Plateforme Intégrée de Biophysique et de Biologie Structurale

Plateau de microscopie en champ proche (AFM)

Scientific manager : This email address is being protected from spambots. You need JavaScript enabled to view it.This email address is being protected from spambots. You need JavaScript enabled to view it.

Technical manager: This email address is being protected from spambots. You need JavaScript enabled to view it.

 

Instruments and techniques:

instrument1 instrument2 instrument3
 JPK Nanowizard 4 coupled to Super Resolution Fluorescence Microscopy
 
 JPK Nanowizard coupled to Epifluorescence Microscopy
 
 High-Speed Atomic Force Microscope
(Developed in collaboration with Pr.Toshio Ando)
 
instrument4 instrument5 instrument6
 Bruker Multimode Nanoscope VIII
 
 
 Bruker multimode Nanoscope III
 
 
 X-Ray-AFM that can be installed in Synchrotron X-Ray beamlines. Equiped with complete Nanonis SPECS control unit.

Atomic Force Microscopy (AFM):
The technique allows characterizing sample morphology at nanoscale. Images are acquired by scanning a nanometric tip in gentle contact or intermittent contact with the sample. The tip is positioned at the end of a micrometric force transducer, the AFM cantilever: it records the variation of sample topography due to changes of tip-sample interaction during scanning operation. In addition, by measuring the tip-sample interaction force as a function of the tip-sample distance, AFMs can evaluate sample elastic and viscous properties.

Objectives:

  • Characterize biological samples topography at nanoscale in dry or liquid environment. Image acquisition can be performed in static or dynamic modes.
cholera1  cholera2 

Cholera Toxin B-oligomers (left) bound to GM1 domains within a DOPC-DPPC (1:1) model membrane (right) as observed by AFM. Milhiet, Pierre Emmanuel, et al. "AFM characterization of model rafts in supported bilayers." Single molecules 2.2 (2001): 109-112.

 

  • Correlate biomolecules activity observed by Fluorescence (Epi and TIRF) or Super Resolution Microscopy (PALM/STORM) with the associated sample topology observed by AFM. Our two fluorescence-AFM correlative setups are mounted on top of Zeiss inverted microscopes. Fast AFM imaging (1 frame in few seconds) can be performed simultaneously with the fluorescence microscopy operation.

figure corrigee

Amphiphatic Lipid Packing Sensors (ALPS, left), and DOPE (Rhodamin marked, center) TIRF image correlated with the AFM image of the model membrane + biomolecules on the right. Scan size = 10 µm.

 

  • Real-time visualization of biomolecules activity at high spatial-temporal resolution by High-Speed AFM (HS-AFM). Our HS-AFM can acquire AFM images up to 10 images/seconds, allowing for dynamic tracking of slow biomolecules activity.

protofibril

Elongation of single protofibril (white arrows) is shown in time lapses. Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils Using High-Speed Atomic Force Microscopy. Milhiet, P. E., Yamamoto, D., Berthoumieu, O., Dosset, P., Le Grimellec, C., Verdier, J. M., ... & Ando, T. (2010). PLoS One, 5(10), e13240. Speed = 1 image/second.

 

  • Evaluation of mechanical properties of biomolecules and cells. Making use of the AFM tip as a nanometric indenter, we can characterize the extrinsic and, if possible, the intrinsic rigidity of materials with high spatial resolution.
DOPC1  DOPC2 

Left: DOPC-DPPC (1:1) model membrane morphology. Right: corresponding Young modulus evaluated modeling the tip-sample mechanical contact with the Hertz theory.

 

Software and analysis:


We make use of both commercial and entirely/partial custom-made software to control our instruments. Data analysis is performed both with C++/Matlab home-made programs as well as with open source software such as ImageJ and Gwyddion or software provided by manufacturers.

Connexion