Fluorescence nanoscopy. Methods and applications

Requejo-Isidro, José

doi:10.1007/s12154-013-0096-3

Cited by 34 publications

(22 citation statements)

References 217 publications

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“…Most importantly,the intensity of the C-Naphox fluorescence signal remained virtually unchanged, even after recording five images (Figure 3a), whereas as ignificant decrease in signal intensity was detected for cells labelled with Alexa 488 during the measurement (Figure 3b). [17] Thepolarity-responsive fluorescence with large Stokes shifts should impart C-Naphox with the potential for multi-color imaging and thus increase its value even further. To our knowledge,C -Naphox is thus the first fluorescent dye that permits repeated STED imaging at such al ow degree of photodegradation.…”

Section: Methodsmentioning

confidence: 99%

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

et al. 2015

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Section: Methodsmentioning

confidence: 99%

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

et al. 2015

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“…Thee nhancement of the fluorophore stability under STED imaging conditions is absolutely imperative for the practical application of STED imaging in the visualization of various biological events.The remarkable photostability of C-Naphox should thus open ap reviously unattainable avenue to record prolonged live STED imaging, potentially enabling the visualization of various biological events with high spatial resolution. [17] Thepolarity-responsive fluorescence with large Stokes shifts should impart C-Naphox with the potential for multi-color imaging and thus increase its value even further.…”

Section: Methodsmentioning

confidence: 99%

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

et al. 2015

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The development of stimulated emission depletion (STED) microscopyr epresented am ajor breakthrough in cellular and molecular biology.H owever,t he intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes,w hich significantly limits the performance and practical utility of STED microscopy. We herein developed ap hotoresistant fluorescent dye C-Naphoxa sapractical tool for STED imaging.Withexcitation using either a l = 405 or 488 nm laser in protic solvents,C -Naphox exhibited an intense red/orange fluorescence (quantum yield F F > 0.7) with alarge Stokes shift (circa 5900 cm À1 ). Even after irradiation with aX elamp (300 W, l ex = 460 nm, full width at half maximum (FWHM) = 11 nm) for 12 hours,99.5 %ofC-Naphoxremained intact. The high photoresistance of C-Naphoxa llowed repeated STED imaging of HeLa cells.Even after recording 50 STED images, 83 %o fthe initial fluorescence intensity persisted.Super-resolution nanoscopic techniques,s uch as stimulated emission depletion (STED) microscopy,h ave evolved continuously over the past decade and have allowed scientists to visualize structural details in biological systems. [1][2][3][4][5][6][7][8][9] In STED imaging,afluorescent dye is exposed to irradiation from two different laser sources,namely an excitation laser and aSTED beam, where the STED beam is adonut-shaped beam for the depletion of excited molecules.T he spatial resolution of STED imaging increases with the I STED /I sat ratio,where I STED is the STED beam intensity and I sat represents the saturation intensity at 50 %depletion.[10] An increase of the I STED value, however, simultaneously accelerates the photobleaching of the dye,mainly through multiphoton absorption, [11] and even the most advanced photostable fluorophores currently available suffer from this limitation. Substantially enhancing the photostability of af luorescent dye has therefore been ap rimary concern in order to employ STED microscopy for live imaging.H erein, we report promising fluorescent small molecules which exhibit outstanding photostability,e ven in living cells and under continuous STED imaging conditions, relative to currently available photostable fluorophores.T his compound class is thus ap owerful tool for the advancement of STED microscopy,with potential applications,for instance, in repeated time-lapse STED imaging and live video imaging. Our molecular design was based on ac ore scaffold of (N,N-diphenylamino)phenyl-substituted benzophosphole Poxide (Bphox;F igure 1), [12] which offers several distinct advantages as af luorescent probe.F irst, the combination of the electron-donating properties of the amino moiety with the electron-accepting properties of the benzophosphole P-oxide leads to compounds with intense fluorescence emissions, which can retain high fluorescence quantum yields (F F )even in polar and protic solvents.S econd, this donor-acceptor combination gives rise to an intramolecular charge transfer (ICT) character of the excited s...

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“…Finally, it is interesting to consider that modulation of FP emission is the core concept of localisation-based 'superresolution' light microscopy techniques including PALM and STORM [27]. It would be a logical step to investigate Fig.…”

Section: -25]mentioning

confidence: 99%

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

et al. 2015

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Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dualmodality probes for ILEM when preserved using the inresin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on recreation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single 'multi-colour' integrated microscope system.

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Fluorescence nanoscopy. Methods and applications

Cited by 34 publications

References 217 publications

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

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