Label-Free Chromatin-DNA Imaging by Circular Polarized Light Scattering Scanning Microscopy

Circular Dichroism (CD) resolved scanning microscopy has proven to be an interesting label free technique to study chiral biological systems. This technique is based on the measurement of the difference of intensity between circular left and right polarization states after interaction with the sample. It was demonstrated in early works that far from the absorption bands, the CD signal is attributed only to scattering effects and is thus referred to as Circular Intensity Differential Scattering (CIDS), which is sensitive to the higher-order chiral conformation of biopolymers.We have developed a scanning microscope based on the measurements of the CIDS signal, using a 50 kHz Photoelastic Modulator (PEM) and two channels detection via a lock-in amplifier. In this way, a differential image extracted from the Mueller Matrix formalism gives access to information at the single molecular level using a high-numerical aperture objective (Nikon 100X oil objective) in a time compatible with the scan time (a few seconds for a complete 512x512 image). As a proof of principle of the technique, the CIDS configuration has been coupled with a modified confocal scanning microscope allowing a multimodal acquisition of the CIDS and the fluorescence contrasts simultaneously. We have studied the chromatin-DNA organization of fixed isolated Hek-cell nuclei, which has shown our capability to distinguish the different order of compaction in DNA, i.e. the more compacted heterochromatin areas from the euchromatin areas.