Microscopy Flashcards
uses of microscopes
view objects/specimens not visible to the naked eye
parts of a microscope
- light source: e.g. sunlight, halogen lamps
- light conditioning system: how we allow light source to reach the specimen, e.g. Kohler illumination
- specimen: on cover glass
- objective: magnifying glass
- detector: e.g. eyes, cameras, photomultiplier tubes to increase signal
what needs to be kept constant in light microscopy?
cover slip needs to be between 0.17 & 0.18 mm
purpose of incubator box when imaging live specimens
- prevents temperatures from changing
- maintains O2 & CO2 to keep specimen alive
examples of experimental timescales
- if looking at embryonic development, will capture image for days
- if looking at motility of microtubules, need to capture images within seconds
problems with timescales
need to take images fast for short timescales
need to monitor conditions for longer timescales
what is the triangle of frustration?
a compromise between 3 different factors
- Temporal resolution: how long and fast images need to be taken
- Spatial resolution: pixel number
- Sensitivity: pick up images in lower light conditions
what is numerical aperture?
the ability of objective to resolve two points that are very close together
- higher the aperture - greater the resolution
what is working distance?
distance objective can work from the sample
immersion mediums
light travels differently through different mediums, if the objective says oil but you put it in water, light may not travel through
light microscopy
modify the intensity & direction of the light source
Brightfield
ensures all the light reaches the sample in light microscopy
DIC
condense the light through a smaller area, which allows for 3D in light microscopy
Phase contrast
uses a phase ring to play with contrast; less detail but more definition
Uses of microscopy
- Histology
- Phase contrast
- Time-lapse microscopy
Using light microscopy for histology
pros: general idea of tissue
cons: lacks details, hard to distinguish between cells
phase contrast light microscopy
helps you see margins of cells where collagen has been denatured
time-lapse light microscopy
can see cell migration
electron microscopy
- light source is an electron source
- collect the energy from the electrons and transform it into an image
transmission electron microscope
- not 3D
- beams of electrons passed through ultra-thin specimen, interacting with the specimen
scanning electron microscope
- sample treated with reagents
- 3D
fluorescence microscopy
- modify proteins to respond to specific wavelengths
- specimen absorbs light & releases energy
what is stokes shift?
the differences between excitation and emission wavelengths
what is photobleaching?
- The amount of light that you put in the excitation may break the continuous cycle of absorption, excitation & emission
uses of fluorescent proteins
can be fused with other proteins & introduced in cells via transfection, allowing the live study of flourescent tags
how to use antibodies to see a protein of interest?
o Secondary antibodies recognise the primary antibody
The secondary antibody has a fluorophore that that is excited and then emits at a particular wavelength, so you can see your protein of interest in colour
difference between conofocal and widefield microscopes
- light source for confocal is a laser that can emit the wavelength
- emission will be enhanced by photomultiplier
- therefore higher resolution & reduced out-of-focus blur
- images are crisp and clear
- only small volumes can be visualised
