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

ABSTRACT 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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    • "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.
    Microscopy Research and Technique 01/2015; 78(1). DOI:10.1002/jemt.22434 · 1.15 Impact Factor
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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.
    Frontiers in Physiology 07/2014; 5:273. DOI:10.3389/fphys.2014.00273 · 3.53 Impact Factor
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    • "Other implementations of the RESOLFT concept are (Hell 2007) SPEM (Saturated Patterned Excitation Microscopy) (Heintzmann et al. 2002) and SSIM (Saturated Structured-Illumination Microscopy) (Gustafsson 2005); however, in contrast to STED and GSD microscopy, mathematical postprocessing is required (Rittweger et al. 2009b). A similar method uses temporally modulated illumination and Fourier analysis to extract high frequency components resulting from saturation effects (Fujita et al. 2007). A special form of RESOLFT microscopy is DSOM (Dynamic Saturation Optical Microscopy) (Enderlein 2005), which makes explicit use of the fluorescence dynamics. "
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    ABSTRACT: Diffraction plays a crucial role in microscopy as it prevents the recording of arbitrarily sharp images with conventional light microscopes. Many names are connected with the notion of diffraction and the definition of resolution. An overview over the contributions of the different scientists to the recognition and definition of the diffraction barrier in the past centuries is given and the recent developments that led to breaking this barrier are portrayed.
    Optical Nanoscopy 11/2012; 1(1). DOI:10.1186/2192-2853-1-8
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