Deciphering macromolecule organization in situ by means of multi-messenger optical microscopy.
Diaspro A., Le Gratiet A., Marongiu R., Bianchini P., Vicidomini G., Castello M., Piazza S., Tortarolo M., Bendandi A., Koho S., Sheppard C.J.R.
V - Biofisica e fisica medica
GSSI Ex ISEF - Aula B - Mercoledì 25 h 16:30 - 19:00
» Download abstract
Polarized light scanning microscopy has proven to be a label free approach to study the organization of macromolecules in biological systems. Polarization changes due to sample properties can be detected at different scattering angles. It was demonstrated in early works that differential scattering of circularly polarized light having opposite rotation, named as Circular Intensity Differential Scattering (CIDS), is sensitive to the properties of chiral conformation of biopolymers. We have developed a scanning microscope based on the measurements of the CIDS signal, using 50 kHz modulation of polarized states and detection channels coupled to a lock-in amplifier. Differential images computed by using the Mueller Matrix formalism give access to information at the single molecular level using a high-numerical aperture objective. As a proof of principle of the technique, the CIDS configuration has been coupled with a modified confocal laser scanning microscope allowing a multimodal acquisition of the CIDS and the DNA fluorescence signals demonstrating the capability to distinguish heterochromatin and euchromatin regions. The development of a spectroscopic image scanning approach was also shown.