Overview
Phimask is a super resolution imaging tool for turning a 2D SMLM setup into a 3D image.
This phase mask is meant for bioimaging analysis to track native cellular processes in intact tissues using phase encoded-positioning.
It can be adapted to any microscope.
Visit our
glossary
for a definition of super-resolution microscopy.
A technology designed by
Pierre Bon
and p
ublished in
Nature Methods
Images
Protocol
Adding 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...).
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)
Talk by 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!
Story
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 webinar proposal - and therefore Pierre stands as our first ever talk guest!"
