fluorescence fluctuation spectroscopy – a tool for the detection of nanometer sized particles in...
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Fluorescence Fluctuation Spectroscopy –
A tool for the detection of nanometer sized
particles in living cells
Michael Edetsberger
Max F. Perutz Laboratories, Department for Biomolecular
Structural Chemistry, University of Vienna
Introduction
Nanoparticles are extensively used in biotechnology
and medicine
Nanoparticles originated from industrial or combustion
processes
Nanoparticles play an important role in environmental
biology, job safety and medicine
Standard techniques
Microscopy (Fluorescence, Laser Scanning)
Good spatial distribution
Diffraction limited
No information about dynamics and concentration
Electron Microscopy (TEM, EELS)
Good spatial distribution
Not diffraction limited
Cells have to be fixed and extensively stained
No information about dynamics and concentration
Outline
Fluorescence Fluctuation Spectroscopy (FFS)
Technical set up and physical models
Simulations to demonstrate the principle of FFS
Translocation of fluorescent 20nm particles
Laser Scanning Microscopy
Fluorescence Fluctuation Spectroscopy
Model for translocation
Confocal set up for Fluorescence
Fluctuation Spectroscopy
Confocal set up for Fluorescence
Fluctuation Spectroscopy
Fluorescence Fluctuation
Spectroscopy (FFS)
Fluorescence Correlation Spectroscopy (FCS):
length and number of bursts
shape of the Correlation curve
Photon Counting Histogram(PCH):
intensity and number of bursts
shape of the PCH curve
Principles
Auto Correlation FunctionPhoton Counting Histogram
• hydrodynamic size• concentration
• specific brightness• concentration
Individual diffusion – different size and
equal brightness
or ormean intensity
Combined diffusion – different size and
equal brightness
mean intensity
black and red together
double concentrated dye (black)single concentrated
Individual diffusion – equal size and
different brightness
mean intensity
oror
Combined diffusion – equal size and
different brightness
mean intensityincreases
black and red togetherdouble concentratedcarriers
Convolutions
species 1 (1kHz and 270nM)
species 2 (700kHz and 2.7nM)
species 3 (1700kHz and 0.9 nM)
convolutions
measurements in the cytoplasm
of a native HeLa cell
Information obtainable by Fluorescence
Fluctuation Spectroscopy
Equally bright species can be differentiated by their diffusion
time and their concentrations can be estimated by the
amplitude-fraction of the Auto Correlation Function
Species of different brightness can be differentiated only by
their diffusion times but no estimation of their concentration
is possible
Only PCH gives the possibility to extract information about
concentration and brightness
Results
Incubation of HeLa cells with 20nm
green fluorescent negatively charged
polystyrene particles
Laser Scanning Images
after 20 minutes incubation
native HeLa cell Genistein treated HeLa cell
Fluorescence Correlation
Spectroscopy (1)
Fluorescence Correlation
Spectroscopy (2)
Fluorescence Intensity
Distribution Analysis (1)
1 minute after adding the particles
5 minutes after adding the particles
15 minutes after adding the particles
30 minutes after adding the particles
60 minutes after adding the particles
Fluorescence Intensity
Distribution Analysis (2)
Summary
Isolated particles are detected with a very short time-delay
Particles are detected in the cytoplasm or the nucleus
whether the cell is native or not
Bigger and brighter particles are detected with a time-delay
exclusively in the cytoplasm of native HeLa cells
Aggregates range from 200 to 600nm and suggest a 5-20%
sphere packing
Model for translocation
Thank you
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