Measuring single molecules’ 3D orientations simultaneously with their 3D spatial localization is currently a topic of intense efforts in optical design. We have developed different methods based on polarized imaging, that are capable to report both 3D spatial localization and 3D orientation (including wobbling) from single molecules. We present how the design of such schemes can be adapted to account from experimental constraints versus accuracy and precision of the estimation of both orientation and localization parameters. We illustrate such performances on actin structural STORM imaging in complex and dense meshworks in fixed cells.
KEYWORDS: Modulation, Polarization, Real time imaging, Tissues, Luminescence, Chromium, Structural imaging, Signal to noise ratio, Second-harmonic generation, Raman spectroscopy
Polarized nonlinear scanning imaging using two photon fluorescence, harmonic generation or coherent Raman has been extensively developed to image molecular orientational organization in tissues. Current approaches using fast modulation (electro-optic) are however still limited to seconds time scales due to their required voltage shape modulation. To reach higher frequencies, we have developed an interferometric frequency shift-based method using Acousto-Optic Modulation. We present degree-precision performances in the retrieved information even at low signal to noise level and sub-second dynamics. The method is applied to real time actin structural imaging in cells as well as collagen and lipids organization imaging in tissues.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.