Single molecule light field microscopy
Ruth R. Sims, Sohaib Abdul Rehman, Martin O. Lenz, Sarah I. Benaissa, Ezra Bruggeman, Adam J. Clark, Edward W. Sanders, Aleks Ponjavic, Leila Mureşan, Steven F. Lee, Kevin O’Holleran
Abstract
We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views, in combination with a bespoke fitting algorithm, enable the 3D positions of single fluorophores to be determined from parallax. SMLFM achieves up to 20 nm localization precision throughout an extended <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"> <mml:mn>6</mml:mn> <mml:mspace width="thinmathspace"/> <mml:mspace width="thinmathspace"/> <mml:mtext>µ</mml:mtext> <mml:mrow class="MJX-TeXAtom-ORD"> <mml:mi mathvariant="normal">m</mml:mi> </mml:mrow> </mml:math> depth of field. The capabilities of SMLFM are showcased by imaging membranes of fixed eukaryotic cells and DNA nanostructures below the optical diffraction limit.