Novel red fluorophores with superior performance in STED microscopy

Wurm, Christian A.; Kolmakov, Kirill; Göttfert, Fabian; Ta, Haisen; Bossi, Mariano L.; Schill, Heiko; Berning, Sebastian; Jakobs, Stefan; Donnert, Gerald; Belov, Vladimir N.; Hell, Stefan W.

doi:10.1186/2192-2853-1-7

Cited by 100 publications

(97 citation statements)

References 15 publications

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“…The measured diameter relative to the size of the whole nuclear pore complex confirmed that the antibody was indeed bound to Nups in the center of the NPC 29 . The resolution of 19 nm is one of the new record of STED super-resolution in biological applications 32–36 . The resolution improvement is largely because two-fold resolution enhancement can be obtained without increasing the depletion laser power.…”

Section: Resultsmentioning

confidence: 99%

Mirror-enhanced super-resolution microscopy

et al. 2016

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Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.

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Section: Resultsmentioning

confidence: 99%

Mirror-enhanced super-resolution microscopy

et al. 2016

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“…(d) Higher-order photobleaching from the first excited electronic state S 1 , as exemplified for the organic dyes pDI and pTDI and for eGFP. Absorption spectra of S 0 (blue) and S 1 (red) and fluorescence development of new bright and photostable dyes, often specialized for STED applications, has and continuously will enhance the applicability and flexibility of experiments using this superresolution technique Kolmakov et al 2010aKolmakov et al , 2010bKolmakov et al , 2012Mitronova et al 2010;Wurm et al 2012).…”

Section: Photo-physical and -Chemical Considerations In Sted Nanoscopymentioning

confidence: 99%

Lens-based fluorescence nanoscopy

Eggeling

Willig

Sahl

et al. 2015

Quart. Rev. Biophys.

137

139

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The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or 'nanoscopy' offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent 'on' and 'off' states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.

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“…The only requirement is the proper adjustment of imaging conditions to their photophysical properties. Live cell imaging with fl uorescent proteins has been demonstrated (Morozova et al 2010 ), and posttranslational labeling with fl uorescence dyes emitting in the red and near-IR and more suitable for STED appears to be a promising alternative (Lukinavičius et al 2013 ;Wurm et al 2012 ).…”

Section: Stimulated Emission Depletion (Sted) Microscopymentioning

confidence: 99%

Sensing Inside the Living Cells

Demchenko

2015

Introduction to Fluorescence Sensing

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Novel red fluorophores with superior performance in STED microscopy

Cited by 100 publications

References 15 publications

Mirror-enhanced super-resolution microscopy

Mirror-enhanced super-resolution microscopy

Lens-based fluorescence nanoscopy

Sensing Inside the Living Cells

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