Fluorescence microscopy Flashcards
applications (3)
detection of proteins:
1- immunolabelling (antibodies- cells without expressing protein or RNAi) - western blot
2- GFP revolution (Aequoria vitoria) - live cell imaging (large molecule changes dynamics - controls: western blot, compare N- and C-, Ab, different tags, protein activity assay
detection of DNA and RNA: in situ hibridization (FISH)
detection of low copy number RNAs (ZZ)
microscopes (4) and photobleaching
wide field: less contrast, more spatial resolution (upright or inverted)
confocal laser scanning: apperture only allows light for the focal plane, optical sectioning (pixel by pixel, layer by layer) - slow, no camera
Spinning disk: multiple laser line, speed (2000 images/second) but limited area (50ms exposure time)
Light sheet: objective moves laser line up and down (optical sectioning), more speed, larger samples, less photoxicity and bleaching, sensitive - single cell detection
Fluorescence Recovery After Bleaching (FRAP): dynamics and binding of proteins (immobile fraction vs mobile fraction)
difraction limited microscopes
Resolution
Rayleigh (x,y) = 0,61y/ NA (numerical aperture of objective)
different colour resolutions (y) - blue < green < red
find equilibrium between resolution and damage
How to improve resolution? formula
Foster Resonance Energy Transfer (FRET): when molecules are less than 10nm from each other energy is transfered with less energy and damage but also bleaches
r= 6\/(1/FRET eff-1) x R0
FRET eff= ((Dpost - Dpre)/ Dpost) / (1- Apost)/ Apre
R0 - foster radius with 50% transfer efficiency
CLEM (correlative light and EM)
Nanoscopy/super resolution
STED: fluorescence supressed by emission, electrons forced to ground state without emitting light only in the centre spot (~30nm)
Localization: dark state, don´t emit light or just a few with a short light pulse
MINIFLUX: STED and dark state
Super resolution radial fluctuations (SRRF)
Advantages (5) and Disadvantages (4)
+
localization of proteins, organelles, DNA, RNA
very sensitive (single molecules)
macro to nano
in vivo
many different techniques and imaging platforms
-
fluorescent label
damaging
time
quantitative imaging challenging
