Structure, dynamique et fonction des biomolécules par RMN

Single-molecule Fluorescence


Photo-Activable Localization Microscopy (PALM/ STORM)

Associated to the group of Marcelo Nollmann

The image that a point source makes on a camera is called the point-spread function (PSF). The PSF is limited by diffraction to be no less than approximately half the wavelength of light. Until very recently, the spread of the PSF defined the maximum resolution attainable by conventional fluorescence microscopy.

PALM and dSTORM are imaging techniques achieving resolution well below the diffraction limit, the 2D resolution being usually between 30 and 50nm (see Betzig et al. 2006 – Hess et al. 2006 – Gould et al. 2009). These techniques rely on the fact the position of single fluorescent emitters can be calculated very precisely by measuring the shape of their PSF, the localization error being inversely proportional to the square-root of the number of photons collected. Super-resolution is achieved using fluorescent labels that are stochastically activated during the measurement. Therefore, at a given time, only a small subset of dyes is emitting, the majority remaining in a “dark” state. Single fluorescent events are detected and their mean positions calculated. After repeating this activation/emission/bleaching cycle many times, a reconstructed high-resolution image of the sample can be calculated using the position of all the molecules detected during the measurement.

PALM usually refers to super-resolution microscopy using photo-activable fluorescent proteins (PAFP) such as mEos2 or Dronpa (see Lippincott-Schwartz et al. 2009 - Fernández-Suárez et al. 2009). PALM is particularly well suited for in vivo imaging. However, the preparation of a fusion between the protein of interest and a PAFP is required.

On the other hand, dSTORM uses synthetic dyes such as Cy5 or Atto647 (Heilemann et al. 2009 – van de Linde et al. 2011). In that case, the activation relies on the formation of a complex between the dye and thiolated compounds, sending the fluorophore into a dark state. These techniques are very well suited for in-vitro experiments or with fixed cells. Immunostaining and specific switching buffers are necessary for this technique.


Applications and developments

Our PALM microscope allows for the investigation of assembly and dynamical movement of fluorescently labeled protein complexes in fixed or live cells such as Bacillus subtilis or Drosophila melanogaster.

Our setup

Inverted microscope Zeiss with a 100x oil-immersion objective (NA = 1.45). Four wavelengths of excitation and activation are available: 641nm (100mW), 561nm (100mW), 488nm (50mW) and 405nm (50mW). Both Epifluorescence and TIRF illuminations are possible on the setup. The focus is controlled in real time by Back Focal Plan Reflection of a 785nm IR laser. Below is a list of the filters available on the setup:

  • 525 +/- 25nm
  • 600 +/- 25nm
  • 700 +/- 37nm

New Developments

New developments are in course to actively stabilize our PALM microscop, adapt it for 3D-PALM, and concentrate the excitation light onto the area of interest to reduce background excitation and increase the power density of the excitation beam. To this aim, we will control the shape and three-dimensional position of the excitation beam in the sample plane by using a spatial light modulator (SLM), a computer controlled optical device that allows for the phase and intensity modulation of the light beam. This device allows for the formation of arbitrary shapes in or out of the focal plane and will permit us to excite an arbitrary region of the cell body and thus considerably reduce background fluorescence.


MARS research facility and European Bio-Imaging initiative

technologies home research members

The CBS biophysics platform offers access to state-of-the art imaging technologies for quantifying protein interactions in live cells, or in vitro. In particular we specialize in fluorescence fluctuation and single molecules approaches for quantitative spatial and temporal quantification of protein structure, interactions and dynamics. These capacities are made available to scientific groups in the Montpellier area and elsewhere in France, Europe and the rest of the world, via a direct collaboration with the CBS research teams, via European Bio-Imaging (see below), or via the MARS research facility.

Indeed, the MARS research facility is a collaboration between the CBS and MRI and aims at offering to the scientific community a wide access to a selection of advanced microscopy technologies, generally not available commercially in the present form (namely super-resolution and fluctuation microscopies, see below). It offers access, training, and support to custom-made optical setups. For some selected projects, our research staff can also develop new approaches meeting your needs, on a collaborative basis.

All the resources described below, together with MRI's high-throughput cloning and microscopy facility, have been selected as a “proof of concept” site of the large-scale pan-European research infrastructure project “Eurobioimaging” (EBI).


MARS Scientific Committee: Catherine Royer, Edouard Bertrand, Emmanuel Margeat, Marcelo Nollmann, Pierre-Emmanuel Milhiet

FBI node coordinators: Catherine Royer, Edouard Bertrand

CBS Participating Scientists: Emmanuel Margeat, Marcelo Nollmann, Catherine Royer, Pierre-Emmanuel Milhiet

User Coordinator: Caroline Clerté (Caroline.clerte at Support and Research Staff : Jean Bernard Fiche, Nicole Lautredou, Patrice Dosset

Microscopy Methods available

These various methods allow for:

  • the determination of cellular structures at super-resolution (PALM/STORM, AFM)
  • the study of dynamics of biomolecules (SPT, FCS, SPT-PALM)
  • the quantification and spatial localisation of biomolecular interactions (N&B, FCCS, FLIM, PALM/STORM)
  • the determination of structures and conformational dynamics at angstrom-resolution (smFRET, AFM).

How to apply

MARS research platform - APPLY NOW !! -

Applicants are invited to fill an application form, that will be reviewed by the MARS scientific committee. After discussion with the applicants, the committee will decide whether the research project can be conducted directly on the MARS platform, whether it implies a close collaboration with the CBS for technical developments, or whether it is out of the scope of the MARS research platform.
For more information, please contact our user coordinator Caroline Clerté (Caroline.clerte at
For microscope reservation, click here
Any reservation on the Mars resources needs to be approved by Caroline Clerte

Via Eurobioimaging

From January to July 2012, Euro-BioImaging conducts a series of proof-of-concept studies and therefore offers free access to European advanced biological and biomedical imaging facilities. Proposals will be evaluated by a panel of reviewers composed by experts of the Euro-BioImaging consortium covering the different imaging technologies and the responsible heads of the participating imaging facilities. Please apply directly on the Eurobiomaging website. Our platform is Facility n°38

Direct collaboration with the CBS teams

Please contact directly Caroline Clerté (Caroline.clerte at, our user coordinator.

How to reach us

To get to CBS/MARS, go here.

Fluctuation microscopy

2 photon excitation Fluorescence Correlation Spectroscopy Microscopies

Associated to the group of Cathy Royer

1- Fluorescence Correlation and Cross Correlation Spectroscopy (FCS & FCCS)

2- Raster Imaging Correlation Spectroscopy (RICS)

3- Number and Brightness analysis (N&B)

and give information in vitro and in vivo on:
  • Diffusion Coefficients
  • Molecular Concentrations
  • Molecular interactions.
  • Processes of diffusion and binding
The specific technique of choice depends on the time-scale of the dynamics, the constraints of the sample systems, and the specific questions of interest.

Our setup

  • Two photon excitation source (Spectra Physics Mai Tai HP) femtosecond tunable IR laser (from 700-1020 nm).
  • Galvo mirrors for laser Raster Scanning imaging.
  • Zeiss Axiovert 200 motorized microscope.
  • XY Piezo electric microscope stage.
  • Dual Channel ISS Alba Fluorescence correlation detector for FCCS.
  • Dichroic filters and band pass filters wheel:
|position |dichroic |filter CH1 |filter CH2| |1 |Empty |Empty |Empty| |2 |Empty |525/70m-2p |500/100| |3 |580 LP |675/50 |Empty| |4 |565 LP |653/95 |525/70m-2p| |5 |565 LP |610/75 |Empty| Others dichroic and filters available: 565 LP//empty// 525/50; 630 LP//empty// 585/40 and 505dcxr//empty//HQ455/100.

Further information on the different FCS techniques

2 Photons excitation provides both high axial resolution and extremely small detection volume, on the order of 0.1 fL. Therefore, 2 PE microscopy ensures that no photo damage happen outside the excitation volume but also a better spectral separation and the possibility to excite 2 distinct fluorophores.

Fluorescence Correlation Spectroscopy (FCS) is based on the observation of the amplitude and speed of fluorescence fluctuations with time occurring in the small excitation volume. The FCS experiments are performed on samples at nanomolar range concentrations so that very few fluorescent molecules diffuse in and out of the laser beam giving rise to important fluctuations. The rate of fluctuation depends on the rate of fluorophore diffusion and the height of the curve is inversely proportional to the average number of fluorophores being observed. 

Fluorescence Cross-Correlation Spectroscopy (FCCS) is based on detection of diffusing molecule labeled with 2 different colors. If a Green molecule diffuses into the volume excitation there is a burst of photons in the Green channel, and similarly for a Red molecule into the Red channel. If these two molecules interact with each other, they will co-diffuse into the excitation volume and a burst of Green & Red photons is observed in both channels at the same time.  

Scanning FCS techniques: Point FCS experiments often come with photo-bleaching problems of the sample and can damage the cells. Therefore, different scanning FCS techniques have been developed to avoid damaging samples. Scanning FCS is a powerful method for accurate measurements of slower diffusion and binding events with notable advantages: i) Less photo damage of the cells; ii) Multiple points FCS simultaneously; iv) Distinguish moving from immobile fraction; v) Spatial cross correlation. Among these scanning FCS techniques we offer Circular Scanning FCS, Raster Imaging Correlation Spectroscopy (RICS) and Number & Brightness (N&B)

RICS was developed by Digman M. and Gratton E. You can refer to the PDF file here. RICS extract information about the molecular diffusion and concentrations from raster scan images of living cells. As the laser performs the raster scanning movement, it creates a space-time matrix of pixels within the image. Therefore, the images contain information on the microsecond time-scale for pixel to pixel on the horizontal scanning axis, millisecond time scale along the vertical scanning axis and on the second time-scale between successive images. 

Number & Brightness : N&B was develop by Digman M. and Gratton E. You can refer to the PDF file here explaining the theory of N&B. (

The moment analysis is based on the determination of the average intensity (1st moment) and the variance (2nd moment) of the fluorescence intensity fluctuation within the raster scan images. The sampling time must be faster than any diffusion time of the studied system. Given 2 series of equal average fluorescence intensities, the larger is the variance, the less molecules contribute to the average (see figure below).

The analysis provides a map of number and a brightness for every pixel in the image.