A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

Görlitz, Frederik; Hoyer, Patrick; Falk, Henning J.; Kastrup, Lars; Engelhardt, Johann; Hell, Stefan W.

doi:10.2528/pier14042708

Cited by 46 publications

(60 citation statements)

References 26 publications

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“…This clearly demonstrates that the choice of markers in relation to the detection windows plays a crucial role. The number of distinguishable markers could be further increased by combining the present hyperSTED setup with other means of discrimination, for example excitation multiplexing21, fluorescence lifetime imaging2223 or photostability18, as previously reported. This is feasible without changes to the hardware, keeping the setup simple and stable.…”

Section: Discussionmentioning

confidence: 79%

“…Evaluating the promise of the hyperSTED approach as presented here, simulations20 indicate that six-colour separation of Abberior Star600, Atto594, DyLight633, Abberior Star635P, KK1441 and CF680R should be possible when adding the lifetime information from ratios of signals in two time gates22, if the assumed spectra and lifetimes of these dyes are as stated in Table 1. Note that for such a sample only one excitation wavelength of 612 nm and a single STED beam would be required.…”

Section: Discussionmentioning

confidence: 98%

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Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

Winter

Loidolt

Westphal

et al. 2017

Sci Rep

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The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam. Unmixing of the different marker signals based on the simultaneous readout of all channels is performed with a non-negative matrix factorization algorithm. We illustrate the approach showing four-colour nanoscopy of fixed and living cellular samples.

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Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 98%

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

Winter

Loidolt

Westphal

et al. 2017

Sci Rep

Self Cite

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“…Three different microscope setups have been used for the study: Laser Induced Fluorescence (LIF) microscopy (3.1) [10], Raman microscopy (3.2) [11] and Coherent Anti-Stokes Raman Scattering (CARS) microscopy (3.3) [12]. Various hydrophilic and hydrophobic intraocular lens materials were studied (3.4).…”

Section: Methodsmentioning

confidence: 99%

Chemical basis for alteration of an intraocular lens using a femtosecond laser

Bille¹,

Engelhardt²,

Volpp³

et al. 2017

Biomed. Opt. Express

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Abstract:The chemical basis for the alteration of the refractive properties of an intraocular lens with a femtosecond laser was investigated. Three different microscope setups have been used for the study: Laser Induced Fluorescence (LIF) microscopy, Raman microscopy and coherent anti-Stokes Raman Scattering (CARS) microscopy. Photo-induced hydrolysis of polymeric material in aqueous media produces two hydrophilic functional groups: acid group and alcohol group. The spectral signatures identify two of the hydrophilic polar molecules as N-phenyl-4-(phenylazo)-benzenamine (C 18 H 15 N 3 ) and phenazine-1-carboxylic acid (C 13 H 8 N 2 O 2 ). The change in hydrophilicity results in a negative refractive index change in the laser-treated areas.

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“…At present, however, simultaneous recordings of more than two fluorophore species still require complex setups and/or analysis algorithms34. Here, we utilize a pulsed fibre laser emitting at 620 nm as the STED light source and demonstrate its versatility in a novel three-colour STED microscopy scheme.…”

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confidence: 99%

Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses

Sidenstein

D’Este

Böhm

et al. 2016

Sci Rep

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108

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Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels (“multilevelSTED”) optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.

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A STED MICROSCOPE DESIGNED FOR ROUTINE BIOMEDICAL APPLICATIONS (Invited Paper)

Cited by 46 publications

References 26 publications

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection

Chemical basis for alteration of an intraocular lens using a femtosecond laser

Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses

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