In vivo dual-color high resolution SIM of optically thick samples with background subtraction

International audienceOne of the main advantages of Structured Illumination Microscopy (SIM) is its ability to provide enhanced spatial resolution within a full-field approach, while requiring only low illumination and reduced acquisition of data and still providing good temporal resolution. Although the resolution enhancement achieved with SR-SIM cannot rival that of scanning or point-by-point approaches, SIM stands as a technique of choice for imaging dynamic processes in live samples. However its current use for biological applications still requires major technical improvements in order to combine lateral super resolution with video rate imaging and optical sectioning in living samples. We present a structured illuminated microscope by fringe projection together with an original and efficient reconstruction algorithm that only requires four acquired images (instead of 9) to obtain a super resolution one. But this high acquisition speed can only be reached when observing two-dimensional (thin) samples. Indeed when observing thick samples, the out offocus background limits the visibility both of the in focus fringes and of the resulting Moiré effect, thus hindering the reconstruction process. It is thus highly desirable, when imaging thick samples, to remove the out-of focus signal from the raw SR-SIM images. We have already demonstrated that by using our reconstruction algorithm and a sliding recombination of the raw images, it is possible to create single-color super resolution movies with a quarterof the information renewed in each reconstructed image. Here we present an extension of this method to combine high speed SIM with direct optical sectioning allowing imaging of in depth phenomena inside thick samples in two colors. This new approach allows making dynamic SIM movies of dual-labelled live cells with high temporal resolution