IN VIVO DUAL-COLOR HIGH RESOLUTION SIM OF OPTICALLY

IN VIVO DUAL-COLOR HIGH RESOLUTION SIM OF OPTICALLY THICK
SAMPLES WITH BACKGROUND SUBTRACTION
Pierre Vermeulen1, François Orieux2, Jean-Christophe Olivo-Marin3, Hong Zhan4, Zsolt
Lenkei5, Vincent Loriette1, and Alexandra Fragola1
1: LP2N, CNRS UMR 5298, Université Bordeaux 1, 33405 Talence, France.
2: Institut Astrophysique de Paris, UMR 7095, 98 bvd Arago, 75014 Paris, France.
3: Unité d’analyse d’images biologiques, Institut Pasteur – CNRS URA 2582, 25, rue du
Docteur Roux 75015 Paris, France
4: Institut de Biologie de l’Ecole Normale Supérieure Paris, INSERM U1024, 46 rue
d’Ulm, 75005 Paris, France.
5: Laboratoire de neurobiologie, CNRS UMR 7637, ESPCI, 10 rue Vauquelin, 75005
Paris, France.
6: Laboratoire de physique et d’étude des matériaux, CNRS UMR 8213, ESPCI, 10 rue
Vauquelin, 75005 Paris, France.
KEY WORDS: super-resolution imaging, dual-color, live imaging, thick sample, structured
illumination microscopy
One 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 of
focus 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 quarter
of 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.