Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution

Abstract

Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.super resolution ͉ moiré ͉ resolution extension ͉ saturation T he fluorescence microscope has become a ubiquitous imaging tool in cell biology through its unique ability to image the 3D interior of a living specimen with multicolor molecular labels of extreme specificity, a combination of strengths not shared by higher-resolution techniques such as electron microscopy and scanned-probe methods. It is therefore unfortunate that its spatial resolution is subject to a hard limit caused by diffraction.Recently, ways have been found to bypass the diffraction limit. 2D resolution in the 30-nm range has been realized by using stimulated emission depletion (STED) (1). STED is based on saturated stimulated emission using two synchronized ultrafast laser sources (2, 3); the underlying concept has been generalized to encompass a class of reversible saturable phenomena (4). STED and other proposed methods (5-7) were conceived in the context of laser-scanning microscopy and are designed to directly minimize the size of a scanned focal point. This article demonstrates an alternative approach that brings theoretically unlimited resolution to a wide-field (nonscanning) microscope by using a nonlinear fluorescence response together with a periodic illumination pattern that fills the field of view.Both structured illumination light and optical nonlinearity, of course, are established ideas. Patterned light, for example, has been used for measuring surface shapes (8) and deformations (9) and for enhancing the sensitivity of fluorescence-recovery-afterphotobleaching experiments (10). Axially structured light has been used to enhance axial resolution in standing-wave fluorescence microscopy (11), 4Pi microscopy (12), and I 5 M (13). Lukosz and Marchand (14) suggested in 1963 that lateral light patterns could be used to enhance resolution, and such patterns have been used for both axial (15) and lateral (16-19) resolution enhancement. They can be more effective than point scanning at retrieving high-resolution information (17,19). Nonlinear fluorescence is the basis for multiphoton fluorescence microscopy (20), and several other c...

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