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  • 1
    Keywords: CELLS ; CELL ; Germany ; MICROSCOPY ; imaging ; SUPPORT ; SYSTEMS ; SAMPLE ; SAMPLES ; RESOLUTION ; SUFFICIENT ; FREQUENCY ; FIELD ; FREQUENCIES ; DISPLAY ; REGION ; LIVE CELLS ; FLUORESCENCE ; max ; SECTIONS ; LIGHT ; 2-PHOTON EXCITATION ; 3-DIMENSIONAL TRANSFER-FUNCTIONS ; AXIAL RESOLUTION INCREASE ; COHERENT USE ; CONFOCAL MICROSCOPES ; ELECTROMAGNETIC DIFFRACTION ; FLUORESCENCE MICROSCOPY ; IMAGE-RESTORATION ; OPPOSING LENSES ; three-dimensional imaging
    Abstract: Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three-to fivefold resolution increase in 4Pi-.uorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at; 80 nm axial resolution
    Type of Publication: Journal article published
    PubMed ID: 15377532
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  • 2
    Keywords: CELLS ; CELL ; MICROSCOPY ; PROTEIN ; PROTEINS ; RESOLUTION ; DYNAMICS ; BIOLOGY ; CYCLE ; SIGNAL ; MITOCHONDRIA ; FLUORESCENCE ; GREEN FLUORESCENT PROTEIN ; FEASIBILITY ; INTERFERENCE ; AXIAL RESOLUTION INCREASE ; COHERENT USE ; FLUORESCENCE MICROSCOPY ; OPPOSING LENSES ; microtubule ; 4PI-CONFOCAL MICROSCOPY ; confocal ; EXCITATION ; multiphoton ; two-photon ; FLUORESCENT PROTEIN ; MOLECULAR-SPECTROSCOPY ; NANOMETER ; PtK2 ; QDot
    Abstract: The most prominent restrictions of fluorescence microscopy are the limited resolution and the finite signal. Established conventional, confocal, and multiphoton microscopes resolve at best similar to 200 nm in the focal plane and only 〉= 500 nm in depth. Additionally, organic fluorophores and fluorescent proteins are bleached after 10(4)-10(5) excitation cycles. To overcome these restrictions, we synergistically combine the 3- to 7-fold improved axial resolution of 4Pi microscopy with the greatly enhanced photostability of semiconductor quantum dots. Co-localization studies of immunolabeled microtubules and mitochondria, demonstrate the feasibility of this approach for routine biological measurements. In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail. (c) 2006 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17045487
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