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Your Next Dimension

TCS SP8 STED 3X

STED (STimulated Emission Depletion) microscopy provides fast, intuitive, and purely optical access to study subcellular architecture and dynamics at the nanoscale. STED super-resolution meets the requirements of daily research and enables to discover minute details with live cell imaging capabilities. TCS SP8 STED 3X, allows the whole spectrum of visible light and opens the door to super-resolution in all dimensions.

Histone H3-Alexa 568 in HeLa cells: Surface rendered 3D reconstruction after Huygens deconvolution of z stack (91 planes). Strucutres of the same color are localized to the same z-plane.

New Technologies that Strengthen Your Science

  • Tunable and direct super-resolution in x,y and z reveals smallest details.
  • Multiple STED lines open up the full spectrum of visible light.
  • Gated detection improves resolution and increases live cell capabilities.
  • STED WHITE objective offers optimal color correction for the full spectrum.
  • Auto beam alignment provides stability and reliability.
  • Modular concept based on TCS SP8 allows upgrades at any time.
  • Smart STED Wizard intuitively controls your experiments.
  • Huygens deconvolution gets more from your raw data.

STED 3X made a quantum leap by expanding into new dimensions – multicolor super-resolution imaging of the cell will surely revolutionize cell biology.

Dr. Yasushi OkadaRIKEN Quantitative Biology Center, Osaka, Japan

STED in the 3rd Dimension

Multiple STED light paths

With TCS SP8 STED 3X, two light paths are available at the instrument that generate different STED patterns. The vortex phase mask creates the classic STED donut which improves resolution only in x and y.
A second light path with a different phase mask forms the z donut that increases resolution also in z. STED light can be steplessly distributed between the two paths.

Engineer your PSF

You can choose between best lateral resolution, best vertical resolution or anything in between to get optimal results. Ultra-thin optical sections reveal unknown details. TCS SP8 STED 3X gives you the possibility to match the resolution of your microscope in all dimensions to your question and specimen.

Adding the third dimension and an additional STED line to STED imaging allows us to see things that were impossible to see before.

Dr. Timo ZimmermannCenter for Genomic Regulation, Barcelona, Spain
Surface rendered 3D reconstruction after Huygens deconvolution of z stack (91 planes). The STED data (green) show a clear lateral resolution increase when STED is used to maximize resolution in xy (green sector in the back) or in all dimensions (green sector in front) compared to the confocal result (red). Clearly the best representation of the observed structure is obtained by 3D STED.

Select Your Resolution

Resolution improvements by allocating the light to the two STED pathways: best lateral resolution is achieved by the classic vortex donut, best vertical resolution by the novel z donut. It can also be optimized for smallest focal volume.

Please switch to ON and move the slider up and down to see the possible light paths and their effect on the focus.

Multicolor Super-Resolution

Cover the whole spectrum of visible light

Multicolor applications give access to detailed information about the interrelationships of various structures. The STED 3X module offers multiple STED laser lines in one instrument: two continuous wave lasers at 592 nm and 660 nm and a pulsed laser at 775 nm cover the whole spectrum of visible light and give access to many applicable fluorophores.

More colors make the difference!

Spectral distribution of accessible STED lasers

The White Light Laser, the AOBS (acousto optical beam splitter) and the tunable spectral detector synergistically enable you to image any kind of fluorophore combination and give you the highest flexibility for your multicolor super-resolution experiment.

STED means for me: Seeing The Essential Details!

Dr. Stephan SigristFU Berlin, Germany

STED image of triple immunostaining in HeLa cells: Green: NUP153-Alexa 532, red: Clathrin-TMR, white: Actin-Alexa 488.

Gated STED - Enter Your Gateway to Live Cell Super-Resolution

Gated STED substantially extends the functionality of the proven STED CW, giving you the option of higher resolution or lower laser lower. More images are obtained and smaller details revealed.

In STED CW imaging resolution is highly dependent on the lifetime of the fluorophore. The STED laser silences fluorophores with long lifetimes more efficiently than short-living ones. The longer a fluorophore stays on average in the excited state, the higher is the chance for stimulated emission and therefore the better the resolution. The lifetime is not evenly distributed at the focal spot and long-living states are found in the center, whereas short-living states are located at the periphery where the STED laser is active. Observing only the long-lived states shrinks the effective spot further.

Live cell imaging: ANF GFP labeled dense core vesicles moving along axons ca. 10 μm deep inside an intact anaesthetized drosophila larva. A confocal and a STED image were recorded every 0.45 seconds. Movie above: Particle tracking performed by TrackMate (Fiji). Figure below: individual confocal and STED frames of such a movie at given time points. Courtesy of Prof. Sigrist, FU Berlin, Germany

Confocal

STED


Resolution Below 50 nm with HyD™ and White Light Laser

The HyD™ detectors together with the White Light Laser as pulsed excitation source give the possibility to detect only in a certain time gate after the excitation pulse. By shifting the time gate away from the excitation pulse a resolution far below 50 nm can be reached. With the same laser power, gated STED achieves a more than 50% higher resolution than STED CW. Smaller details can be observed without the need to apply more STED light, increasing live cell capabilities.

Lifetime distribution of fluorophores in the effectiv focal spot of a STED CW microscope. Long-living states (red) are located at the center whereas short living ones (blue) at the periphery.

Use the slider to adjust the time gate and see the resulting resolution improvement

Software Solutions
for TCS SP8 STED 3X

Know your resolution

STED microscopy is the fast and direct way to super-resolution. The LAS AF (Leica Application Suite Advanced Fluorescence) grants you the greatest possible comfort. A sketch of the estimated effective PSF gives you direct and online visual feedback on the effects of your chosen technical parameters.

Intuitive workflow

As an additional innovation the Smart STED Wizard is implemented into LAS AF. The intuitive workflow allows you to control the instrument with three simple sliders. Assisted by the sketch of the effective PSF you define the general level of super-resolution you need. For optimal outcome the wizard adjusts all necessary settings like STED laser intensity, pixel size, z step size, the pinhole, gate settings and averaging.

Powerful Huygens STED deconvolution

With every TCS SP8 STED 3X system you exclusively get a powerful Huygens STED deconvolution package by SVI. Huygens increases contrast dramatically and also enhances resolution in x, y and z. The outcome can be directly compared with the raw data and avoids being mislead by image processing artifacts. Leica and SVI have enabled Huygens to handle 3D STED data and have developed LAS AF<->Huygens data exchange that facilitates the interplay of the two software packages. One mouse click sends acquired data to Huygens for deconvolution. Vice versa, deconvoluted pictures are just as easily sent back to LAS AF.

The ability to do STED in 3D will bring our research to the next level – right on spot of the researcher‘s needs.

Dr. Christian EggelingUniversity of Oxford, UK

Smart STED workflow

  1. Adjust the desired effective PSF with the STED and 3D slider
  2. Choose between signal-to-noise and number of images with the dosage slider
  3. Define the area of interest during a confocal live scan and adjust excitation accordingly
  4. Collect your super-resolved data by capturing an image or starting a series

STED WHITE Objective
for the Whole Spectrum of Light

Chromatic Abberation of STED WHITE

Objectives are the eyes of every microscope and critical for determining the resolving power of a confocal system. Transmission and color correction of an objective influence excitation and detection efficiencies.

Based on the excellent Leica CS2 objectives, Leica Microsystems has designed a new objective with optimal chromatic correction and transmission for STED 3X. The Leica HC PL APO 100x/1.40 OIL STED WHITE enables you to perform STED microscopy in the full spectrum of visible light.

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