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Deep nanoscopy 45-65 µm inside cleared adult kidney sample of a rat. 20 µm xyz stack confocal/3D STED. Nephrin visualized with STAR 635P. Clearing by modified CLARITY protocol. STED lens: HC PL APO 93X/1.30 GLYC motCORR – STED WHITE. Sample courtesy of David Unnersjö-Jess, KTH, Stockholm, Sweden.

You would like to start with STED imaging?

There’s no need to invest in a dedicated instrument to see life’s details. The new Leica TCS SP8 STED ONE 592 and STED ONE 660 nanoscopes are your strategic entry into nanoscopy on the basis of the highly versatile innovative confocal platform Leica TCS SP8. Spectral detection in combination with the sensitivity of Leica HyD hybrid detectors and the excitation freedom offered by White Light Laser make a STED nanoscope by Leica Microsystems a tool of maximal flexibility not only for nanoscopy.

If you need access to as many fluorophores and colors as possible, the Leica TCS SP8 STED 3X enables you to use up to three STED lines facilitating STED over the full visible spectrum.

Technologies that strengthen your science

  • Tunable and direct nanoscopy in x,y and z reveals smallest details
  • STED WHITE objectives offer optimal color correction for the full spectrum
  • motCORR STED objectives for deep live imaging
  • Multiple STED lines open up the full spectrum of visible light
  • Gated detection improves resolution and increases live cell capabilities
  • Auto beam alignment provides stability and reliability
  • Smart STED Wizard intuitively controls your experiments
  • Modular concept based on TCS SP8 allows upgrades at any time

"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 Okada, RIKEN Quantitative Biology Center, Osaka, Japan

The “R&D 100 Award 2014” and the “Scientist Top 10 Innovations Award 2014” recognize the Leica TCS SP8 STED 3X among the most significant high-technology products introduced in 2014.
STED image of triple immunostaining in HeLa cells: Green: NUP153-Alexa 532, red: Clathrin-TMR, white: Actin-Alexa 488.

Multicolor nanoscopy

Cover the whole spectrum of visible light

Multicolor applications give access to detailed information about the interrelationships of various structures. Colocalization studies are routinely performed with STED ONE nanoscope. The multiple STED laser lines of STED 3X  at 592 nm and 660 nm and the pulsed laser at 775 nm, which reaches resolutions below 30 nm, cover the whole spectrum of visible light and give access to many applicable fluorophores.

More colors make the difference!

The White Light Laser, the AOBS (Acousto Optical Beam Splitter) and the tunable spectral detectors synergistically enable you to image any kind of fluorophore combination and give you the highest flexibility for your multicolor experiment.

"STED means for me: Seeing the essential details!"

Prof. Stephan Sigrist, FU Berlin, Germany

Nanoscopy empowered by cutting-edge optics

STED WHITE – don‘t compromise on optics

When it comes to resolving very small details, it’s crucial to start with the best optics. The chromatic correction of the three STED WHITE objective lenses ensures optimal overlay of excitation and STED PSF in z all over the visible spectral range. It's possible to use the white light laser as excitation source for STED at 592/660/775. Enjoy the freedom to choose from more fluorophores than ever for STED nanoscopy.

STED objective lens for deep live cell nanoscopy

Standard fixed samples are easily imaged with the STED WHITE oil objective lens (HC PL APO 100x/1.40 OIL). It delivers highest NA and resolution. For complex experiments, the STED WHITE Glycerin objective lens (HC PL APO 93X/1.30 GLYC motCORR) creates new opportunities. The motorized correction collar allows precise and swift adjustment of optical lenses to varying cover glass thickness, changes in temperature and specimen inhomogeneity. Shed light on details deep inside your specimen with 3D STED not only at room temperature, but also at 37°C.

  • "The lens is a must, everybody should have it!" - Dr. Yasushi Okada, RIKEN Quantitative Biology Center, Osaka, Japan
  • "The 93x STED objective with glycerol immersion opens up a new range of applications that are important for biological imaging." - Timo Zimmermann, Center for Genomic Regulation, Barcelona, Spain

NEW! STED WHITE objective lens for aqueous media

The HCS PL APO 86x/1.20W motCORR objective lens delivers the highest performance in water-based experiments. The motCORR technology combined with the best refraction index matching make this lens the ideal companion in aqueaous media applications.

Chromatic aberration of STED WHITE objectives. Depth of field depicted in gray dashed line.

Gated STED – your gateway to live cell nanoscopy

Live cell STED nanoscopy: 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).

gSTED – more images with smaller details

Gated STED substantially extends the functionality of the proven STED CW, giving you the option of higher resolution or lower laser power.

More images are obtained and smaller details revealed.

"In fact, of all STED modalities reported so far, g-STED provides the sharpest images with the lowest peak power."

Vicidomini et al., Nature Methods 2011

Living T cell in suspension. 3D reconstruction of confocal and STED stacks. Maximum projection. Courtesy of Marco Fritsche, Mathias Clausen and Christian Eggeling, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK.

Resolution below 50 nm with HyD and White Light Laser

The Leica HyD hybrid detectors together with the White Light Laser as pulsed excitation source allow the detection of signals to be limited to 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 effective focal spot of a STED CW microscope. Long-living states (red) are located at the center, whereas short-living ones (blue) are at the periphery.

STED in the 3rd dimension

Multiple STED light paths

With TCS SP8 STED 3X and TCS SP8 STED ONE, two light paths for xy and z are available at the instrument that generate different STED patterns. STED light can be steplessly distributed between the two paths.

Engineer your PSF

You can choose between best lateral resolution, best axial resolution or anything in between to get optimal results. Ultra-thin optical sections reveal unknown details. STED 3X and STED ONE enable you 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 Zimmermann, Center for Genomic Regulation, Barcelona, Spain

Surface rendered 3D reconstruction 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: the best lateral resolution is achieved by the classic vortex donut, the best axial resolution by the z donut. It can also be optimized for the smallest focal volume.

Investigate molecular dynamics: SP8 STED with FCS

STED provides a powerful tool to continuously tune the size of the observation area down to the nanoscale. For this reason, STED-FCS (fluorescence correlation spectroscopy) reports on the diffusion time, concentration, and binding constant of molecules even at higher concentrations (> 100 nM) than those possible with confocal FCS approaches.

STED-FCS can be combined with fluorescence lifetime imaging (FLIM) and related cross-correlation techniques (STED-FLCS, STED-FLCCS) enabling the study of different molecules whose fluorescence spectra may overlap.

STED calibration performed on lipid bilayers prepared with KK114. The autocorrelation curves obtained at increasing STED intensities (0%, 10%, 20%, 50% and 100%) are normalized at 1 μs. The shift of the autocorrelation curves indicates a decrease in the effective observation volume with STED. Courtesy of Christian Eggeling, University 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 nanoscopy data by capturing an image or starting a series.

Software solutions for STED – know your resolution

STED microscopy is the fast and direct way to nanoscopy. The LAS X gives you the greatest possible convenience. A sketch of the estimated effective PSF gives you direct and online visual feedback on the effects of your chosen technical parameters.

Intuitive workflow

The Smart STED Wizard allows you to control the instrument with three simple sliders. Assisted by the sketch of the effective PSF, you define the general level of resolution below the diffraction limit you need. For optimal results the wizard adjusts all the necessary settings, like STED laser intensity, pixel size, z step size, pinhole, gate settings and averaging.

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

Dr. Christian Eggeling, University of Oxford, UK

Interested to know more?

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