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

Harvard-Massachusetts Institutes of Technology, Division of Health Sciences and Technology, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Biophysical Journal (Impact Factor: 3.97). 10/2007; 93(5):1747-57. DOI: 10.1529/biophysj.106.097907
Source: PubMed


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.

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    • "Total Internal Reflection Fluorescence (TIRF) microscopy provides an extremely thin emitting region, which can be treated as 2D for SIM purposes. SIM has already been used in TIRF [9] [10] [11], but not for time series imaging of live samples. Here we demonstrate live TIRF SIM at 100 nm resolution, with 3.7 to 11 Hz frame rates over fields of view of 32×32 to 8×8 µm. "
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