Abstract
We report how a recently developed polarization imaging technique, implementing micro-wave photonics and referred to as orthogonality-breaking (OB) imaging, can be adapted on a classical confocal fluorescence microscope, and is able to provide informative polarization images from a single scan of the cell sample. For instance, the comparison of the images of various cell lines at different cell-cycle stages obtained by OB polarization microscopy and fluorescence confocal images shows that an endogenous polarimetric contrast arizes with this instrument on compacted chromosomes during cell division.
Highlights
Over the last decades, the relentless development of novel optical imaging techniques addressing specific issues of the biology or biomedical community has given rise to a huge number of unconventional imaging techniques
The first implementations of this original approach on synthetic samples have permitted to confirm its interest, as OB polarimetric sensing offers a mean to access some specific polarization/anisotropy parameters and from a single measurement [23,24,25,26]. We report how such a technique has been implemented on a commercial fluorescence confocal microscope set up in order to provide the instrument with a complementary polarization imaging channel, and how we applied it to cellular imaging
The left column shows the transillumination image obtained with a ×10 objective from the measured DC signal of the avalanche photodiodes (APD), while the center and right column respectively display the orthogonality-breaking contrast (OBC) amplitude and phase maps obtained from Eq (2)
Summary
The relentless development of novel optical imaging techniques addressing specific issues of the biology or biomedical community has given rise to a huge number of unconventional imaging techniques Several of these were granted with worldwide commercial and scientific success such as Optical Coherence Tomography, advanced confocal fluorescence microscopy, non-linear microscopy, phase microscopy, etc. As for any fluorescence-based method, confocal microscopy requires the labeling of the sample using either fluorescent compounds displaying specific localizations whithin the cells, dyes coupled to antibodies or recombinant chimeric constructs composed of a fluorescent protein fused to a protein of interest. This labeling step has proven to be usable in multiple contexts, it suffers some limitations. Being able to monitor such cellular structures can prove useful to quickly identify proliferating cells within a biological sample or to identify cells displaying altered architecture of their genomic material, a feature that is shared by multiple tumor cells [27]
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