Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

Bossi, Mariano L.; Fölling, Jonas; Belov, Vladimir N.; Boyarskiy, Vadim P.; Medda, Rebecca; Egner, Alexander; Eggeling, Christian; Schönle, Andreas; Hell, Stefan W.

doi:10.1021/nl801471d

Cited by 233 publications

(291 citation statements)

References 47 publications

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“…For instance, the emission maxima of MitoTracker Red and ER-Tracker Red differ by 16 nm. They can be distinguished using a ratiometric method by splitting their emission into a shortand a long-wavelength channel; the probe identity can then be determined from the intensity ratio of these two channels (30,32,40).…”

Section: Imaging Mitochondria With Cationic Rosamine and Carbocyaninementioning

confidence: 99%

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Shim

Xia

Zhong

et al. 2012

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

461

455

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Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.nanoscopy | diffraction limit | photoswitchable dye | stochastic optical reconstruction microscopy | photoactivation localization microscopy

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Section: Imaging Mitochondria With Cationic Rosamine and Carbocyaninementioning

confidence: 99%

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Shim

Xia

Zhong

et al. 2012

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

461

455

View full text Add to dashboard Cite

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“…The fluorescence was further cleaned by inserting a band pass filter (532/211; AHF Analysentechnik) to minimize the out-of-band background. Image acquisition, localization, spectral assignment, image reconstruction and simulations of two-dimensional histograms were performed as described before 41 . Image acquisition was started after switching off the protein ensemble in a preselected cell region until clear signatures of single emitters were observed.…”

Section: Analysis Of the Influence Of Irradiation On Cell Viabilitymentioning

confidence: 99%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

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VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

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“…Nanoscopic imaging of multiple fluorophores in the same sample (multicolour PALM or STORM) based on the different emission or activation wavelength was readily achieved after these techniques had been first demonstrated with either two or a single imaging wavelength [45][46][47][48][49]; up to four fluorophores can be discriminated with these strategies [50,51]. Combinatorial pairing of photoswitchable tags with fluorophores that enhance photoactivation enabled subdiffraction multiparameter detection of six different probes [52].…”

Section: Localisation Microscopymentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

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Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffractionlimited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

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Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

Cited by 233 publications

References 47 publications

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

Fluorescence nanoscopy. Methods and applications

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