lecture 2 -FRET Flashcards
What is Fluorescence resonance energy transfer ?
energy transfer that an excited donor transfers energy (not an electron) to an acceptor group through a non-radiative process.
How does FRET work ?
Cyan & yellow FP
CFP-donor
YFP-acceptor
if the excitation spectrum of 1 fluorphore (CFP) overlaps with YFP then then YFP will fluoresce
shorter wavelength–>longer wavelength
higher eneregy–>to lower energy
CYP–>YFP
only if there close -100 A
Advantages of FRET ?
make fusion proteins in which fluoresent protin covalently linked through covaletly to protein to be studied
attached by atag instead of immumofluroesence which attaches and ends up killing the cell
-tells dynamically when and where proteins can interact -FRET activity increases
Applications of FRET ?
- Vesicle fusing
2.Detect protein cleaving in 2
3.Detect confirmational change e.g N & C terminal interacting
4.Common pairing is CYP & YFP
What is FRAP ?
Fluoresence recovering after photobleaching
* Bleach small area with fluorophores inactivating fluorophores
light energy applied–>photofluroeence recovery
* if molecules move they will swap places with fluoresent neighbours
* region will fluoresce again
meaures dynamic of proteins in cells
What is Fluoresence microscopy ?
works well in layer of cultured cells
thick tissue slices or organisms the unfocused emmitted light from outside the focal plane blurs the image
ways to remove:
-Deconvoltion
-laser scanning confocal
-spinning disk confocal
How does Confocal microscopy work ?
in thick samples the light from outside the focal plane blurs the whole image
to get thin section:
using laser to pinpoint source of light and put through a pin hole to illuminate a limited zone as focus image is imaged
Confocal as pinhole hole in a conjugate plane with a point on the specimen
One in front of the light source and one in front of the detector
What is Laser confocal ?
Emit light in a narrow beam
Mirrors then scan this beam across and up/down the specimen
- galvanometers
what is laser scanning microscope ?
photmultipliers detect the light
computers neede to record this and form a screen image
works with laser display
it remembers where the laser point has hit and remebers
laser scanning vs spinning disk micrsoscopes
?
The point-scanning confocal microscope light path of a single-wavelength point of light from an appropriate laser reflects off a dichroic mirror and bounces off two scanning mirrors and passes through the objective lens to illuminate a spot in the specimen. The scanning mirrors rock back and forth in such a way that the light scans the specimen in a raster fashion (see green lines in the specimen). The fluorescence emitted by the specimen passes back through the objective lens and bounces off the scanning mirrors onto the dichroic mirror passing through a pinhole, which excludes light from out-of-focus focal planes.
(b) The spinning disk confocal microscope light path from the laser is spread to illuminate pinholes on the coupled spinning disks, the first consisting of microlenses to focus the light on pinholes in the second disk. The excitation light passes through the objective lens to provide point illumination of a number of spots in the specimen. The fluorescence emitted passes back through the objective lens and through the holes in the spinning disk, and is then bounced off a dichroic mirror into a sensitive digital camera. The pinholes in the disk are arranged so that as it spins, it rapidly illuminates all parts of the specimen several times. As the disk spins as fast as 3000 rpm, very dynamic events
2 photon imaging technique ?
uses 2 photons to excite the fluorphore; further reduces out of focal plane excitation; good for deep imaging > 1mm in live animals e.g. mouse
What is TRIF ?
illuminates fluorphores right next to the coverslip only. Good for imaging where cells or molecules are adhering to the coverslip e.g. motors on microtubues, cell adhesion, vesicles and exocytosis
What is light sheet ?
two objectives at right angles; sample illuminated in a sheet in one focal plane. Sheet moved by stepping objectives so each focal plane imaged
Define Resolution ?
of light microscopy is 200 nm 0.2µm
Fluorophore are point sources of light – if illuminated correctly
What is point spread function ?
– location of fluorophore lies at the centre of distribution of light observed
