Fluorescence Lifetime Imaging in TIRF Microscopy
P. Blandin1,2, S. Lévêque-Fort1, F Druon2, M. Hanna2, V. Couderc3, P. Georges2, R.
Briandet4, Z. Lenkei5 and M.P. Fontaine-Aupart1
Laboratoire de Photophysique Moléculaire, UPR 3361 du CNRS, 91400 Orsay, France
Laboratoire Charles Fabry de l'Institut d'Optique, UMR 8501 du CNRS, 91400 Orsay, France
Xlim, Université de Limoges, UMR CNRS 6172, 87060 Limoges, France
Unité de Bioadhésion et Hygiène des Matériaux, INRA, 91300 Massy, France
Laboratoire de Neurobiologie, ESPCI - CNRS UMR 7637 75005 Paris, France
Keywords: FLIM, TIRF, bioadhesion, receptors
Total internal reflection fluorescence microscopy (TIRFM) is a powerful optical
technique to observe single molecule fluorescence at surfaces . Associated with
fluorescence lifetime imaging, TIRFM enables to measure contrasts independent of
fluorophores concentration and reveal probe environment (pH imaging, ion mapping,…) with
subwavelength axial resolution.
We develop a home made device based on an Olympus IX 71 in the through the objective
TIRF configuration. The entrance of the laser beam on the back aperture of the high
numerical aperture objective (Olympus TIRFM 1,45) is finely controlled to switch easily from
epifluorescence configuration to TIRF. For the excitation, we use a commercial tunable
pulsed laser with a low repetition rate (few tens MHz), which permits to observe lifetime until
several nanoseconds. A high rate imager (HRI Kentech) read by a cooled CCD camera (Orca
EG Hamamatsu) allows to record time gated fluorescence images to obtain FLIM map for
large field of view (typically 100µm x 100µm).
In order to measure longer lifetimes , like for Q-dots , we develop in parallel a lower
repetition rate (4 MHz) laser tunable in wavelengths in the visible range (400-800 nm). With
such a source, a large part of the commonly used fluorophores can be excited, and lifetime till
several tens nanoseconds can be measured. We thus develop a diode-pumped passively mode-
locked solid state laser, with a long cavity, injected in new photonic crystal fibers with
particular geometries to improve non linear effects.
After a complete characterization of the setup, we will demonstrate its capability to
tackle biological problems near an interface (adhesion mechanism, membrane traffic …).
One of the applications of such original device is the study of bacterial biofilm in flow cell.
The formation of biofilms, microbial communities attached to surfaces, is an extremely
common phenomenon associated with significant problems in medical, industrial and
environmental areas. TIRF gives access to the very first layers of the biofilm, and so permits,
associated with lifetime imaging, to apprehend their attachment processes to solid surface,
responsible of their persistence.
Neuropharmacology is another wide domain of application which can directly benefit of the
restricted depth of excitation field, as the events of the plasma membrane can be separated
from endosomal events. Previous studies show that sub-neuronal localization profoundly
modifies activation- induced trafficking of the type 1 cannabinoid receptor (CB1R) receptors.
Preliminary TIRF-FLIM images of CB1R in a physiologically relevant cellular context will
also be presented.
 D. Axelrod, Total internal reflection fluorescence microscopy in cell biology,
BIOPHOTONICS, PT B METHODS IN ENZYMOLOGY 361: 1-33 2003
 Leveque-Fort S, Papadopoulos DN, Forget S, Balembois F, Georges P, Fluorescence
lifetime imaging with a low-repetition-rate passively mode-locked diode-pumped Nd : YVO4
oscillator ,OPTICS LETTERS 30 (2): 168-170 JAN 15 2005