Fast monitoring of in-vivo conformational changes in myosin using single scan polarization-SHG microscopy

Abstract

Fast imaging of molecular changes under high-resolution and label-free conditions are essential for understanding in-vivo processes, however, current techniques are not able to monitor such changes in real time. Polarization sensitive second harmonic generation (PSHG) imaging is a minimally invasive optical microscopy technique capable of quantifying molecular conformational changes occurring below the diffraction limit. Up to now, such information is generally retrieved by exciting the sample with different linear polarizations. This procedure requires the sample to remain static during measurements (from a few second to minutes), preventing the use of PSHG microscopy from studying moving samples or molecular dynamics in living organisms. Here we demonstrate an imaging method that is one order of magnitude faster than conventional PSHG. Based on circular polarization excitation and instantaneous polarimetry analysis of the second harmonic signal generated in the tissue, the method is able to instantaneously obtain molecular information within a pixel dwell time. As a consequence, a single scan is only required to retrieve all the information. This allowed us to perform PSHG imaging in moving C. elegans, monitoring myosin's dynamics during the muscular contraction and relaxation. Since the method provides images of the molecular state, an unprecedented global understanding of the muscles dynamics is possible by correlating changes in different regions of the sample.

Keywords: (180.4315) Nonlinear microscopy; (190.2620) Harmonic generation and mixing.

Fig. 1

Scheme of the experimental setup showing the excitation with circular polarization, the coordinates system used, the 3 PMTs detecting the SHG signal at 0°, 45° and 90° and an example of the generated images using starch.

Fig. 2

Results for a) starch (SHG active molecule is amylopectin), b) collagen, c) muscle (active molecule is myosin) and d) mitotic spindles (active molecule is α-β tubulin heterodimer). Every panel from a) to d) shows the three acquired images (i.e. from each of the PMTs) required for the SS-PSHG analysis (top row), the retrieved main axis orientation, anisotropy parameter and effective orientation of the nonlinear dipole of the macromolecule (center) and the corresponding image histograms (bottom)..

Fig. 3

SS-PSHG analysis of moving C. elegans worm. The analyzed region corresponds to the pharynx with the most posterior lobe (terminal bulb) at the bottom of the image, framed by the body walls (the two lateral lines). Eight consecutive frames at 1 frame per second, with a size of 500x500 pixels are shown. a) Total emitted SHG signal. b) Mapping of the thick filaments orientation in every pixel measured with respect the vertical axis. c) Mapping of the anisotropy parameter. Note the change in color in the last frame (red square) due to the contraction of the left worm body wall.

Fig. 4

Monitoring of muscular contraction and relaxation with SS-PSHG. The images (500x500 pixels) of the posterior lobe of a C. elegans worm waking up after anesthesia are analyzed for 30 seconds. a) The total detected SHG signal (left), the thick filament orientation (center) and the anisotropy parameter (right) with the two region of interest (ROI) are shown ( Media 1). b) Five frames for ROI 1 showing the changes on the thick filaments orientation (top) and anisotropy parameter (bottom). Note the change in color at second 4. c) Evolution of average values for the thick filaments orientation (top) and anisotropy parameter (bottom) in ROI 1 (left) and ROI 2 (right). Note that an increase on the anisotropy parameter is associated to a contraction process, while a decrease is associated to a relaxation process. Error-bars corresponds to standard deviation within the ROI.