Showing papers on "Photoactivated localization microscopy published in 2003"
TL;DR: A family of concepts has emerged that overcomes the diffraction barrier altogether and, relying on saturated optical transitions, these concepts are limited only by the attainable saturation level.
Abstract: For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis Recently, microscopes have been reported that provide three- to sevenfold improved axial resolution in live cells Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever
983 citations
TL;DR: Recent progress on the technical implementation ofSpectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging.
520 citations
TL;DR: This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation.
Abstract: Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.
486 citations
TL;DR: This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on the laboratory's interest in single-molecule microscopy and spectroscopy.
Abstract: ■ Abstract Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory’s interest in single-molecule microscopy and spectroscopy.
230 citations
TL;DR: The basic physical principles behind two‐photon excitation are described and the advantages and limitations of its use in laser‐scanning microscopy are discussed, including recent technological advances.
Abstract: Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application.
201 citations
Journal Article•
TL;DR: In this article, the imaging performance in single-photon (l-p) and two-p (2p) fluorescence microscopy is compared in terms of the three-dimensional (3D) point spread function and the 3-D optical transfer function.
Abstract: Summary The imaging performance in single-photon (l-p) and two photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of.the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled •. giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.
97 citations
TL;DR: In this article, the authors demonstrate sub-diffraction lateral resolution of 28±2 nm in far-field fluorescence microscopy through stimulated emission depletion effected by an amplified laser diode.
Abstract: We demonstrate sub-diffraction lateral resolution of 28±2 nm in far-field fluorescence microscopy through stimulated emission depletion effected by an amplified laser diode. Measurement of the optical transfer function in the focal plane reveals a 6-fold enlargement of the spatial bandwith over the diffraction limit. The resolution is established by imaging individual fluorescent molecules on a surface. Corresponding to 1/25 of the responsible wavelength, the attained resolution represents a new benchmark in far-field microscopy and underscores the viability of fluorescence nanoscopy with visible light, conventional optics and compact laser systems .
91 citations
TL;DR: Pronounced separation (750 nm) between two individual fluorescence spots in a novel super-resolution microscopy based on a two-color up-conversion fluorescence depletion technique has been investigated.
Abstract: Pronounced separation (750 nm) between two individual fluorescence spots in a novel super-resolution microscopy based on a two-color up-conversion fluorescence depletion technique has been investigated. This microscopy has the potential to achieve a spatial resolution (<300nm) of 1/2 the diffraction limit.
78 citations
TL;DR: It is demonstrated that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores through consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction.
Abstract: We demonstrate that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast in fluorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolar solvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction. The reverse-contrast images also provide clear indication of nanotube location.
59 citations
TL;DR: The combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA are focused on.
Abstract: A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.
20 citations
TL;DR: In this paper, the authors report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM), which was calibrated with standard fluorophore specimens and was shown to have high accuracy and reproducibility.
Abstract: We report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM). The system was calibrated with standard fluorophore specimens and was shown to have high accuracy and reproducibility. We demonstrate the applicability of this instrument in living cells for measuring the effects of protein targeting and point mutations in the protein sequence, which are not obtainable by conventional intensity-based fluorescence microscopy methods. We discuss the relevance of such time-resolved information in quantitative energy transfer microscopy and in measurement of the parameters that characterize the intracellular physiology.
01 Jan 2003
TL;DR: In this paper, the basic principles of multi-photon microscopy are reviewed and an overview of some cutting edge developments such as spectral resolved imaging techniques and high throughput image cytometry is provided.
Abstract: Multi-photon microscopy is an emerging technique with far reaching impact in biological and medical analysis of tissues, cells and biomolecules. This paper reviews the basic principles of multi-photon microscopy and provide an overview of some cutting edge developments such as spectral resolved imaging techniques and high throughput image cytometry.
14 Oct 2003
TL;DR: An overview on some advanced techniques of fluorescence spectroscopy and fluorescence microscopy is given in this paper, including time-resolved, energy transfer and total internal reflection (TIRFM) methods.
Abstract: An overview on some advanced techniques of fluorescence spectroscopy and fluorescence microscopy is given. These techniques include time-resolved fluorescence spectroscopy, energy transfer spectroscopy (FRET), total internal reflection fluorescence microscopy (TIRFM) and fluorescence lifetime imaging (FLIM). The principle of these methods is explained, and numerous applications are described.
TL;DR: A new image processing technique for estimating cell numbers and contours in three-dimensional (3-D) microfabricated scaffolds based on a statistical approach, and utilizes the extreme value theory, which assumes that cells in the image field are rare, high-intensity events.
Abstract: This paper presents a new image processing technique for estimating cell numbers and contours in three-dimensional (3-D) microfabricated scaffolds. The method is based on a statistical approach, and utilizes the extreme value theory, which assumes that cells in the image field are rare, high-intensity events. Confocal microscopy images of fibroblasts on 3-D structures were processed using the method, and the resulting data on cell numbers was compared with countings obtained using a Burker chamber. The results were identical to within a few percent.
TL;DR: This unit will describe the use of total internal reflection fluorescence microscopy (TIR‐FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane within 50 nm of the cell surface.
Abstract: The wavelength of light imposes a physical limit of approximately 400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR-FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR-FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal-to-noise ratio but also minimizes the photodamage to the cell.
Journal Article•
TL;DR: In this article, the authors developed a high sensitivity two-photon scanning microscope with a mode-locked titanium-sapphire laser and achieved a spatial point spread function of 0.3 μm radially and 0.9 μm axially for an 1.25 N.A. objective.
Abstract: We have developed a high sensitivity time-resolved two-photon scanning microscope. At an excitation wavelength of 960 nm, a spatial point spread function of 0.3 μm (FWHM) radially and 0.9 μm (FWHM) axially is measured for an 1.25 N.A. objective. The light source is a mode-locked titanium-sapphire laser. The time resolution is 400 ps with common chromophores used in microscopy. Time resolution is obtained using the frequency-domain heterodyning technique in which the laser is synchronized at a very high cross-correlation frequency to the rest of the electronics. We demonstrate spatial and time resolution using well-characterized fluorescent microspheres. We show two applications of two-photon time-resolved fluorescence microscopy: time-resolved imaging of multiple dye labeled cells and quantitative cellular calcium concentration using a lifetime indicator.
TL;DR: This chapter describes the filters and mirrors required for some of the current methods of fluorescence microscopy, and the properties of Fluorescence, a phenomenon in which a substance absorbs light of a specific wavelength, and rapidly radiates light of another wavelength.
Abstract: Publisher Summary This chapter describes the filters and mirrors for applications in fluorescence microscopy. The chapter serves as a short summary of fluorescence; applications in fluorescence; and, specifically, the filters and mirrors required for some of the current methods of fluorescence microscopy. One of the most prominent changes in microscopy is the recent explosion of applications associated with fluorescence microscopy. Fluorescence is a phenomenon in which a substance (atom or molecule) absorbs light of a specific wavelength, and rapidly radiates light of another (usually longer) wavelength. The substances that exhibit the properties of fluorescence are called fluorescent molecules, fluorochromes, or fluorophores. The absorption of the initial photon is termed “excitation,” and functions to move the fluorophore from the ground state to an excited state. From this intermediate, excited singlet state, there are three principal options available: (1) intersystem crossing, which can lead to a prolonged emission of light called phosphorescence; (2) nonradiative conversion; or (3) emission of a photon as the electron falls back to the ground state.
TL;DR: In this paper, a biphotonic effect was demonstrated to contribute to fluorescence under usual conditions for multiphoton fluorescence microscopy, and that it might be exploited in fluorescence imaging under conditions where usual mechanisms are inapplicable.
Abstract: We have observed fluorescence from Coumarin 334 in solution using light of twice an actinic wavelength for excitation. The effect is demonstrated to be biphotonic, but is observed at optical powers below those required for resonant biphotonic excitation, and with an incoherent source. We infer operation of HRS. By extrapolation we propose that this effect should contribute to fluorescence under usual conditions for multiphoton fluorescence microscopy, and that it might be exploited in fluorescence microscopy under conditions where usual mechanisms are inapplicable.
13 Oct 2003
TL;DR: In this article, a theoretical modeling of turbid medium images formed by two-photon fluorescent microscopy technique is proposed, and the properties of the calculated images, such as the registered fluorescence intensity dependence on the focusing depth and the influence of the scattering in the medium, have been studied.
Abstract: We propose a theoretical modeling of a turbid medium images formed by two-photon fluorescent microscopy technique. On the basis of this model, general properties of the calculated images, such as the registered fluorescence intensity dependence on the focusing depth and the influence of the scattering in the medium, have been studied. A comparison of image properties for single-photon and two-photon microscopy is given.
06 Jun 2003
TL;DR: In this article, the authors measured the optical response of fluorescent beads to sharp metallic and semiconducting probes, revealing several underlying near-field interactions, and suggested that /spl sim/10 nanometer optical resolution with spectroscopic chemical sensitivity is possible, and bear strongly on molecular-scale biological imaging.
Abstract: We measured the optical response of fluorescent beads to sharp metallic and semiconducting probes, revealing several underlying near-field interactions. Our results suggest that /spl sim/10 nanometer optical resolution with spectroscopic chemical sensitivity is possible, and bear strongly on molecular-scale biological imaging.
15 Dec 2003
TL;DR: In this article, the use of an annular aperture in two-color excitation fluorescence microscopy is shown to be useful because the side maxima is effectively suppressed and large area detection of two photons imaging is also possible.
Abstract: In this paper we show that the use of an annular aperture in two-color excitation fluorescence microscopy is interest because the side maxima is effectively suppressed and large area detection of two photons imaging is also possible.