Abstract
Fluorescence imaging is used to study the dynamics of a wide range of single molecules in solution or attached to a surface. Two key challenges in this pursuit are to image immobilized single molecules in the presence of a high level of fluorescent background, and to image freely diffusing single molecules for long times. We present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The Convex Lens Induced Confinement (CLIC) system restricts molecules to a wedge-shaped gap of nanoscale depth. The shallow depth of the imaging volume leads to up to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. The elimination of out-of-plane diffusion leads to approximately 10,000-fold longer diffusion-limited observation time per molecule than is achieved with confocal detection in free solution. The restriction of freely diffusing molecules to gaps whose depth is greater than the molecular diameter provides a simple measure of molecular size. The CLIC system may be implemented using a minor modification to a standard flow cell, and does not require any nanofabrication, nor any custom optics, electronics, or computer control. We demonstrate the advantageous properties of CLIC measurements by imaging singly labeled surface-immobilized DNA molecules in the presence of a high concentration of free dye (up to 2 micromolar); by counting the transmembrane proteins in freely diffusing lipid vesicles; and by measuring the sizes of freely diffusing proteins and DNA with diameters ranging from 3nm to 200 nm. Further, we apply CLIC to probe weak intermolecular interactions in a range of systems.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.