Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning
Exciting developments: Switching individual photochromic and fluorescent rhodamine amides enables 3D far‐field optical microscopy with nanoscale resolution, excellent signal‐to‐noise ratio, and fast acquisition times. The rhodamine amides can be switched on using two photons, which enables 3D detailed imaging of thick and densely stained samples (such as 5‐μm silica beads (see image) and living cells) to be constructed.
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- "Continued development of techniques for 3D SMF tracking and imaging is therefore very important. Methods to extend SMF imaging to 3D include astigmatism (Huang et al., 2008), multiplane methods (Juette et al., 2008), optical sectioning (Fölling et al., 2007), interferometry (Shtengel et al., 2009) and double-helix point-spread function microscopy (Pavani et al., 2009). Interestingly, 3D superresolution images of protein superstructures in live C. crescentus cells have recently been achieved with a doublehelix point spread function microscope (Lew et al., 2011) and an astigmatic lens (Biteen et al., 2012). "
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ABSTRACT: Single-molecule fluorescence microscopy enables non-invasive, high-sensitivity, high-resolution imaging, and this direct, quantitative method has recently been extended to understanding organization, dynamics and cooperativity of macromolecules in prokaryotes. In this issue of Molecular Microbiology, Bakshi et al. (2012) examine fluorescently labelled ribosomes and RNA polymerase (RNAP) in live Escherichia coli cells. By localizing individual molecules with 30 nm scale accuracy, they resolve the spatial distribution of RNAP (and thus of the E. coli nucleoid) and of the ribosomes, measure diffusion rates, and sensitively count protein copy numbers. This work represents an exciting achievement in terms of applying biophysical methods to live cells and quantitatively answering important questions in physiologically relevant conditions. In particular, the authors directly relate the positions, dynamics, and numbers of ribosomes and RNAP to transcription and translation in E. coli. The results indicate that, since the ribosomes and the nucleoid are well segregated, translation and transcription must be predominantly uncoupled. As well, the radial extension of ribosomes and RNAP to the cytoplasmic membrane is consistent with the hypothesis of transertion (simultaneous insertion of membrane proteins upon translation).
Molecular Microbiology 05/2012; 85(1):1-4. DOI:10.1111/j.1365-2958.2012.08089.x · 4.42 Impact Factor
Available from: Ricardo Henriques
- "A side effect of using this method means that thousands of images may need to be acquired to generate one super-resolution dataset. This feature has become the backbone of techniques such as photoactivation localization microscopy (PALM) , fluorescence photoactivation localization microscopy (FPALM) , stochastic optical reconstruction microscopy (STORM)  and PALM with independently running acquisition (PALMIRA)   . For the remainder of this review we focus on this method and how it has emerged as one of the most tractable approaches for any laboratory wishing to generate super-resolved data. "
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ABSTRACT: Super-resolution imaging allows the imaging of fluorescently labeled probes at a resolution of just tens of nanometers, surpassing classic light microscopy by at least one order of magnitude. Recent advances such as the development of photo-switchable fluorophores, high-sensitivity microscopes and single particle localization algorithms make super-resolution imaging rapidly accessible to the wider life sciences research community. As we take our first steps in deciphering the roles and behaviors of individual molecules inside their living cellular environment, a new world of research opportunities beckons. Here we discuss some of the latest developments achieved with these techniques and emerging areas where super-resolution will give fundamental new "eye" sight to cell biology.
Biotechnology Journal 06/2009; 4(6):846-57. DOI:10.1002/biot.200900024 · 3.49 Impact Factor
Available from: Dominik Wildanger
- "This configuration is optimal for lateral resolution enhancement and typically provides a resolution of 20– 40 nm in the focal plane (Donnert et al., 2006), but the axial resolution remains at the confocal level (>500 nm) resulting in the fact that small and dark structures remain difficult to discern if other bright features are present within the confocal axial section. This limitation holds equally for the combination of single-molecule photoswitching microscopy with two-photon excitation (Fölling et al., 2007), which also provides lateral super-resolution with 3D optical sectioning. Increasing the axial resolution beyond its diffraction limit requires more demanding approaches, such as the use of a STED-4Pi microscope or a combination of several depletion beams (Harke et al., 2008). "
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ABSTRACT: Tackling biological problems often involves the imaging and localization of cellular structures on the nanometer scale. Although optical super-resolution below 100 nm can be readily attained with stimulated emission depletion (STED) and photoswitching microscopy methods, attaining an axial resolution <100 nm with focused light generally required the use of two lenses in a 4Pi configuration or exceptionally bright photochromic fluorophores. Here, we describe a simple technical solution for 3D nanoscopy of fixed samples: biological specimens are fluorescently labeled, embedded in a polymer resin, cut into thin sections, and then imaged via STED microscopy with nanoscale resolution. This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes.
Microscopy Research and Technique 09/2008; 71(9):644-50. DOI:10.1002/jemt.20602 · 1.15 Impact Factor
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