Single molecule light field microscopy

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.