Precise nanometer localization analysis for individual fluorescent probes
TL;DR: A localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations), and results show good agreement with the derived precision equation.
About: This article is published in Biophysical Journal.The article was published on 2002-05-01 and is currently open access. It has received 2390 citations till now. The article focuses on the topics: Point spread function & Background noise.
Citations
More filters
TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Abstract: We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
7,924 citations
TL;DR: A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores that can, in principle, reach molecular-scale resolution is developed.
Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
7,213 citations
TL;DR: This paper introduces the localized surface plasmon resonance (LSPR) sensor and describes how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events and changes in molecular conformation.
Abstract: Recent developments have greatly improved the sensitivity of optical sensors based on metal nanoparticle arrays and single nanoparticles. We introduce the localized surface plasmon resonance (LSPR) sensor and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events and changes in molecular conformation. We then describe recent progress in three areas representing the most significant challenges: pushing sensitivity towards the single-molecule detection limit, combining LSPR with complementary molecular identification techniques such as surface-enhanced Raman spectroscopy, and practical development of sensors and instrumentation for routine use and high-throughput detection. This review highlights several exceptionally promising research directions and discusses how diverse applications of plasmonic nanoparticles can be integrated in the near future.
6,352 citations
Cites background from "Precise nanometer localization anal..."
...This spectral super-resolution is analogous to spatial super-resolution used to track single fluorescent molecules to nanometre resolution (far better than either the pixel spacing or the diffraction-limited spot size...
[...]
Book•
01 Jan 2006TL;DR: In this paper, the authors proposed a method for propagating and focusing of optical fields in a nano-optics environment using near-field optical probes and probe-sample distance control.
Abstract: 1. Introduction 2. Theoretical foundations 3. Propagation and focusing of optical fields 4. Spatial resolution and position accuracy 5. Nanoscale optical microscopy 6. Near-field optical probes 7. Probe-sample distance control 8. Light emission and optical interaction in nanoscale environments 9. Quantum emitters 10. Dipole emission near planar interfaces 11. Photonic crystals and resonators 12. Surface plasmons 13. Forces in confined fields 14. Fluctuation-induced phenomena 15. Theoretical methods in nano-optics Appendices Index.
3,772 citations
TL;DR: A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit, and suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
3,437 citations
Cites background from "Precise nanometer localization anal..."
...Although the PSF of a high numerical aperture lens is not strictly Gaussian (34,35), for distances less than the 1/e(2) radius, a Gaussian is a reasonable approximation of the PSF, and is frequently used to fit the image of a point-like fluorescent object (16,26,27)....
[...]
...20 mm) recommended for optimal localization precision in the presence of background (27)....
[...]
References
More filters
01 Jan 1990
TL;DR: Methods for Three-Dimensional Imaging and Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen.
Abstract: Foundations of Confocal Scanned Imaging in Light Microscopy -- Fundamental Limits in Confocal Microscopy -- Special Optical Elements -- Points, Pixels, and Gray Levels: Digitizing Image Data -- Laser Sources for Confocal Microscopy -- Non-Laser Light Sources for Three-Dimensional Microscopy -- Objective Lenses for Confocal Microscopy -- The Contrast Formation in Optical Microscopy -- The Intermediate Optical System of Laser-Scanning Confocal Microscopes -- Disk-Scanning Confocal Microscopy -- Measuring the Real Point Spread Function of High Numerical Aperture Microscope Objective Lenses -- Photon Detectors for Confocal Microscopy -- Structured Illumination Methods -- Visualization Systems for Multi-Dimensional Microscopy Images -- Automated Three-Dimensional Image Analysis Methods for Confocal Microscopy -- Fluorophores for Confocal Microscopy: Photophysics and Photochemistry -- Practical Considerations in the Selection and Application of Fluorescent Probes -- Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy -- Confocal Microscopy of Living Cells -- Aberrations in Confocal and Multi-Photon Fluorescence Microscopy Induced by Refractive Index Mismatch -- Interaction of Light with Botanical Specimens -- Signal-to-Noise Ratio in Confocal Microscopes -- Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging -- Blind Deconvolution -- Image Enhancement by Deconvolution -- Fiber-Optics in Scanning Optical Microscopy -- Fluorescence Lifetime Imaging in Scanning Microscopy -- Multi-Photon Molecular Excitation in Laser-Scanning Microscopy -- Multifocal Multi-Photon Microscopy -- 4Pi Microscopy -- Nanoscale Resolution with Focused Light: Stimulated Emission Depletion and Other Reversible Saturable Optical Fluorescence Transitions Microscopy Concepts -- Mass Storage, Display, and Hard Copy -- Coherent Anti-Stokes Raman Scattering Microscopy -- Related Methods for Three-Dimensional Imaging -- Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen -- Practical Confocal Microscopy -- Selective Plane Illumination Microscopy -- Cell Damage During Multi-Photon Microscopy -- Photobleaching -- Nonlinear (Harmonic Generation) Optical Microscopy -- Imaging Brain Slices -- Fluorescent Ion Measurement -- Confocal and Multi-Photon Imaging of Living Embryos -- Imaging Plant Cells -- Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells -- Automated Confocal Imaging and High-Content Screening for Cytomics -- Automated Interpretation of Subcellular Location Patterns from Three-Dimensional Confocal Microscopy -- Display and Presentation Software -- When Light Microscope Resolution Is Not Enough:Correlational Light Microscopy and Electron Microscopy -- Databases for Two- and Three-Dimensional Microscopical Images in Biology -- Confocal Microscopy of Biofilms — Spatiotemporal Approaches -- Bibliography of Confocal Microscopy.
4,121 citations
TL;DR: It is found that kinesin moves with 8-nm steps, similar to biological motors that move with regular steps.
Abstract: Do biological motors move with regular steps? To address this question, we constructed instrumentation with the spatial and temporal sensitivity to resolve movement on a molecular scale. We deposited silica beads carrying single molecules of the motor protein kinesin on microtubules using optical tweezers and analysed their motion under controlled loads by interferometry. We find that kinesin moves with 8-nm steps.
1,829 citations
"Precise nanometer localization anal..." refers methods in this paper
...…to within 2 nm at 30 frames/s (Gelles et al., 1988), in a DIC-based experiment to localize a hair cell cilia to 1 pm/Hz1/2 (Denk et al., 1989; Denk and Webb, 1990), and in conjunction with optical trapping to localize latex beads in experiments on motor proteins (Svoboda et al. 1993; Wang, 1999)....
[...]
TL;DR: Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion, including directed motion, confined motion, and anomalous diffusion, which requires a revision of existing views of membrane structure and dynamics.
Abstract: Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is observed, but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.
1,818 citations
"Precise nanometer localization anal..." refers background or methods in this paper
...Although a simplified theoretical analysis predicts an Airy disk for the point-spread function (Saxton and Jacobson, 1997), a Gaussian is mathematically more tractable, and the differences between the two are minor in practice....
[...]
...Reviews of the field are given by Cherry et al. (1998) and Saxton and Jacobson (1997)....
[...]
TL;DR: It is found that cross-correlation is the most accurate algorithm for large particles, however, for point sources, direct Gaussian fit to the intensity distribution is the superior algorithm in terms of both accuracy and precision, and is themost robust at low signal-to-noise.
908 citations
"Precise nanometer localization anal..." refers methods in this paper
...In addition, Cheezum et al. (2001) have written a comparison of four algorithms used in particle tracking....
[...]
...Although this has been found true of the full least-squares fitting algorithm (Cheezum et al., 2001), it was necessary to test it for the Gaussian mask algorithm....
[...]
...The first was a full least-squares fit to a Gaussian distribution that incorporated both photon-counting noise from the particle being measured and a general background noise (see also Cheezum et al., 2001)....
[...]
TL;DR: Measurements reveal basic mechanical features of kinesin-driven movements along the micro-tubule lattice, and place significant constraints on possible molecular mechanisms of movement.
Abstract: Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work1–3. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport4–10, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro4,9. Under certain conditions, a few (≤10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1–2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the micro-tubule lattice, and place significant constraints on possible molecular mechanisms of movement.
838 citations
"Precise nanometer localization anal..." refers methods in this paper
...Such strategies have been used to localize latex beads and colloidal gold to within 2 nm at 30 frames/s (Gelles et al., 1988), in a DIC-based experiment to localize a hair cell cilia to 1 pm/Hz1/2 (Denk et al., 1989; Denk and Webb, 1990), and in conjunction with optical trapping to localize latex…...
[...]
...This procedure for measuring the localization precision is similar to computing the standard deviation of the distance between two different spots and dividing by 21/2 (Gelles et al., 1988), but instead uses the distance between the same spot measured on two separate occasions....
[...]
...Monte Carlo results for the precision of localization were generated by computing a large number of images with random positions, fitting each image, and computing the statistical errors in the fits....
[...]