
-gene fusion proteins
vital dye
distinguishes living cells from dead cells
- calcein AM = can pass thru living & dead cells & will go green
- propidium iodide = only pass thru damaged/dead cells & will go red
immunofluorescence
uses antibodies + fluorochrome to attach to cell surface illuminating it / then use fluorescent microscopy
confocal microscopy
scans thru different planes of focus in the specimen to construct a 3D image from multiple 2D images
-non focused light is blocked out (less damaging to specimen) & can be used on live specimens
-used in combo with fluorescent tagging (live) or dyes (dead)
3 types of electron microscopes and what are EMs
EMs use electrons to visualize an image
- transmission electron microscope (TEM)
- scanning electron microscope (SEM)
- cryo-electron microscope (cryo-EM)
resolution of an electron microscope and why is it better than light
limit of 0.1-0.2nm
unlike light which is 200nm
ultrastructure
structures that are too small to see with a light microscope (less than 200nm)
TEM (5)
transmission EM
- must be fixed
- sliced extremely thin
- stained with heavy metals & so kills cells
- electrons pass THRU specimen
- under vacuum
how is TEM better than a light microscope / comparison
TEM:
100,000x mag
less than 0.5nm res
how does a TEM image appear in regard to regions
electrons pass thru less dense regions = areas appear brighter
dense regions scatter more electrons = appear darker in image
shadowing
adds a shadow to specimen by adding heavy metals at an angle
freeze etching
rapidly chills and shatter cells forming jagged edges which show partial 3D structure
immunolabelling
heavy metals (ex.gold) are bound to antibodies specific to a protein of interest
SEM (6)
scanning EM
- fixed
- coated with heavy metals
- scanned over surface of specimen
- mag 10,000x 5nm res
- easy 3D whole image is obtained
- no slices need
why might it be good to visualize the outside surface of a virus?
to see surface proteins / detection of virus particles
are EM images produced in color ?
NO all start out grey but color can be added
cryo EM
- uses a combo of rapid cooling & packing proteins tightly into a sample to allow TEM to visualize individual 3D protein structures
what would i use to view a live specimen (2)
- dark field
- fluorescence
what would i use if i want a 2D image (3)
- bright/ dark field
- epiflourescence
- TEM
what would i use if i want a 3D image (4)
- confocal
- TEM with shadowing or freeze etching
- SEM
- cryo EM
Is the thing I want to look at hard to distinguish (e.g. a protein of interest)?
(2) what could you use
- fluorescence tagging
-immunogold labelling
