Showing papers on "Photoactivated localization microscopy published in 2004"

Nanometer-localized multiple single-molecule fluorescence microscopy.

Abstract: Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based ( 100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.

On the fundamental imaging-depth limit in two-photon microscopy

Abstract: The limit on imaging-depth in two-photon microscopy depends on parameters of the imaging system such as available power, wavelength, numerical aperture, and fluorescencecollection field-of-view and on properties of the sample such as scattering and absorption cross-sections and fluorophore distribution. These dependencies are discussed and strategies for optimizing the imaging depth are presented.

Image calibration in fluorescence microscopy

Abstract: A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non-uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach-rate-related imaging practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. As the illumination and imaging properties of a microscope can be evaluated separately, the methods presented are also valuable for general microscope testing and characterization.

Two-color two-photon 4Pi fluorescence microscopy.

Abstract: In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength.

Single molecule spectroscopic characterization of GFP-MUT2 mutant for two-photon microscopy applications

Abstract: Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.

Optical methods and sensors for in situ histology in surgery and endoscopy.

Abstract: For endoscopic in situ histology, cellular structures have to be imaged non-invasively with sufficient resolution and contrast. Confocal, multi-photon or coherence-gated microscopy are able to reject multiply scattered photons and thus provide sufficient contrast at microscopic resolution. They are the basis for imaging methods such as reflection microscopy, fluorescence microscopy, autofluorescence microscopy, optical coherence microscopy and tomography and multi-photon microscopy, which will be explained and compared. Finally, current concepts for miniaturized optical sensors aiming to implement these imaging methods endoscopically will be presented.

Two-Point-Separation in Far-Field Super-Resolution Fluorescence Microscopy Based on Two-Color Fluorescence Dip Spectroscopy

Abstract: The two-point-resolution of a novel two-color far-field super-resolution fluorescence microscopy has been evaluated. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase modulation technique for the laser beam. The two-point-resolution of the microscopy has been evaluated by using the fluorescent beads of which the size is specified by the observation with AFM. By introducing two color lasers, the observed fluorescence image shrunk down beyond the diffraction limit. In the observation of neighboring two fluorescence beads, pronounced separation be- tween two individual fluorescence spots has been investigated. By taking the focal depth into consideration, FWHM and profile of the fluorescence images were well reproduced. It is proved that our technique breaks the diffraction limit and the spatial resolution of the optical fluorescence microscope can be improved by 2 times higher than the diffraction limit.

Internet-Based Education in Confocal and Widefield Fluorescence Microscopy

Abstract: Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

In vivo fluorescence microscopy of neuronal activity in three dimensions using voltage-sensitive dyes

Abstract: We report three-dimensional in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The depth-dependent differential fluorescence optical sections of activity were consistent with known cortical architecture.