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Bringing Light into the chaos: a general introduction to optics and light microscopy. Part 1: The root of all evil. Part 2:. Fluorescence microscopy and special applications. Contrasting techniques - a reminder…. Brightfield -absorption Darkfield -scattering
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Part 1: The root of all evil
and special applications
excitation and emission spectra of EGFP (green) and Cy2 (blue)
No filter can separate these wavelengths!Excitation / emission
You can plot and compare spectra and check spectra compatibility for many fluorophores using the following Spectra Viewers.
Invitrogen Data Base
BD Fluorescence Spectrum Viewer
University of Arizona Data Base
NCI ETI Branch flow Cytometry
upright Zeiss microscopes, fluorescence tissue culture microscopes, timelapse microscopes
Wide-field image confocal image
Molecular probes test slide Nr 4, mouse intestine
3D reassembly possible
Careful: increasing image size (more pixels) does not mean that the objective can resolve the same!!! (resolution determined by NA, a property of the objective)
You can do timelapse movies with the confocal.
Mainly for fast processes
Be aware that not all our confocals have incubation chamber and CO2!
Two Leica confocals and one Olympus FV 1000
Excitation: long wavelength (low energy)
Each photon gives ½ the required energy
Emission: shorter wavelength (higher energy) than excitation
Use for deep tissue imaging
new La Vision microscope (live mouse imaging, will be installed in the new building)
FRET situation: Excitation of the donor (GFP) but emission comes from the acceptor (RFP)
Energy transfer, no emission!
ways to measure:
Detect the emission of the acceptor after excitation of the donor, e.g. excite GFP with 488 but detect RFP at 610 (GFP emission at 520)
(with overlapping excitation/emission
Excitation of many electrons at the same time count the different times when they are falling back down (i.e. photons are emitted)
lifetime = ½ of all electrons are fallen back
fused GFP-RFP expressed in COS 1 cell: average lifetime of 2108 psExample of FLIM-FRET measurement
Joan Grindlay, R7
You still need: a suitable FRET-pair with the right orientation of the π-orbitals
Interaction of proteins is not enough, because fluorophores have to be close enough and in the right orientation!
Use of FLIM:measurements of concentration changes (Ca2+), pH change etc, Protein interactions
FRET: Leica confocal 2 or Olympus FV 1000
FLIM: Leica confocal 1 and soon LIFA system from Lambert Instruments
Olympus FV 1000FRAP (Fluorescence Recovery After Photobleaching)
Pictures taken from a activation movie: activation of a line trough the lamellipodia of the cell, activated GFP_F diffuses quickly
Olympus FV 1000