Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Wiedenmann, Jörg; Nienhaus, G. Ulrich

doi:10.1586/14789450.3.3.361

Cited by 84 publications

(82 citation statements)

References 68 publications

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“…In this article, we describe a method whereby a thin layer of photoactivatable fluorescent proteins (15) can selectively be activated in a location several micrometers deep in a cellular sample. This thin layer is then excited and imaged using the PALM technique with a demonstrated resolution of better than 50 nm.…”

mentioning

confidence: 99%

Multilayer three-dimensional super resolution imaging of thick biological samples

Vaziri

Tang

Shroff

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

167

120

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Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 m deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of Ϸ10 m in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.nanoscopy ͉ PALM microscopy ͉ 3D imaging ͉ temporal focusing I nitial studies, based on defocusing (1), astigmatism (2), and others (3), have extended super resolution imaging (2, 4-13) to three dimensions (3D); however, only up to a few hundred nanometers in depth in biological samples. This is mainly due to the following: In photoactivated localization microscopy (PALM) (6, 12) and stochastic optical reconstruction microscopy (STORM) (8), a sufficient signal-to-noise ratio is required to discriminate single molecules from the background. In the defocusing approaches, background typically increases with imaging depth and the signal is further reduced as it is spread out across more pixels, making the signal to noise ratio insufficient for molecules that are more than a few hundred nanometers away from the focal plane. In addition, PALM is inherently a wide-field technique that is not well suited to point-scanning methods. The reason for this is 2-fold: First, in PALM, single molecules need to be imaged, that is, their emission has to be spread out across several pixels (6) and second, data are most efficiently collected when emissions from spatially segregated molecules are recorded in parallel-point-scanning methods are inherently serial, and are thus slower than wide-field methods (14).Thus, super resolution imaging would benefit greatly by adopting a wide-field technique and combining it with optical sectioning for enhanced signal to noise ratio at depth.In this article, we describe a method whereby a thin layer of photoactivatable fluorescent proteins (15) can selectively be activated in a location several micrometers deep in a cellular sample. This thin layer is then excited and imaged using the PALM technique with a demonstrated resolution of better than 50 nm. A succession of thin layer images can be combined to produce a volume image several micrometers in dep...

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mentioning

confidence: 99%

Multilayer three-dimensional super resolution imaging of thick biological samples

Vaziri

Tang

Shroff

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

167

120

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“…Remarkably, the fluorescence properties of some FPs can be modified by irradiation with light of specific wavelengths (18). In GFP variants, irradiation with intense light around 400 nm results in transformation of the p-HBI chromophore from a nonfluorescent, neutral form to the fluorescent anionic state.…”

Section: Light-induced Activation Of the Chromophorementioning

confidence: 99%

“…Shortly thereafter, the first genes of GFP-like proteins, including the red emitter dsRed, were isolated from different anthozoa species (14)(15)(16). Characterization of the novel proteins revealed that more than 700 million years of molecular evolution created diverse properties with exciting application potential, including an entire rainbow of fluorescence colors and the possibility to control the emission intensity or color by targeted light irradiation (8,(17)(18)(19)(20). Unfortunately, adverse properties such as oligomerization or slow maturation may hamper the use of these proteins in some applications (16).…”

Section: Introductionmentioning

confidence: 99%

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

Wiedenmann¹,

Oswald²,

Nienhaus³

2009

IUBMB Life

215

162

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SummaryThe green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP-like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse molecular properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes.

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“…For these experiments, photoactivatable fluorescent proteins (PAFPs) have been shown to be very convenient, as they can be used as fusion markers for a particular protein under study. EosFP is a popular photoactivatable fluorescent protein, which changes its fluorescence irreversibly from green to red upon illumination with ∼400 nm light (Wiedenmann et al 2004;Nienhaus et al 2006;Wiedenmann and Nienhaus 2006). By appropriate adjustment of the 400-nm activating laser intensity, only a few molecules are converted to red emitters and registered in the red color channel during acquisition of a CCD image for 30-100 ms.…”

Section: Localization-based Super-resolution Microscopymentioning

confidence: 99%

Optical imaging of nanoscale cellular structures

Hedde

Nienhaus

2010

Biophys Rev

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Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Cited by 84 publications

References 68 publications

Multilayer three-dimensional super resolution imaging of thick biological samples

Multilayer three-dimensional super resolution imaging of thick biological samples

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

Optical imaging of nanoscale cellular structures

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