Lecture 5 Flashcards
Light microscope
Limit of about 0.5um, beyond which details are obscured by the effects resulting from the wavelike nature of light. Light is focused on a sample by lenses, which focus an image of the illuminated sample in the eye.
colour contrast in light microscopy
colour contrast - stained portion of cell will absorb light of some wavelengths, which depends on the stain, but allow others to pass thru; coloured image visible in normal bright field microscope.
bright field microscope
Light passing thru cell in culture forms the image directly.
Dark field microscopy
exploits the fact that light rays can be scattered in all directions by small objects in their path. Creates a bright image against a black background.
Can watch the movements involved in processes like mitosis and cell migration.
Phase contrast microscope
increases the phase differences of light waves so that the waves are more nearly out of phase, producing amplitude differences when the sets of waves recombine, creating an image of the cell’s structure. –> visualise living cells.
Same for differential interference contrast microscope.
Can watch the movements involved in processes like mitosis and cell migration.
fluorescent microscope
The illuminating light is passed thru two sets of filters - one to filter the light before it reaches the specimen and another to filter the light obtained from the sample. The first filter passes only the wavelengths that excite the particular fluorescent dye, while the second filter blocks out this light and passes only those that are emitted when the dye fluoresces.
Used to detect specific proteins or molecules in a cell and tissues.
Can image cell dynamics.
immunofluorescence
Very sensitive because many molecules of the secondary antibody recognise each primary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable - commonly fluorescent probes.
GFP
Emission of 500nm of light.
What’s the advantage of tagging a protein with GFP, rather than using immunofluorescence?
dynamics
What’s the advantage of using immunofluorescence, rather than tagging a protein with GFP?
higher signal-to-noise ratio
Confocal microscope
Generally used with fluorescent optics, but illuminates onto a single point at a specific depth in the sample, rather than the whole sample. Need source of pinpoint illumination via a laser whose light passes thru pinhole. Can use it to reeve structure of complex 3D objects/networks etc. Better for thicker specimens with high levels of out-of-focus light.
TIRF
Laser light shines onto cover slip surface at the precise critical angle at which total internal reflection occurs. Light does not enter the sample and the majority of fluorescent molecules are not illuminated. Just those that are in the layer closest to the surface of the coverslip become excited, so their emitted light does not compete with out-of-focus light from overlaying molecules and can now be detected. Can detect single fluorescent molecules. Restricted to thin layer within 100-200nm of cell surface.
Also, strictly passive observation method.
What limits spatial resolution in a
conventional light microscope
Diffraction
Size of kinesin step
8 nm
Is it possible to resolve individual steps of kinesin using a conventional light microscope?
yes
The position of a single fluorescent molecule
can be determined with high accuracy. The center of the point spread function can be determined within ~1nm for bright samples.
Expansion microscopy
Advantage: high spatial resolution
Diadvantage: No temporal resolution
