12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

MEASURING NUMBERS OF FLUOROPHORES LABELING cDNA IN SOLUTION,
WITH FLUORESCENCE CORRELATION SPECTROSCOPY AND CONTINUOUS
PHOTOBLEACHING.
Antoine Delon * , IrèneWang * , Emeline Lambert *** , Silva Mache *** , Régis Mache *** , Jacques
Derouard * , Vincent Motto-Ros ** , Rémi Galland *
* Univ. Grenoble I / CNRS, LSP, BP 87, 38402 Saint Martin d'Hères
** Univ. Lyon I / CNRS, LaSIM, 69622 Villeurbanne Cedex
*** Univ. Grenoble I / CNRS, PCV, BP 53, 38041 Saint Martin d'Hères
rgalland@spectro.ujf-genoble.fr
We developed a Total Internal Reflection Fluorescence Microscopy experiment in order to
detect very small amount of cDNA strand labeled with Alexa647 fluorophores. However the
interpretation of this kind of measurement needs to accurately characterize the labeling of the
cDNA strand, i.e. to know precisely the number of fluorescent bases per cDNA strand and
their brightness.
In the present study we present different approaches that aim at determining the brightness
and the number of Alexa647 molecules labeling the C bases of two sequences of cDNA,
corresponding to two transcripts of different sizes, a short and a long transcript (123 and 306
base long, with resp. 45 and 74 dCTP deoxynucleotides). In each case, the dCTP-Alexa647
labeled bases have been incorporated during reverse-transcription.
Two kinds of experiments have been performed and combined: continuous photobleaching
and Fluorescence Correlation Spectroscopy (together with the factorial cumulant analysis
method).
Continuous photobleaching measurement was realized in exciting small quantities of cDNA
strand confined in micrometric well under continuous illumination. As a result we show that
there is almost no interactions between fluorophores on the same cDNA strand and that the
photobleaching cross section of Alexa incorporated in cDNA strand is about half that of free
Alexa in aqueous solution.
On the other side, Fluorescence Correlation Spectroscopy measurement enable us to measure
the concentration, the diffusion constant and especially the brightness of the labeled cDNA
strand and the free Alexa. We thus observe that the cDNA strand brightness is about twice the
brightness of free Alexa providing information about the interplay between the radiative, non
radiative and photobleaching decay rate in relation with their photobleaching cross section.
We then studied the brightness and concentration evolution of labeled cDNA strand during
the solution photobleaching. Factorial cumulant analysis of the fluorescent fluctuations put
into evidence the fact that the brightness of cDNA strands is not uniform, due to the
distribution of the number of Alexa647 fluorophores labeling cDNA. We thus propose that
the number of fluorescent labels per cDNA follows a Poisson distribution, with a mean value
of about 2 for the long cDNA strands.