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Zebrafish are powerful animal models for understanding biological processes and the molecular mechanisms involved in different human diseases. Advanced optical techniques based on fluorescence microscopy have become the main imaging method to characterize the development of these organisms at the microscopic level. However, the need for fluorescence probes and the consequent high light doses required to excite fluorophores can affect the biological process under observation including modification of metabolic function or phototoxicity. Here, without using any labels, we propose an implementation of a Mueller-matrix polarimeter into a commercial optical scanning microscope to characterize the polarimetric transformation of zebrafish preserved at different embryonic developmental stages. By combining the full polarimetric measurements with statistical analysis of the Lu and Chipman mathematical decomposition, we demonstrate that it is possible to quantify the structural changes of the biological organization of fixed zebrafish embryos and larvae at the cellular scale. This convenient implementation, with low light intensity requirement and cheap price, coupled with the quantitative nature of Mueller-matrix formalism, can pave the way for a better understanding of developmental biology, in which label-free techniques become a standard tool to study organisms.

Mueller matrix microscopy is an advanced imaging technique providing a full characterization of the optical polarization fingerprint of a sample. The Lu-Chipman (LC) decomposition, a method based on the modeling of elementary polarimetric arrangements and matrix inversions, is the gold standard to extract each polarimetric component separately. However, this models the optical system as a small number of discrete optical elements and requires a priori knowledge of the order in which these elements occur. In stratified media or when the ordering is not known, the interpretation of the LC decomposition becomes difficult. In this work, we propose a new, to our knowledge, representation dedicated to the study of biological tissues that combines Mueller matrix microscopy with a phasor approach. We demonstrate that this method provides an easier and direct interpretation of the retardance images in any birefringent material without the use of mathematical assumptions regarding the structure of the sample and yields comparable contrast to the LC decomposition. By validating this approach through numerical simulations, we demonstrate that it is able to give access to localized structural information, resulting in a simple determination of the birefringent parameters at the microscopic level. We apply our novel, to our knowledge, method to typical biological tissues that are of interest in the field of biomedical diagnosis.

Circular intensity differential scattering (CIDS) is based on the analysis of circular polarized light scattering and has been proven to be an interesting label-free microscopy technique sensitive to the chiral organization at the submicroscopic level. However, this approach averages the localized contrasts related to the sample polarimetric properties in the illumination volume. Additionally, the detection sensitivity suffers from the confinement of the mixture of structures, and it becomes an arduous task to discriminate the source of the signal. In this work, we show that a phasor map approach combined with CIDS microscopy has provided an intuitive view of the sample organization to recognize the presence of different molecular species in the illumination volume. The data represented in terms of polarization response mapped to a single point called a phasor also have the potential to pave the way for the analysis of large data sets. We validated this method by numerical simulations and compared the results with that of experimental data of optical devices of reference.

Expansion microscopy (ExM) is a novel preparation method enhancing the optical resolution by expanding uniformly the relative distance between fluorescence molecules on a sample placed inside a polymerized gel matrix. However, a skilled operator is needed for fluorescent labeling protocols and a high light dose is required for measurement. In this work, we couple ExM with a label-free differential circular polarization microscopy technique, demonstrated to be sensitive to the chiral organization of biopolymers. We show that by improving the distance between chiral groups, the new imaging contrast gives access to a better resolution of the chromatin-DNA organization in situ.

Circular Intensity Differential Scattering (CIDS) provides a differential measurement of the circular right and left polarized light and has been proven to be a gold standard label-free technique to study the molecular conformation of complex biopolymers, such as chromatin. In early works, it has been shown that the scattering component of the CIDS signal gives information from the long-range chiral organization on a scale down to 1/10th–1/20th of the excitation wavelength, leading to information related to the structure and orientation of biopolymers in situ at the nanoscale. In this paper, we review the typical methods and technologies employed for measuring this signal coming from complex macro-molecules ordering. Additionally, we include a general description of the experimental architectures employed for spectroscopic CIDS measurements, angular or spectral, and of the most recent advances in the field of optical imaging microscopy, allowing a visualization of the chromatin organization in situ.

Circular dichroism imaging has proved a powerful and simple method for extracting information on chiral molecules without specific fluorescent labels. Numerous mathematical models show that outside the absorption band, the circular dichroism signal comes from the scattering interaction and brings additional information about the organization of biopolymers. With this article, we propose a fast method to control the polarization states without moving parts, by means of a photoelastic modulator. We implemented the technique on a modified commercial confocal microscope realizing a multimodal configuration. We demonstrate its imaging capabilities by studying the organization of chromatin DNA inside isolated cell nuclei.