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Application Note

Absolute Diffusion Coefficients:Compilation of Reference Data for FCSCalibration

Peter Kapusta, PicoQuant GmbH, Jul !"#", Re$%#

Conventional FCS can provide absolute diffusionconstant and concentration values only when theexact shape and size of the confocal volume is

known. In practice, the shape is approximated bya 3 !aussian function. Size and elon!ation of thevolume is obtained by calibration measurement ofa reference compound with a known diffusionconstant.

In order to !et correct confocal volume para"meters, the experimental conditions durin!calibration #wavelen!ths, excitation power, cover

slip thickness, solvent and immersion medium$must be the same as durin! the measurement.

%recise diffusion coefficients of several referencecompounds suitable for calibration purposes aresummarized in the followin! table&

Fluorophore'(m

maximumin nm

iffusion coefficientin water at !&'C (!)*%#& K+

in )*+ cm-s")ethods and references

 /tto00"maleimid 12.* 4 *.)*2.* 4 *.*52.*5 4 *.*

-fFCS 6)7, %F8"9: 6)7-fFCS 62, 17pmFCS 627

 /tto00"carboxylic acid 10 2.- 4 *.*1 -fFCS 6),37, %F8"9: 6),37

 /tto00"9;S esther 10 2.-0 4 *.* -fFCS 617

Cy0 * 3. 4 *.) -fFCS 617

 /lexa 2 0 3.3 4 *.) -fFCS 617

 /lexa 33 2 3.2 4 *.) -fFCS 617

:hodamine 8 00*2.)2 4 *.*02.3 4 *.2

2.)2 4 *.*)

-fFCS 6), 17%F8"9: 67

%

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9ote that the diffusion coefficient is not a constant.It is temperature dependent accordin! to theStokes"(instein relationship,

 D T =  kT 

6T r 

where  D  is the diffusion coefficient, k   is the(.).**23--

637 ertin!er ?., %acheco ., von der ;ocht I., ;artmann :., 8re!or I., (nderlein D.& Two-focus fluorescence correlationspectroscopy: A new tool for accurate and absolute diffusion measurements.Chem%hysChem, * #-**$ 233, http&==dx.doi.or!=)*.)**-=cphc.-****31

627 Eorlann O., ertin!er ?., ichalet P., Jeiss S., (nderlein D.& Measuring diffusion with polarization-modulation dual-focusfluorescence correlation spectroscopy.>ptics (xpress, #- #-**1$ 2*5, http&==dx.doi.or!=)*.)32=>(.).*)2*5

607 ertin!er ?., (wers