Odoo • Image et Texte

Phimask

powered by the SELFI technology 


Designed by Dr. Pierre Bon

"I have discovered Phimask by chance ! (as usual?)...
By the end of my PhD I was adjusting  in a very unconventional manner a grating-based interferometer normally adapted to white-light microscopy. I observed fringes when imaging an incoherent light source, where theoretically I shouldn't have observed anything. This made me realize that the interferometer could be adapted to fluorescence sources and 6 years after it was working on a single molecule based fluorescent microscope!"  

  Book one of the first Phimasks!




    EXAMPLES OF RESULTS

     APPLICATIONS

      TECHNOLOGY FEATURES

      METHODS

    PUBLICATIONS

  A 1rst TALK FOR THE PHIMASK COMMUNITY, WITH PIERRE BON

May 7, 2020. Pierre Bon shared his insights on Phimask before answering scientific questions from his research community. We also learnt about the best implementation of Phimask on a microscope, as well as the algorithm to extract the 3D position of each emitter. A great moment sharing scientific know-how together!

 
 

  EXAMPLES OF RESULTS

The phase-only diffraction grating designed for optimal super-resolution PSF sampling. Phimask allows 3D localization deep in tissue with a quasi-isotropic precision and limited photon loss (<20%).

Odoo • Image et Texte
How to build a SELFI-based interferometer using Phimask for fluorescence super-localization? (Up) The optical setup and expected point spread function (PSF) with SELFI. (Down) SELFI-PSF evolution long the z axis.
Image credit: extracted from Bon et al. "Self-interference 3D super-resolution microscopy for deep tissue investigations", Nature Methods, 2018
Phimask used with dSTORM for 3D super-resolution imaging. 3D super-resolved reconstruction using Phimask combined with dSTORM. 60x NA=1.3, Phalloidin-A647, human fibroblasts (WI-38).
Image credit: extracted from Bon et al. "Self-interference 3D super-resolution microscopy for deep tissue investigations", Nature Methods, 2018
Odoo • Image et Texte
Phimask used with PALM for 3D super-resolution imaging. 3D super-resolved imaging using Phimask combined with PALM. 60x NA=1.3, mEOS2 fused with Paxillin or Talin-C, mouse embryonic fibroblasts (MEF).
Image credit: extracted from Linarès-Loyez et al. "Self-Interference (SELFI) Microscopy for Live Super-Resolution Imaging
and Single Particle Tracking in 3D", Frontiers in Physics, 2019

  APPLICATIONS

Domains:

  • Deep 3D localization fluorescence microscopy
  • Compact intererometry adapted to fluorescence
  • Tracking of fluorescent particles in 3D

Techniques:

  • Any optical microscopy techniques capable of furnishing single fluorescent objects

  • dSTORM

  • PALM

  • Single particle imaging

  • (u)PAINT

  TECHNOLOGY FEATURES

Adapted to any microscope: The Phimask is inserted between the microscope image plane and the camera. It is thus compatible with any kind of fluorescent microscope.

Quasi-isotropic single-shot 3D localization: In one frame, one can extract the 3D localization of a single particle with a quasi-isotropic precision.

Very limited photon loss: Phimask is capable of generating interferences with limited photon loss (<20%).

A non degraded lateral 2D resolution: Since the PSF has very limited broadening and almost all photons contribute to the image, the lateral resolution is not degraded.

  METHODS

Protocol of use  

  • Adding the Phimask on the experiment:
    The PhiMask has to be placed a in front of the sensor of the camera you want to use. If the sensor is mechanically inaccessible, you can use an imaging relay.
    The Phimask produces interferences (fringes) which are sampled by your camera. The fringes carry the particule localization in 3D.

  • Retrieving the 3D localization:
    By using fluorescent smaller than the resolution of the microscope (e.g. 100 nm beads for a NA=1.4 microscope objective) and a z-axis motorized microscope, it is possible to perform a calibration of the SELFI-PSF along the z axis. This calibration can be used as a look-up-table to determine the axial z localization of each emitter during an experiment.
    The lateral localization can be obtained by applying a low-pass filter to the image to remove the fringe modulation. This leads to the signal envelope which is exactly the same as recording the image with the PhiMask; regular state-fo-the-art 2D localization algorithms can be applied (ex. 2D Gaussian fitting, Wavelets...).

  PUBLICATIONS

Linarès-Loyez J, Ferreira JS, Rossier O, Lounis B, Giannone G, Groc L, Cognet L and Bon P. Self-Interference (SELFI) Microscopy for Live Super-Resolution Imaging and Single Particle Tracking in 3D. Front Phys. 2019 May;7:68. https://www.frontiersin.org/articles/10.3389/fphy.2019.00068/full

Bon P, Linarès-Loyez J, Feyeux M, Alessandri K, Lounis B, Nassoy P, Cognet L. Self-interference 3D super-resolution microscopy for deep tissue investigations. Nat Methods. 2018 Jun;15(6):449-454. https://www.nature.com/articles/s41592-018-0005-3

Kellermayer B, Ferreira JS, Dupuis J, Levet F, Grillo-Bosch D, Bard L, Linarès-Loyez J, Bouchet D, Choquet D, Rusakov DA, Bon P, Sibarita JB, Cognet L, Sainlos M, Carvalho AL, Groc L. Differential Nanoscale Topography and Functional Role of GluN2-NMDA Receptor Subtypes at Glutamatergic Synapses. Neuron. 2018 Oct;10;100(1):106-119.e7. https://www.cell.com/neuron/fulltext/S0896-6273(18)30785-2?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0896627318307852%3Fshowall%3Dtrue