Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells

Rice University, Houston, Texas, United States
Journal of Biomedical Optics (Impact Factor: 2.86). 11/2006; 11(6):064013. DOI: 10.1117/1.2372457
Source: PubMed


We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

1 Follower
14 Reads
  • Source
    • "A standing wave embodiment of this technology has been Correspondence to: Pascal Vallotton. Tel: +61 (0)2 9325 3208; fax: +61 (0)2 9325 3200; e-mail: reported that allows improving the lateral resolution down to 100 nm (Gliko et al., 2006). Gordon et al. measure nanometre range distances between two single identical fluorophores by relying on the analysis of the Airy-like intensity patterns, before and subsequent to the photo-bleaching of one of the fluorophores (Gordon et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Considerable efforts have been deployed towards measuring molecular range distances in fluorescence microscopy. In the 1-10 nm range, Förster energy transfer microscopy is difficult to beat. Above 300 nm, conventional diffraction limited microscopy is suitable. We introduce a simple experimental technique that allows bridging the gap between those two resolution scales in both 2D and 3D with a resolution of about 20 nm. The method relies on a computational approach to accurately correct optical aberrations over the whole field of view. The method is differential because the probes of interest are affected in exactly the same manner by aberrations as are the reference probes used to construct the aberration deformation field. We expect that this technique will have significant implications for investigating structural and functional questions in bio-molecular sciences.
    Journal of Microscopy 12/2008; 232(2):235-9. DOI:10.1111/j.1365-2818.2008.02094.x · 2.33 Impact Factor
  • Source
    • "This makes it feasible to optimize the software suite to generate model morphologies for nonpyramidal neurons (although no modification was necessary for test cases of medium spiny neurons of the striatum). Furthermore, as advanced optical instruments are under continual development, the modifiable nature of ORION can adapt to expanded imaging capabilities (e.g., Gliko et al. 2006; Reddy et al. 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We have developed a fully automated procedure for extracting dendritic morphology from multiple three-dimensional image stacks produced by laser scanning microscopy. By eliminating human intervention, we ensure that the results are objective, quickly generated, and accurate. The software suite accounts for typical experimental conditions by reducing background noise, removing pipette artifacts, and aligning multiple overlapping image stacks. The output morphology is appropriate for simulation in compartmental simulation environments. In this report, we validate the utility of this procedure by comparing its performance on live neurons and test specimens with other fully and semiautomated reconstruction tools.
    Journal of Neurophysiology 09/2008; 100(4):2422-9. DOI:10.1152/jn.90627.2008 · 2.89 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
Show more

Olga Gliko