Phimask

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%).

Features Protocol Story

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What is it for? Use it if turning your 2D SMLM single molecule localization microscopy images into 3D images is a fantasy for you.

Designed by Pierre Bon

Published in Nature Methods

Visit our glossary for a definition of super-resolution microscopy.

Features

Adapted to any excitation modality

Phimask is inserted between the microscope image plane and the camera, in the emission path

Compatible with multiple colors

Phimask is compatible with up to 3 colors by default in the 500 – 750 nm range

Quasi-isotropic single-shot 3D localization

Extract the 3D localization of a single particle in 1 frame, 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

Compatible with any optical microscopy technique

as long as it is capable of providing single fluorescent objects (dSTORM,PALM, single particle imaging, uPAINT)

Gallery

Talk

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!

Installation & image analysis protocol

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...).

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Phimask, Pierre Bon and us

"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!"

Contact Pierre Bon about his technology.

Pierre Bon

Esther Graudens
New projects at Idylle

"Our relationships with Pierre started even before Idylle was born! We were closely following his work and his papers. When we started our operations, it was just natural to invite him to get onboard. He kindly replied with enthusiasm and our story with Phimask began.
Since then, we have lived a few premieres together. We were lucky that Pierre is that kind of man who says yes first. We share the same love for new (sometimes crazy?) ideas. An example? He immediately jumped on our webi nar proposal - and therefore Pierre stands as our first ever talk guest!"

Contact us if you want to know more about the best way to set up Phimask on your microscope - or discuss your research project.

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