High-Resolution Confocal Microscopy by Saturated Excitation of Fluorescence

RIKEN, Вако, Saitama, Japan
Physical Review Letters (Impact Factor: 7.51). 12/2007; 99(22):228105. DOI: 10.1103/PhysRevLett.99.228105
Source: PubMed


We demonstrate the use of saturated excitation in confocal fluorescence microscopy to improve the spatial resolution. In the proposed technique, we modulate the excitation intensity temporally and detect the harmonic modulation of the fluorescence signal which is caused by the saturated excitation in the center of the laser focus. Theoretical and experimental investigations show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensity and contributes to improve the spatial resolution in three dimensions beyond the diffraction limit of light.

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    • "High excitation intensities are required to induce the saturation. Similarly to SAX microscopy [5], the saturation of CARS can be used to effectively reduce the point-spread function by extracting the saturated CARS emission at harmonic frequencies, thereby, increasing the spatial resolution of CARS microscopy beyond the classical limit. "
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    ABSTRACT: We demonstrate a vibrational microscopy technique with subdiffraction spatial resolution by the use of saturation of coherent anti-Stokes Raman scattering (CARS). The saturated CARS signals effectively produce a reduced point-spread function at harmonic frequencies, which is extracted by temporal modulation of the pump beam and demodulation of the CARS signal. An increase in spectral resolution and suppression of the nonresonant background signal accompany the spatial- resolution enhancement. Our simple, enhanced CARS technique promises to be useful in studying molecules in gas and liquid phases as well as soft condensed-matter systems.
    No preview · Article · Jul 2015 · Physical Review Applied
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    • "SSIM explained the resolution enhancement with higher harmonic generation. Similarly, the saturation-harmonics generation can be modulated and detected in time-domain (Fujita et al. 2007; Yamanaka et al. 2013), which may open up a new horizon for current spatial modulation approaches. "
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    ABSTRACT: The recent work by Chmyrov et al. (Nature Methods 2013) presents a capstone for the current major super-resolution microscopy techniques. In optical super-resolution microscopy, two pathways are commonly taken: targeted illumination modulation, or stochastic single-molecule localization. Incoherent cross-standing microscopy has utilized the concept of structured illumination in generating patterns, to generate effective 100,000 "doughnuts" as with STED; and used the photo-switchable dye to decrease the requirement of modulation intensity. It has combined the key elements of all these major super-resolution techniques.
    Full-text · Article · Jan 2015 · Microscopy Research and Technique
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    • "This approach is known as RESOLFT (REversible Saturable OpticaL Fluorescence Transitions) and was first proposed and demonstrated by STED (STimulated Emission Depletion), which exploits the stimulated emission phenomenon of a fluorescent dye (Hell and Wichmann, 1994; Klar and Hell, 1999). RESOLFT can also be realized by other photoreactions, including those from a ground-state transition phenomenon (GSD: Ground State Depletion) (Hell and Kroug, 1995; Bretschneider et al., 2007), the saturation of fluorescence excitation (SAX: SAturated eXcitation) (Fujita et al., 2007), or from reversibly photoswitchable fluorescent proteins (Hofmann et al., 2005). RESOLFT can also be combined with structured illumination microscopy (SIM) (Heintzmann and Cremer, 1999; "
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    ABSTRACT: Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies.
    Full-text · Article · Jul 2014 · Frontiers in Physiology
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