Two-Dimensional Standing Wave Total Internal Reflection Fluorescence Microscopy: Superresolution Imaging of Single Molecular and Biological Specimens

Abstract

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or approximately 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.

Label-free super-resolution imaging of adenoviruses by submerged microsphere optical nanoscopy

“…Digital or computational holography has the advantage of acquiring high-contrast three-dimensional pictures of un-labeled cells. 27 The use of two-dimensional evanescent standing wave illumination has been shown by Chung et al 27 to improve the image resolution of a standard total internal reflection fluorescent optical microscope to approximately 100 nm for cell imaging. A continuous-wave 532-nm laser was used for the illumination.…”

Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution

“…For superresolution techniques, the acquisition time requirements become more stringent because the resolution length scale is much smaller, underscoring the need for development of both faster hardware and more photostable photoswitchable probes. To address the former concern, in recent years we and others (27,40) have developed methods for electronic pattern generation based on spatial light modulators. As yet, this technology has been used only for livecell linear SIM, but similar technology could be applied toward live-cell NL-SIM.…”

Overcoming the Resolution Limit Using Striped Patterns: Structured Illumination Microscopy

“…Standing wave-Total internal reflection fluorescence (SW-TIRF) [16,17] Harmonic excitation light microscopy-total internal reflection fluorescence (HELM-TIRF) [12] TIRF SIM [25] Nonlinear Structured illumination microscopy Deliberate saturation of fluorophores' excited state < 50 nm Even further resolution enhancement.…”