Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells
ABSTRACT 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.
- SourceAvailable from: Peter Kner[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 abstract] [Hide abstract]
ABSTRACT: Most structured illumination microscopes use a physical or syn-thetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. In this study, we designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination pat-terns of arbitrary orientation and period, limited only by the illumination aperture (NA = 1.45), the response time of a fast, piezo-driven tip-tilt mirror (10 ms) and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrate the performance of our instrument by imaging mitochondrial dynamics in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope provides a versatile, compact and cost-effective arrangement for super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles - important questions in both cell biology and neurophysiology.Optics Express 11/2013; 21(22):26162-73. DOI:10.1364/OE.21.026162 · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The ability to image beyond the diffraction limit is the central tenet of the burgeoning field of superresolution fluorescence microscopy, also referred to as optical nanoscopy. The advent of superresolution has revolutionized biological fluorescence microscopy and the field at large. However, much of that excitement has been tempered by prohibitive imaging requirements. Achieving superresolution entails certain sacrifices, namely imaging speed, choice of fluorophore, ease of multicolor and three-dimensional imaging, and generally more complex instrumentation as compared to standard widefield imaging techniques. Several techniques utilizing structured illumination occupy an intriguing middle ground between the ease of use associated with traditional fluorescence microscopies and the unprecedented resolving power of modern superresolution methods, resulting in undeniably robust imaging techniques. Presented here is a review of the conceptual basis of structured illumination and its implementation, including its performance in comparison to other nanoscopies and the most recent developments in the field.ChemPhysChem 02/2014; 15(4). DOI:10.1002/cphc.201301086 · 3.36 Impact Factor