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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Available from: Euiheon Chung, Oct 08, 2015
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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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    ABSTRACT: Linear Structured Illumination is a powerful technique for increasing the resolution of a fluorescence microscope by a factor of two beyond the diffraction limit. Previously this technique has only been used to image fixed samples because the implementation, using a mechanically rotated fused silica grating, was too slow. Here we describe a microscope design, using a ferroelectric spatial light modulator to structure the illumination light, capable of linear structured illumination at frame rates up to 11Hz. We show live imaging of GFP labeled Tubulin and Kinesin in Drosophila S2 cells.
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    ABSTRACT: A novel scheme for two-dimensional (2D) standing wave fluorescence microscopy (SWFM) using acousto-optic deflectors (AODs) is proposed. Two laser beams were coupled into an inverted microscope and focused at the back focal plane of the objective lens. The position of each of two beams at the back focal plane was controlled by a pair of AODs. This resulted in two collimated beams that interfered in the focal plane, creating a lateral periodic excitation pattern with variable spacing and orientation. The phase of the standing wave pattern was controlled by phase delay between two RF sinusoidal signals driving the AODs. Nine SW patterns of three different orientations about the optical axis and three different phases were generated. The excitation of the specimen using these patterns will result in a SWFM image with enhanced 2D lateral resolution with a nearly isotropic effective point-spread function. Rotation of the SW pattern relative to specimen and varying the SW phase do not involve any mechanical movements and are only limited by the time required for the acoustic wave to fill the aperture of AOD. The resulting total acquisition time can be as short as 100 µs and is only further limited by speed and sensitivity of the employed CCD camera. Therefore, this 2D SWFM can provide a real time imaging of subresolution processes such as docking and fusion of synaptic vesicles. In addition, the combination of 2D SWFM with variable angle total internal reflection (TIR) can extend this scheme to fast microscopy with enhanced three-dimensional (3D) resolution.
    Proceedings of SPIE - The International Society for Optical Engineering 01/2008; DOI:10.1117/12.761891 · 0.20 Impact Factor
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    ABSTRACT: Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.
    Biophysical Journal 07/2008; 94(12):4957-70. DOI:10.1529/biophysj.107.120345 · 3.97 Impact Factor
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