Lecture 14 Fluorescent Microscopy Flashcards
Epi Fluorescent light path
Specimen is illuminated with light of specific wavelength(e.g. green) which excited the fluoroohores so that they emit light of a longer wavelength (e.g. red). Image is then magnified and focused on retina/detector
Chromatic beam splitter - reflects and transmits light of different wavelength differently e.g. reflect green/transmit red
Secondary filter - selects wavelength of light reaching the detector
Primary filter - selects wavelength of light used to excite specimen
Specimen is labelled with fluorescent probes
Fluorescent molecules are excited by light of a particular wavelength
1) short light wavelength illumination
2) excites electron to higher shell
3) electron returns to lower shell releasing energy as a proton of a longer light wavelength
Immunofluorescence
1) antigen - molecule present on cell structure e.g. beta tubulin protein on microtubule. Antibodies recognise the antigens
2) primary antibody binds to specific antigen
3) secondary antibody with fluorescent tag attached to primary antibody
(Small polypep bound to actin allows fluoro tag to be attached to antibody actin cannot be directly connected to fluoro tag)
Can be viewed by fluoro microscope
Fluorescent recovery after photobleaching (FRAP)
Used to study molecular mobility - is protein anchored or partially anchored - can it move?
1) molecule of interest e.g. membrane protein tagged/labelled with fluorescent marker
2) small area bleached with a laser
3) fluorescent recovery in sense that bleaching is redistributed - rate of recovery related to rate that molecules move e.g. no recovery shows protein is immobile
F 1/2 time it takes for fluorescence to recover 50%. Relates to different coefficient.
Never fully recovers because
- bleached fluorophores don’t recover
- some molecules are immobile
-some bleaching additionally due to light exposure in imaging
Clarity and resolution improved by
Confocal microscopy - physical filter
Or deconvolution - software filter
Confocal microscopy
Pinhole (in light path by detector) prevents out of focus glare
Lasers produce monochromatic light of specific wavelength
Lasers produce a point source of light
Dichroic mirror reflects or transmits specific wavelengths
Scanning mirrors move the point source of light across the specimen
Pinhole filters out of focus light to optical sections
Image produced is similar to fluorescent microscopy
3D structural illumination microscopy (SIM)
Extra fine resolution calculated by interference patterns generated computationally
Localisation microscopy
photo activated localisation microscopy PALM
Collect thousands of images in each of which only a few GFP molecules are excited. Each GFP emits photons in a Gaussian dist. For which the centre can be calculated to produce an image
Process:
- locate single fluorophores by non permanent bleaching, low wave light to activate only a few fluorescent molecules
- take a series of images with these few bright spots showing then put them in dark state and activate another group
-creates an image by layering collected images depicting exact locations of each independent fluorophore - very ‘crisp’ image
Electron microscopy
SEM - scanning
Electron beam fired at sample, sample emits electrons (back scatter) in response and these are collected by a detector to form an image
TEM - transmission
Electron beam passed through a thin slice of sample
Resolution of EM theoretical ~0.003nm actual ~0.1nm
Resolution of a light microscope
Resolution : The closest 2 objects can be whilst still distinguished
Depends on wavelength (gamma)
Resolution of light microscope =
D= 0.61gamma/N sin alpha
Wavelength of violet light = 380nm
N= numerical aperture of lens
Light collection angle - larger is better
Usually light microscope ~200nm res
Resolution of EM theoretical ~0.003nm actual ~0.1nm
