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  • 1
    Electronic Resource
    Electronic Resource
    Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope (1998)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 73 (1998), S. 1769-1771 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach. © 1998 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.122276
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  • 2
    Electronic Resource
    Electronic Resource
    Two-photon excitation 4Pi confocal microscope: Enhanced axial resolution microscope for biological research (1995)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 66 (1995), S. 1698-1700 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: The applicability of two photon excitation 4Pi confocal fluorescence microscopy to biological imaging is demonstrated. We show that 4Pi confocal microscopy in combination with a simple deconvolution algorithm allows axial localization and quantification with 0.14 μm resolution in a biological sample. The 4Pi-confocal microscope extends the applicability of far field fluorescence microscopy to high resolution three-dimensional imaging and quantification of subcellular structures.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.113897
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  • 3
    Electronic Resource
    Electronic Resource
    Measurement of the 4Pi-confocal point spread function proves 75 nm axial resolution (1994)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 64 (1994), S. 1335-1337 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: In a 4Pi-confocal microscope the specimen is illuminated and observed coherently from above and below such that the numerical aperture is increased [S. W. Hell, European Patent Application 91121368.4 (filed 1990, published 1992), S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992)]. The point spread functions of 4Pi-confocal and confocal microscopes were measured. Our measurements prove a three- to seven-fold increase of axial resolution, thus opening the prospect for a powerful three-dimensional imaging technique with an axial resolution down to 75 nm.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.111926
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  • 4
    Electronic Resource
    Electronic Resource
    Potential of confocal microscopes to resolve in the 50–100 nm range (1996)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 69 (1996), S. 3644-3646 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: We determine the resolution of high-performance confocal microscopes by measuring the three-dimensional point–spread function (3D-PSF) of an optimized confocal setup. The 3D-PSF is standardized by recording the scattered light of pointlike objects. For a wavelength of 543 nm and a specified numerical aperture of 1.4 (oil), we find an axial and lateral focal full width at half-maximum (FWHM) of 460±20 and 145±10 nm, respectively. A high signal-to-noise ratio is obtained by using recording times comparable to those of near-field scanning optical microscopy. We further reduce the effective PSF extent by means of a three-dimensional deconvolution technique exploiting the information gained from the measurement of the focus. We show that it is possible to obtain an axial and lateral FWHM of the far-field effective PSF after deconvolution of 80 and 40 nm, respectively. © 1996 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.117010
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  • 5
    Electronic Resource
    Electronic Resource
    Single sharp spot in fluorescence microscopy of two opposing lenses (2001)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 79 (2001), S. 2321-2323 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: We demonstrate theoretically, experimentally, and in an imaging application the possibility to generate a single predominant sharp diffraction maximum in the effective point-spread function of a fluorescence microscope that coherently uses two opposing lenses. This is achieved through binary pupil filters that preclude the origination of the unfavorable strong interference side maxima that are otherwise present in these systems. Mathematical postprocessing, which has so far been a prerequisite to gain artifact-free images, is now optional or obsolete. © 2001 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.1407303
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  • 6
    Electronic Resource
    Electronic Resource
    Depolarization by high aperture focusing (2000)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 77 (2000), S. 612-614 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: We introduce a method employing ferroelectric monomolecular layers, by which it is possible to measure the light field polarization in the focus of a lens. This method allowed us to noninvasively establish the perpendicularly oriented focal field that is anticipated at high apertures. For a numerical aperture 1.4 oil immersion lens illuminated with linearly polarized plane waves, the integral of the modulus square of the perpendicular component amounts to (1.51±0.2) % of that of the initial polarization. It is proven that depolarization decreases with decreasing aperture angle. Whereas for regular imaging conditions depolarization is largely negligible, it plays a significant role in microscopy of highest resolution, microspectroscopy, and single molecule studies. © 2000 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.127061
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  • 7
    Electronic Resource
    Electronic Resource
    Ultrafast dynamics microscopy (2000)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 77 (2000), S. 597-599 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: We report the three-dimensional imaging of the vibrational, solvent, orientational, and electronic relaxation in organic fluorescent samples at 200–500 nm spatial resolution. This is achieved in steady-state recordings by exciting the fluorophore with a femtosecond pulse and subsequent quenching with a time-delayed, redshifted femtosecond pulse through stimulated emission. Temporal resolution of 380 fs is solely determined by the pulse widths and is further reducible. Images of submicron structures revealing vibrational and solvent relaxation gradients are shown. Furthermore, we introduce contrast modes based on stimulated emission depletion and apply them to cellular imaging. © 2000 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.127056
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  • 8
    Electronic Resource
    Electronic Resource
    Stimulated emission depletion microscopy with an offset depleting beam (2001)
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 78 (2001), S. 393-395 
    Details
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: We demonstrate that an offset stimulated emission depletion (STED) beam breaks the diffraction barrier of fluorescence microscopy in both the lateral and the axial directions. A 2.5-fold axial reduction of the focal spot is accomplished through the ear-shaped lobes of the diffraction maximum of the STED beam. The effect of the minima and side maxima of the STED beam on the lateral and axial resolution is shown to be in remarkable agreement with theory. Conditions are given for which a regular STED beam reduces the axial extent of a confocal spot from 490±36 to 175±18 nm, and simultaneously from 183±12 to 70±8 nm along the direction of the offset. The latter establishes the lowest reported value in far-field fluorescence microscopy. © 2001 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.1338491
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  • 9
    Electronic Resource
    Electronic Resource
    Three-dimensional super-resolution with a 4Pi-confocal microscope using image restoration (1998)
    [S.l.] : American Institute of Physics (AIP)
    Journal of Applied Physics 84 (1998), S. 4033-4042 
    Details
    ISSN: 1089-7550
    Source: AIP Digital Archive
    Topics: Physics
    Notes: The combination of two-photon excitation 4Pi-confocal fluorescence microscopy with image restoration leads to a fundamental improvement of three-dimensional resolution in the imaging of transparent, fluorescent specimens. The improvement is exemplified by randomly dispersed fluorescent beads and with actin filaments in a mouse fibroblast cell. For an illumination wavelength of 810 nm, we obtained lateral and axial full-width at half-maxima of point-like objects of 120–140 nm, and 70–100 nm, respectively. Fluorescent beads that are 150 nm apart are imaged with an intensity dip of ∼25%. This amounts to a ∼sixfold improvement of the axial resolution over standard two-photon confocal microscopy. In the cell, the 3D-images reveal details otherwise not resolvable with focused light. © 1998 American Institute of Physics.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1063/1.368616
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  • 10
    Electronic Resource
    Electronic Resource
    Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit (1995)
    Springer
    Applied physics 60 (1995), S. 495-497 
    Details
    ISSN: 1432-0649
    Keywords: 07.60 ; 87.64
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract We introduce and study a novel concept in farfield fluorescence microscopy fundamentally overcoming the classical diffraction resolution limit. This is accomlished by reducing the spatial extent of the effective focus of a scanning fluorescence microscope. The reduction is achieved by depleting the ground-state energy of the molecules located in the outer region of the focus. Our theoretical study shows that ground-state-depletion fluorescence microscopy has the potential of increasing the resolution of far-field fluorescence microscopy by an order of magnitude which is equivalent to a lateral resolution of 15 NM.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01081333
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