Fluoromicroscopy Flashcards
How do you increase the contrast of brightfield microscope images?
phase contrast enhancement –> increases contrast ; from this you can produce differential inteference contrast. But this is very dependent on the sample
what are the pros and cons of the brightfield microscope?`
pros:
simple and easy to use/set up
no phototoxicity (doesn’t destroy sample)
cons:
phase contrast enhancement is sample dependent
only good for very thin samples
based on light absorbtion so RI low
Why use fluoromicroscopy?
To study structure and function of molecules. You can do live cell imaging - ie view the passage of vesicle down a synapse and into the snyaptic cleft; visualise where drugs of interest bind eg ecstacy and serotonin reuptake receptors
Can tag different molecules in the same sample with different fluorophores
What is fluoresence?
- The result of light absorption
- requires external energy sources to excite fluorophores (UV light)
- electrons are excited to a higher energy state; when they return to ground state the emit fluorescent light of a longer wavelength (stokes shift)
- happens on ns level
What are intrinsic fluorophores? What can be used for genetic labelling?
- they are fluorescent materials with a primary/auto fluoresence, eg GFP family
- GFP jellyfish protein absorbs blue light and emmits green fluoresence
GFP. It can be encorporated into target gene as a reporter gene (eg in tissue-specific KD) so can be used to study gene expression, protein localisation and protein interactions
What are extrinsic fluorophores? Give examples
molecules which possess a fluorescent feature. For exampe, antibodies can be covalently bound to flourophores and then indirectly bind to target molecules (direct 1 antibody, indirect 2 antibody)
Or fluorescent dUTPs can be attachedd to single stranded probes, which hybridize to single stranded DNA from denatured chromosomes and thus fluoresces under a microscope (direct DNA/RNA labelling); indirect labelling requires non-fluorescent probe on the single stranded probe, then you apply a labelled antibody once the probe has hybridised to DNA. The antibody will fluoresce under a microscope.
These DNA/RNA labelling methods can be used for visualising gene expression or record live cell imaging of mitosis.
What features are added to a bright-field microscope to make it a fluorescent microscope?
- fluorescent ight source (UV lasers)
- filter assembly: dichotic mirror, excitation and emission filter
What do the three parts of the filter tube do?
- excitation filter - only allows wavelength of light theough that will excite the fluorophore
- beam splitter/dichotic mirror - refracts the light - acts light a switch for the two filters
- emission filter - only allows light (of lower wavelength) emitted from the sample to pass through the eye piece
Draw the typical fluoromicroscope set up (filter tube)
How do confocal microscopes overcome the problem of blurring often seen in widefield? (compare the two methods)
Confocal is a widefield microscope with the addition of a pinhole, scanning unit and point detector. There requires careful calibration to make sure the pinhole is in the right position. The pinhole influences resolution and detection sensitivity; smaller pinhole = compromise between spatial resoltion and s:n.
If the pinhole is closedk, larger axial res, so signal weaker (smaller signal:noise but larger spatial res)
Essentially electrons pass through pinhole to reach the detector –> this only allows for small part of the image to be captured at a time (filtration) Increases field depth, because there is less background noise. Imaging and scanning across the sample in smaller portions increases resoltuion (scans one particle at a time!) and allows for 3D imaging. However, confocal takes longer and thus more likely to photobleach your sample.
Widefield images particles all at once at different depths –> the combination of on-target and off-target (beneath the focal plane) fluoresence gives blurred image. This results in a lower contrast and lower resolution image, and only images in 2D. However, imaging speed is faster and set up easier and cost lower. Use ths to visualize thinner samples and basic cell structure
What might you image with a confocal microscope?
- genetic matieral (if DNA/RNA labelling)
- colocalisation of cells/proteins - overlay images colocalization implies cell-cell interactions
- cell viability (ratio of dead/alive cells) for example in cytyotoxicity assays or to correlate behaviour to no. of cells - overlay images
- computers will pseudocolour images according to colours corresponding with wavelength of light emitted
What are the two main issues to consider about fluoromicroscopy?
1, photobleaching –> light exposure can destroy fluorophores so they’re unable to fluresce. Limit no images taken of sample and the time the sample is exposed to laser.
2. spectral cross-talk - there’s limites space between spectral curves (wavelengths) so they might overlap, thus you might see singles from one dye in multiple channels. Theefore you need to choose correct filters, dyes have conjugated imaging strategies and you cal do sectral unmixing with a computer. Also you can also label fluorophores with close spectra at different spatial locations
Outline the typical immunofluorescence protocol
- fixation with formaldehyde. This forms crosslinks between AA (DNA proteins, not DNA itself) which freezes the protein in time. Allows for brighter signal and use of 4 fluorophores at a time.
- permeabalisation with detergent. This disrupts the cell membrane and allows the antibodies to enter the cell.
- wash away detergent
- Blocking to make sure that antibodies don’t bind to non-specific binding regions
- primary antibody, and leave in fridge overnight
- wash
- secondary, leave overnight in fridge
- wash
- mount on coverslip, cant apply coverslip directly as this will dehydrate cells so need a medium in betwen
What controls do you need in the DSB repair foci counting experiment?
one group of cells you don’t have any DNA damaging agent - need to know what baseline DSB looks like.
Fluorophores will only mark proteins with DSB –> so by counting the fluorophore youre counting the foci (DSB)
What challengea and weaknesses are associated with foci counting experiments, and how can you overcome these?
- what classifies as a foci? - this is subjective. mayne need a computer to measure fluoresence threshold to be able to distinguish foci from background
- one strong signal or two foci with similar signals close together? - need a computer program that measures fluorescent peak convergence
- losing count? keep score or do digitally
weaknesses of this method is that it is contrained by how durable/good tour anitbodies and lebelling is
can improve efficiency of result by doing blind counting , using different fluorophores and higher number of cells
