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

NEAR-FIELD FCS: DRIVING BIOLOGY BEYOND THE DIFFRACTION LIMIT
Winckler P, Jaffiol R, Plain J and Royer P
Université de Technologie de Troyes, Institut Charles Delaunay FRE CNRS 2848,
Laboratoire de Nanotechnologie et d’Instrumentation Optique, LRC CEA,
12 rue Marie Curie, BP2060, 10010 Troyes cedex, France
contact: rodolphe.jaffiol@utt.fr
For single molecule detection in solution, the issue is to get the probability of finding molecule
in the observation volume significantly inferior to unity. This can be achieved through
diluting the dye of interest or limiting the detection to a very small volume. Classical fluorescence
correlation spectrosocpy (FCS), based on confocal detection, presents two important
limitations for in vivo studies. First, its requires to work with a nanomolar concentration of
molecules, very far from the biologically relevant concentration (i.e µM, or higher). On the
other hand, the background signal such as autofluorescence, is still often important. In order
to overcome the limitations of classical FCS, we propose a new approach to reduce the detection
volume based on a non radiative energy transfer. We present two applications of our
technique to make FCS and fluorescence imaging at the nanoscale, see Fig. 1.
(A)
(B)
Fig. 1. (A) Picture of adhesion areas between cells and substrate. Superposition of two
widefield images, one DIC picture in grey scale and one fluorescence picture in red scale.
(B) Typical autocorrelation curve recorded on an cell adhesion area.
Red curve, fit according to a pure 2-D diffusion of molecules.
12 th <strong>Zeiss</strong> <strong>sponsored</strong> FCS <strong>workshop</strong> – Cargèse, Corsica, France – <strong>12th</strong> -16th October 2009