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