The brain extracellular space (ECS) is a complex network that constitutes a key microenvironment for cellular communication, homeostasis, and clearance of toxic metabolites1. Signaling molecules, neuromodulators, and nutrients transit via the ECS, therefore mediating the communication between cells. Despite the relevance of this important part of the brain, its dynamics and structural organization at the nanoscale is still mostly unknown2. We have recently demonstrated that single-walled carbon nanotubes (SWCNTs) can be used to image and probe live brain tissue, providing super-resolved maps of the brain ECS and quantitative information on the local diffusion environment3,4. Here, we propose an important refinement of this approach by implementing a structured illumination technique (named HiLo microscopy5) to image fluorescently labelled neuronal structures in parallel to SWCNT NIR imaging. This technique is based on speckle illumination and relies on the acquisition of one structured and one uniform illumination image to obtain images deep into tissues with good optical sectioning. Having access to spatially resolved SWCNT diffusivity around specific neuronal structures will provide more precise insights about the heterogeneity of the brain environment.
Co-design consists in optimizing an imaging system taking into account the model of image formation, the properties of the optics and the method of information extraction.
This methodology is used in biology to observe, with nanometric resolutions, single molecules using fluorescence microscope.
This super-resolution imaging technique makes it possible to estimate very accurately the position of an isolated fluorescent particle in the field of the camera beyond the diffraction limit.
Today, biologists try to image thicker and less transparent samples.
There is then, a need to extend depth-of-field of fluorescence microscope so that single-molecule location accuracy is as invariant as possible by defocalisation.
For several years, our team has co-designed phase masks placed in the pupil of a lens to extend its depth-of-field.
These masks produce a relatively blurred image, but its quality is independent of the axial position of the object.
A unique deconvolution process is then applied to reconstruct the objects at all depths.
Optimization of the phase function of the mask is therefore based on an image quality criterion after deconvolution.
We propose here to co-design these phase masks for a fluorescence microscope in order to generate a point-spread-function which location accuracy is invariant along the optical axis.
From a theoretical point of view, the originality lies in the fact that the optimization criterion is no longer the image quality after deconvolution but the localization accuracy of single-molecule in the plane.
Our mask optimization criterion is therefore based on Cramer-Rao lower bound and the associated single-molecule localization post-processing stage is designed such as its accuracy reaches this lower bound.
Our results validate this optimization methodology and show that optimal masks can significantly increase the depth of exploration of single-molecule imaging techniques.