Optical-Scanning Isotropic-Resolution Imaging System – OSIRIS

The optical transmission microscope is the tool of choice for studying biological specimens, thanks to its unique properties for imaging living specimens, in 3-D, possibly over long periods. This explains that in recent years, tremendous efforts have been dedicated to inventing new techniques, to improve the resolution, or the speed of acquisition for example.

One of the limitations in optical transmission microscopy or in fluorescence microscopy is the non-isotropic resolution of the instrument, which limits the quality of the images of small 3-D specimens.

In order to obtain an isotropic resolution, several approaches have been proposed, like 3-D structured illumination or STED microscopy for example. Some are even commercially available. But in their present implementations, while improving the resolution in 2D (STED, PALM/STORM) or in 3D (structured illumination), none permit to obtain an isotropic and improved resolution. The reason is that STED 3D is not yet implemented commercially, and structured illumination or PALM/STORM, while delivering an unsurpassed resolution, require bulky, two-objective setups to deliver an isotropic resolution.

For specimens requiring an isotropic imaging, but not with a resolution in the 10s of nanometers like STED or PALM/STORM, we propose to study an alternate approach, which consists in combining adapted image processing with an adequate specimen micromanipulation, in order to deliver a 3-D, isotropic resolution in the sub-100nm range.

The technique can be applied to fluorescence microscopy, or to tomographic diffractive microscopy, a recently developed approach, which permit to image in 3-D non-labelled specimens. The proposed methods could for example be easily applied to free standing specimens, like red or white blood cells, pollens, diatoms or radiolares or even small organisms like C-elegans, or artificially manufactured 3-D objects like micro-needles, photonic bandgap optical fibres, calibration microbeads, microdropplets, microcristals, micro- and nano fibers,, combustion residue particles, and in general to man-made, fluorescent and/or transparent or semi-transparent micro- and nanoobjects.

The proposed technique combines specimen rotation with specific reconstruction algorithms to combine multiple view of the same object into an improved-, isotropic-resolution image. The goal is to deliver isotropic resolution in the 100nm range in 3-D by a synthetic aperture process using coherent light imaging for transparent objects, and by multikernel deconvolution for fluorescent specimens. The specimen rotation device is common to both imaging modes, so that multimodal imaging under the same microscope will be achievable with the proposed device.