Multicolor multifocus microscopy for multidimensional single molecule imaging

The ability to control the wavefront at the output of an imaging system allows to correct the imaging system optical aberration but also to impose a controlled deformation to encode specific information in the recorded images. Diffractive optical elements (DOE) provide an efficient mean to manipulate light. When placed in the back focal plane of the microscope objective, DOE were shown to generate a controlled PSF shape allowing to retrieve useful information such as the position and orientation of single molecules¹ [1].

In multifocus microscopy (MFM) for instance²³, a DOE is placed in the back focal plane. It plays two roles: first, it splits the emission into a specific array of equal-intensity diffraction orders, and second it applies a specific deformation of the wavefront in each diffraction order. The grating splits the emission into several paths while imposing a specific defocusing or focusing power to each. In MFM multiple imaging planes are thus formed side by side on the same camera allowing a fast and sensitive volumetric imaging of biological samples. Still the wavelength-dependent diffraction properties of DOE can limit the use of MFM for multicolor applications⁴. The purpose of this internship is to explore new methods for the realization of achromatic DOE⁵. In addition, the use of DOE as a mean to detect and identify different fluorescent species with distinct spectral properties will be explored. Such developments will be further tested for single molecule imaging in real biological model systems for validation.

Tasks:

• Simulate and
• Fabricate the DOE in a clean room
• Write the necessary algorithms for image reconstruction and analysis
• Validation on real samples

1. Pavani et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function, PNAS (2009) ↩︎
2. Abrahamson et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy Nature Methods (2013) ↩︎
3. Hajj et al. Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy, PNAS (2014) ↩︎
4. Hajj et al. Highly efficient multicolor multifocus microscopy by optimal design of diffraction binary gratings, Scientific Reports (2017) ↩︎
5. Orange-Kedem et al. 3D printable diffractive optical elements by liquid immersion, Nature Communications (2021) ↩︎