STED

Abberior Instruments Protected STED technique enhances resolution and contrast in live-cell STED microscopy

neuron

3D-protected STED imaging of neuron in living brain tissue. 3D-rendering of a 3D-image stack of a living neuron expressing the AC-rsEGFP2 actin label in a hippocampal brain slice. Protected STED was applied with resolution enhancement in the xy and z directions. The image shows a stretch of dendrite with dendritic spines. The stack corresponds to 56 z slices spaced by 80 nm. Scale bar: 1 µm.

Abberior Instruments Protected STED technique has been highlighted in a new January 2016 paper in Nature Photonics. Full paper available for download here.

Coordinate-targeted Fluorescence nanoscopy with multiple off states

Johann G. Danzl*†, Sven C. Sidenstein†, Carola Gregor, Nicolai T. Urban, Peter Ilgen, Stefan Jakobs and Stefan W. Hell*

Nature Photonics: Published Online 18 January 2016 DOI: 10.1038/NPHOTON 2015.266

Abstract: By exploiting a second off state of a reversibly switchable fluorophore, a general approach that can reduce photobleaching and enhance resolution of coordinate-targeted fluorescence nanoscopy has been demonstrated.

Far-field super-resolution fluorescence microscopy discerns fluorophores residing closer than the diffraction barrier by briefly transferring them in different (typically ON and OFF) states before detection. In coordinate-targeted superresolution variants, such as stimulated emission depletion (STED) microscopy, this state difference is created by the intensity minima and maxima of an optical pattern, causing all fluorophores to assume the off state, for instance, except at the minima. Although strong spatial confinement of the on state enables high resolution, it also subjects the fluorophores to excess intensities and state cycles at the maxima. Here, we address these issues by driving the fluorophores into a second off state that is inert to the excess light. By using reversibly switchable fluorescent proteins as labels, our approach reduces bleaching and enhances resolution and contrast in live-cell STED microscopy. Using two or more transitions to off states is a useful strategy for augmenting the power of coordinate-targeted super-resolution microscopy.

Download and read the full Nature Photonics article here.

Selected Figures from the article appear below:

figure 1

Figure 1 | MOST nanoscopy and protected STED. a, Molecular states for multiple off-state transitions (MOST) super-resolution imaging. Single off transition modalities, such as STED microscopy, (grey box) utilize a single off transition only. In MOST concepts, an additional off state (OFF2) is used for enhancing the on/off contrast and for ?uorophore protection. The system of states can be extended by further off states OFFi. The combination of STED and reversible photoswitching of ?uorophores is termed protected STED. Wavelengths are given for protected STED with rsEGFP variants. b, In conventional STED microscopy, molecules at intensity minima (that is, intensity <IS, here shown forxy doughnut geometry) are allowed to assume the signalling ON state. Molecules further away from the minimum are exposed to relatively high STED beam intensities. c, In the protected STED mode, molecules in regions of high STED intensity are pre-emptively transferred to an inert protective state OFF2. ISOFF1?OFF2 is the intensity for 95% probability of transfer to OFF2. d, Normalized spatial distribution of probabilities for a molecule to reside in OFF1 after photoactivation (POFF1(?OFF2)); to reside in OFF1 after the protective OFF1?OFF2 transition (POFF1(?OFF2)); and to reside in the ON state ( PON) after joint action of both off transitions (driven by the light intensity distributions ION?OFF1 and IOFF1?OFF2) and excitation (OFF1?ON, not shown). d: region where ON state can be assumed, ?: distance from intensity minimum. e, Schematic of setup. DM: dichroic mirror. PM 0–2?: helical phase modulation.

 

figure 3

Figure 3 | Reduced bleaching in protected STED imaging. a, Image series of living cells expressing keratin-rsEGFP2 for STED (7.6 mW STED power) and protected STED (350 µs deactivation, 1.5 mW STED power) with similar resolution and brightness in the ?rst frame (larger images: Supplementary Fig. 7). Scale bar: 1 µm. b, Image brightness as a function of frame number normalized to the ?rst frame in each series for STED (blue triangles), protected STED (red squares), and RESOLFT (black circles, 400 µs deactivation). Mean ± standard deviation of measurements in 10 cells.

figure 5

Figure 5 | 3D-subdiffraction resolution and dual colour imaging. a, Protected STED image of a living CV-1 cell expressing the AC-rsEGFP(N205S) actin label with xy and z resolution increase. The image corresponds to a single optical slice of the cell of subdiffraction width (<150 nm). Individual ?laments are seen to enter or leave this plane from above or below. Bottom: confocal data. Scale bar: 1 µm. Raw data smoothed with 1.3 pixel wide Gaussian (data without smoothing: Supplementary Fig. 18). b, Zoomed view of region indicated in a, showing branching actin ?laments and corresponding line pro?le at the position of the arrowheads. A double Lorentzian ?t yielded 57±8 nm and 40±7 nm FWHM and 90.5nm peak spacing. r, coordinate along line pro?le. c, Zoomed view and corresponding line pro?le with Lorentzian ?t yielding 45±2 nm FWHM. Scale bars b,c: 500nm. d, 3D subdiffraction imaging of keratin ?laments labelled with rsEGFP2 in a living HeLa cell. The z position is colour-coded and an xz section at the position of the arrowheads is given on the right. Scale bar: 1 µm. e, Combination of protected STED (endosomes labelled with rsEGFP(N205S)-Rab5a, colour map ?re) and STED (microtubules decorated with citrineMap2, colour map green) for dual colour imaging. Scale bar: 1 µm. Confocal data is shown in the bottom corner on the left.

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