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.
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.
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.
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