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Home page > Biophotonics Team > Physics/chemistry for imaging (...) > Advanced fluorescence microscopy

Development of advanced fluorescence-based instrumentation

P. Didier, L. Richert

Last update 20 April 2013

As part of our research activities, we develop and use advanced fluorescence-based instruments for spectroscopy and microscopy:
- Quantitative two-photon imaging
- Single molecule microscopy
- Sub-Diffraction Imaging
- Time-resolved fluorescence measurements
- Atomic Force Microscopy
- Peptide synthesis

Quantitative two-photon imaging

We have build-up a two-photon microscope that combines two photon imaging with quantitative microscopy modalities, such as FLIM and FCS

Two photon imaging offers several advantages compared to confocal microscopy, since it induces less photo-damage, it allows higher fluorescence collection efficiency and imaging of thicker samples (due to the better penetration of IR light as compared to visible light)

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Fluorescence Lifetime Imaging Microscopy (FLIM)

FLIM is a technique in which the fluorescence lifetimes of a chromophore are measured at each spatially resolvable element of a microscope image. Protein-protein interactions can be spatially resolved by monitoring the fluorescence lifetime of a donor in Fluorescence Resonance Energy Transfer (FRET) experiments. The major advantage of this technique is that the fluorescence lifetime is an intrinsic parameter that does not depend on the donor concentration and the excitation power of the laser.

Fluorescence Correlation Spectroscopy (FCS)

FCS is a technique that monitors the intensity fluctuations of fluorescent species that diffuse through a femtoliter volume (defined by the laser excitation). The diffusion constant, local concentration and molecular brightness, related to the hydrodynamic and photophysical properties of these species can be determined.

Two focus FCS

To determine with high accuracy the diffusion coefficient of fluorescent species, a two-Focus FCS geometry was built up, using a Michelson interferometer. The precise distance between the two foci allows overcoming the uncertainties relying on the determination of the size and shape of the confocal volume in single focus FCS.

Scanning FCS (sFCS)

With sFCS, the fluorescence signal is collected by scanning the laser beam on the sample. The fluorescence fluctuations contain both temporal and spatial information about the fluorescent species. Different sFCS modes are available depending on the requested parameters (circular FCS, RICS, N&B). The sFCS techniques are particularly suited for slowly diffusing species

[top Page]Single molecule microscopy

Single molecule microscopy provides unique information on heterogeneous populations of molecules by giving access to the complete distribution of observables, allowing discrimination between static and dynamic heterogeneity of their properties, and enabling the detection of rare events or succession of events hidden by ensemble averaging and the impossibility to synchronize molecules. We have build a set-up composed of a widefield microscope with TIRF illumination (Total Internal Reflection Fluorescence) coupled to an EMCCD to image single molecules.

[top Page]Sub-Diffraction Imaging

Until recently, optical microscopy was limited by the diffraction that limited the resolution to  200 nm in the lateral direction and 500 nm in the axial direction. . Recent advances allowed breaking the diffraction limits, introducing the extremely promising era of “nanoscopy”, with resolution ≤50 nm. Among the different nanoscopy techniques, we have developed two setups based on widefield and point scanning microscopy, respectively; which allow imaging nanoobjects with 40 nm resolution.


In the Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) methods, a few molecules are activated, imaged and then switched-off or photo-bleached. A sub-diffraction image can be reconstructed after determining with high accuracy the position of all individual molecules.


In the STED (Stimulated Emission Depletion) technique, the point spread function (PSF) is physically reduced by a doughnut shaped depletion laser that switches-off the fluorophores in the periphery of the excitation spot.

[top Page]Time-resolved fluorescence measurements

We have developed a set-up, based on Time-Correlated Single Photon Counting (TCSPC), working with a picosecond laser excitation and a microchannel plate photomultiplier. This set-up allows measurement of fluorescence lifetimes and rotational correlation times down to 30 ps.

Time-resolved intensity decays provide detailed information about the environment of the fluorophore, as well as about conformations of molecules and excited state reactions. These decays are also ideally suited to monitor resonance-energy transfer and dynamic quenching processes. Time-resolved anisotropy decays can be used to investigate the local and global (size dependent) rotations of a labeled species

[top Page]Atomic Force Microscopy

The laboratory is equipped with a commercial Scanning Probe Microscope (SPM) (NT-MDT solver PRO) that allows AFM measurements in dry and liquid phases. A specific module is also available for Scanning Tunnel Microscopy (STM).

A second setup combining AFM and TIRF microscopy was developed. The AFM microscope works in solid and liquid phase in contact and tapping mode. Correlation and analysis of AFM and fluorescence image are made post processing. STORM (Stochastic Optical Reconstruction Microscopy (STORM)) and SFT (Single Fluorophore Tracking) modes are available too.

Peptide synthesis

An ABI 433 (Applied Biosystem) synthesizer is used to perform solid phase peptide synthesis using the Fmoc method. The peptide size and quantity can be easily tuned with the type of resin used. Peptides ranging from 20 to 96 amino-acids have been synthesized (NCp7, Vpr and Tat from HIV-1, HCV Core protein, HBV capsid protein…) (12, 13). Labelling with non-natural amino-acids, commercial chromophores (fluorescein, rhodamine….) and chromophores synthesized in the laboratory (3-hydroxychromone derivatives) can be easily realized.


fluorescence, microscopy