ZEISS LSM 980 with Airyscan 2

Your Next Generation Confocal for Fast and Gentle Multiplex Imaging

To monitor life as undisturbed as possible requires low labeling density for your biological models—for example, 3D cell culture, spheroids, organoids or even whole organisms—and this calls for 3D imaging that combines optical sectioning with low phototoxicity and high speed. Then there are the repeated experiment runs it takes to get statistically-valid data for your conclusions: it soon becomes apparent you will also need high throughput.

LSM 980 with Airyscan 2 is the ideal platform for your confocal 4D imaging, optimized for simultaneous spectral detection of multiple weak labels up to 900 nm emission with the highest light efficiency. Add Airyscan 2 with its Multiplex mode to get more imaging options for your experiments. Choose the perfect setup to gently image larger fields of view with super-resolution in shorter acquisition times than ever before. A number of software helpers will optimize your workflow and support efficient acquisition and data management.

ZEISS LSM 980 with Airyscan 2

Shaping High-end Imaging for Your Research

  • Employ a wealth of fluorescent labels from 380 nm to 900 nm. 
  • Enjoy spectral flexibility with up to 36 simultaneous channels. 
  • Acquire more information in less time with Airyscan 2 Multiplex.
  • Quickly find regions of interest with AI Sample Finder.
  • Get optimal imaging and detector settings with Smart Setup.
  • Extend your research with NLO, NIR, Cryo, and SIM² imaging.

Get Better Data Faster

Use the Multiplex mode for Airyscan 2 and get more information in less time. Smart illumination and detection schemes let you image your most challenging three-dimensional samples with high framerates beyond the diffraction limit and still treat your sensitive samples gently. By combining the full flexibility of a point scanning confocal with the speed and gentleness of the sensitive Airyscan area detector, it’s now possible to answer your scientific questions up to ten times faster with super-resolution.

Image: HeLa cells stained for DNA (blue, Hoechst 44432), microtubules (yellow, anti-tubulin Alexa 488) and F-actin (magenta, phalloidin Abberior STAR Red). Imaged with ZEISS Airyscan 2 in Multiplex mode for efficient super-resolution imaging of a large field of view. Courtesy of A. Politi, J. Jakobi and P. Lenart, MPI for Biophysical Chemistry, Göttingen, Germany.

HeLa cells stained for DNA (blue, Hoechst 44432), microtubules (yellow, anti-tubulin Alexa 488) and F-actin (magenta, phalloidin Abberior STAR Red)
Cos-7 cells, DAPI (blue) and Anti-Tubulin Alexa 700 (red). Alexa 700 imaged with NIR detector.

Image with More Sensitivity

LSM 980 brings you the best of two worlds to image your most challenging samples. You get the light efficient beam path of the LSM 9 family with up to 36 simultaneous channels for full spectral flexibility up to the near infrared (NIR) range. Plus, when you combine it with Airyscan 2, this revolutionary area detector extracts even more information from your sample in less time. This lets you image faint signals with 4 – 8 times higher signal-to-noise ratio (SNR). You don‘t need to close a pinhole to get super-resolution, which makes your 3D imaging even more light efficient.

Image: Cos-7 cells, DAPI (blue) and Anti-Tubulin Alexa 700 (red). Alexa 700 imaged with NIR detector. Sample courtesy of Urs Ziegler and Jana Doehner, University of Zurich, ZMB, Switzerland

Increase Your Productivity

It’s never been easier to set up complex confocal live cell imaging experiments. ZEN microscopy software puts a wealth of helpers at your command to achieve reproducible results in the shortest possible time.

AI Sample Finder helps you quickly find regions of interest, leaving more time for experiments. Smart Setup supports you in applying best imaging settings for your fluorescent labels. Direct Processing enables parallel acquisition and data processing. ZEN Connect keeps you on top of everything, both during imaging and later when sharing the whole story of your experiment.

ZEN BioApps: From beautiful images to valuable data – analyze your images efficiently.
ZEN BioApps: From beautiful images to valuable data – analyze your images efficiently.

The Airyscan Principle

Principle of airyscan

Classic confocal laser scanning microscopes use point illumination to scan the sample sequentially. The microscope optics transform each point to an extended Airy disk (Airy pattern). A pinhole then spatially limits this Airy disk to block out-of-focus light from reaching the detector. Closing the pinhole gives higher resolution, but at the price of detecting fewer photons – and these photons cannot be brought back by e.g. deconvolution.

Airyscan 2 is an area detector with 32 concentrically arranged detection elements. This allows you to acquire more of the Airy disk at once. The confocal pinhole itself remains open and does not block light, thus more photons are collected. This produces much greater light efficiency while imaging. Airyscan 2 gives you a unique combination of gentle superresolution imaging and high sensitivity.

How the New Multiplex Mode for Airyscan 2 Works

The LSM 9 family with Airyscan 2 from ZEISS now gives you more options to fit imaging speeds and resolution to your experimental needs. You combine a confocal area detector with smart illumination and readout schemes, which let you choose from different parallelization options. The new Multiplex mode uses knowledge about the shape of the excitation laser spot and the location of single area detector elements to extract more spatial information, even during parallel pixel readout. This allows taking bigger steps when sweeping the excitation laser over the field of view, improving your achievable acquisition speeds.

In fact, the high amount of spatial information captured in the pinhole plane allows reconstructing a final image with better resolution than the acquisition sampling. Airyscan 2 in Multiplex mode can acquire up to four superresolution image lines with high SNR in a single sweep. Your LSM 980 with Airyscan 2 allows to stretch the excitation laser spot to image eight lines in parallel. Use this speed advantage for ultrafast time series of single slices, for rapid tiling of large areas or for fast volumetric time-lapse imaging.

Watch the Multiplex mode animation trailer

LSM 980 Airyscan SR Multiplex SR-4Y Multiplex SR-8Y Multiplex CO-8Y
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Antibody labelling, fine structures

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Antibody labelling, tiling

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Live cell imaging

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Near Infrared (NIR) Imaging

Expand Your Spectral Range

Expanding your spectral range into the NIR allows you to use more labels in parallel. Visualize additional structures with more dyes in your multi-color experiments, with the Quasar and NIR detectors efficiently supporting spectral multiplexing experiments. NIR fluorescent labels are less phototoxic for living samples due to the longer wavelength. This allows you to investigate living samples for longer periods of time while limiting the influence of light. Additionally, light of longer wavelength ranges is less scattered by the sample tissue, increasing penetration depth.

For any of the advantages you pursue with NIR labels, the dual-channel NIR detector combines two different detector technologies (extended red GaAsP and GaAs) for optimal sensitivity up to 900 nm.

Typical spectral quantum efficiency (QE) of ZEISS LSM 980 detectors, including NIR.
Typical spectral quantum efficiency (QE) of ZEISS LSM 980 detectors, including NIR.
Cos-7 cells, DAPI (magenta), Anti-tubulin Alexa 568 (blue), Actin Phalloidin-OG488 (yellow) and Tom20-Alexa 750 (red).

Cos-7 cells, DAPI (magenta), Anti-tubulin Alexa 568 (blue), Actin Phalloidin-OG488 (yellow) and Tom20-Alexa 750 (red). Imaged in Lambda mode across the visible and NIR spectrum. Individual signals separated by Linear Unmixing. Maximum intensity projection of a z-stack.
Sample courtesy of Urs Ziegler and Jana Doehner, University of Zurich, ZMB, Switzerland.

Microtubules of Cos-7 cell (Anti-Tubulin AF700). Comparison of the ZEISS LSM 980 MA-PMT and the ZEISS NIR GaAsP detector; excitation with 639 nm laser at same laser power. Emission range for the MA-PMT is set to 660 – 757 nm, and for the NIR detector is 660 – 900 nm,
Sample courtesy of Urs Ziegler and Jana Doehner, University of Zurich, ZMB, Switzerland.

Multiphoton Microscopy with LSM 980 NLO

Multiphoton microscopy (two-photon microscopy, non-linear optical microscopy, NLO) is a preferred method for non-invasive and deep tissue imaging of living or fixed samples, particularly in neuroscience. Multiphoton microscopy capitalizes on the fact that longer wavelengths (600 – 1300 nm) are less absorbed and less scattered by tissues, travelling deeper into the sample while still forming a focal point. The required energy to excite a fluorescent dye is provided not by one photon but by two photons with half the energy each. The probability of two photons to reach the fluorophore at the same time is only sufficient at the focal point. That is why emission light originates from the focal plane and can be efficiently detected, generating an optical section while omitting a pinhole.

Energy diagram of two-photon microscopy
Energy diagram of two-photon microscopy
Combine confocal and multiphoton capabilities

An LSM that shares confocal and multiphoton capabilities gives you access to both technologies in the way that best suits your experiments:

  • Combine deep tissue penetration with enhanced sensitivity, resolution and speed.
  • Reduce light exposure and get clear separations of all emission signals
  • Efficiently collect light with high-sensitive GaAsP NDD detectors close to the signal 
  • Visualize non-stained structures with multiphoton excitation by second or third harmonic generation (SHG, THG).
     
Zebrafish brain vasculature
Zebrafish brain vasculature imaged in coronal orientation. Acquired with the two-photon laser excitation at 1,000 nm. The emission light was captured with the GaAsP BiG.2 non-descanned detector. Color coded of 293 µm z-stack. Sample courtesy of the Fish Facility, Leibniz-Institut für Alternsforschung – Fritz-Lipmann-Institut e.V. (FLI), Jena, Germany.
Mouse brain slice with neuronal cytoplasmic GFP label
Mouse brain slice with neuronal cytoplasmic GFP label. The 100 µm volume was acquired with two-photon laser excitation at 1,000 nm with the GaAsP BiG.2 non-descanned detector. The dataset was color coded for depth and an orthogonal projection was created with ZEN blue. Sample courtesy of Prof. J. Herms, LMU, Munich, Germany.

Applications

ZEISS LSM 980 at Work

Meiosis in starfish oocytes
The depth coding shows a subset of 52 μm. The movie shows the transport of chromosomes, labeled by Histone 1-Alexa 568, in a starfish oocyte undergoing meiosis. A z-stack of 67 μm was acquired every 2.4 seconds with Airyscan CO-8Y mode. Concomitant with chromosome transport, the nucleolus (the large spherical structure) is disassembling.

Meiosis in starfish oocytes
The rendering is a projection of the process along z-axis (maximum intensity) and time (color-coded projection); to illustrate the movement of the chromosomes within the volume of the nucleus. © Courtesy of P. Lenart, MPI for Biophysical Chemistry, Göttingen, Germany.

Oocytes store all the nutrients to support early embryonic development, and are therefore very large cells with a large nucleus. Oocytes need to divide before fertilization. How to make cell division work in this very large cell is the topic investigated by P. Lenart’s lab.
They have shown that, surprisingly, an actin network is required to collect chromosomes scattered in the oocyte nucleus. They are then handed over to microtubules, which capture chromosomes and align them on the spindle. The actin-driven and microtubule-driven transport phases have very different speeds and show other differentiating characteristics that can be distinguished by tracking chromosome motion.

This is a nice imaging challenge, because chromosomes are scattered in the spherical nucleus with a diameter of 80 μm and are transported over a period of approximately 15 minutes. Back in 2005 we could acquire stacks every 45 s, which was sufficient to distinguish actin- and microtubule-driven phases. Using the new, high resolution trajectories shown here we hope to learn about the details of the transport mechanism.

Peter Lenart

tissue explant of ependyma from the ventricular system of a mouse brain

This ZEN Connect project documents the experiment performed with the tissue explant of ependyma from the ventricular system of a mouse brain. All acquired data of the experiment session is kept in context. The overview images by camera and LSM allow to precisely record the localization of the acquired ciliary beating within the sample. The flow map of cilia generated flow along the ependymal wall is added as a reference.

overview of fluorescently labeled motile cilia on ependyma tissue explant from the mouse brain

An overview of fluorescently labeled motile cilia on ependyma tissue explant from the mouse brain is quickly acquired by tiling with Airyscan 2 in Multiplex CO-8Y mode to find regions of interest. Z-Stack displayed in colored depth coding. The exact position of the recorded motile cilia is documented.

Live imaging with 143 frames per second of fluorescently labeled motile cilia of brain ependyma. Acquired with Airyscan CO-8Y mode combining image quality and speed; for detailed analysis of ciliary beating direction and frequency. © Courtesy of G. Eichele, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.


© Courtesy of M. Paoli, Galizia Lab, University of Konstanz, Germany.

The brain, thoracic and abdominal ganglia of the cockroach are joined together by bilateral connective bundles of ascending and descending interneurons forming the ventral nerve cord. In this preparation, left and right connectives were individually labelled (Alexa 488: green, Alexa 647: magenta) posteriorly to the suboesophageal ganglion to observe the extension of their innervation within the different neurophils, and throughout the ipsi- and contralateral parts of the brain (DNA labelled with DAPI: cyan). Imaging was performed using Tiling and Stitching to capture the complete volume (3×2.3× 0.26 mm). 3D animation of the complete dataset was done with arivis Vision 4D, ideal for rendering and analyzing large datasets. The 4D viewer in arivis Vision 4D can be configured to adjust the appearance of individual channels independently to highlight specific features.

Theses settings, along with clipping planes or the varying opacity of individual channels, can be stored into key frames which the software automatically interpolates between to produce a seamless animation. These animations can be previewed and edited prior to producing high resolution video renders.


Zebrafish is a well establish model for studying development of the vascular system. Multiphoton imaging is a great way to capture the intricate vasculature patterns in zebrafish brain at great depth. Additionally, through the Second Harmonic Generation (SHG), structural information of the surrounding tissues can be captured without the need of additional labelling.


Zebrafish brain and eye vasculature (green) and Second Harmonic Generation (grey) in sagittal orientation. A volume of 267 μm was acquired with the two-photon laser at 1,000 nm and emission was detected with the GaAsP BIG.2 detector. SHG allowed the visualization of the tissue structures, such as the retinal cells and ocular muscles. Sample courtesy of the Fish Facility, Leibniz-Institut für Alternsforschung – Fritz-Lipmann-Institut e.V. (FLI), Jena, Germany.


NIR: Expand the number of labels
To capture the complex world of biology, the ability to expand the number of labels is a great advantage. LSM 980 can image simultaneously multiple labels, covering a wide emission range up to 900 nm. These Cos-7 cells were labelled with 4 different fluorophores, two of which have their emission peak in the near infrared range (NIR), Alexa 700 and Alexa 750. Utilizing the flexible LSM 980 Quasar and NIR detectors, all labels were imaged with optimal sensitivity.

Cos-7 cells Anti-TOM20 AF750 (red), Anti-Tubulin AF568 (cyan), Actin Phalloidin-OG488 (magenta), DAPI (yellow). Imaged with LSM 980 including the ZEISS NIR detector in channel mode. The fluorescent signals were separated by Linear Unmixing, facilitating clear separation between the spectrally overlapping dyes Alexa 700 and Alexa 750.

Cos-7 cells Anti-TOM20 AF750 (red), Anti-Tubulin AF568 (cyan), Actin Phalloidin-OG488 (magenta), DAPI (yellow).
Sample courtesy of Urs Ziegler and Jana Doehner, University of Zurich, ZMB, Switzerland.
Cos-7 cells Anti-TOM20 AF750 (red), Anti-Tubulin AF568 (cyan), Actin Phalloidin-OG488 (magenta), DAPI (yellow).

Navigate and correlate with ease
As the world of microscopy transitions gradually to larger samples, it becomes more important to maintain the positional context and keep a record of the areas captured. AI Sample Finder automatically classifies the sample carrier, identifies the sample, finds the focus, and creates a fast overview image using the T-PMT detector or camera. You can freely navigate using the overview image for orientation, and effortlessly move to the structures of interest. Making sure you only spend time imaging regions that hold information for your research. ZEN Connect correlates all data associated with the sample.

In this example, mouse intestinal tissue was labelled with three fluorophores covering an emission spectrum of 500 – 850 nm. AI Sample Finder automatically identified the carrier and created an overview image using the T-PMT to capture the Alexa 488 label. The overview image is used for sample navigation and identification of regions of interest. The ZEISS LSM 980 Quasar and NIR detectors were used to acquire images of the visible and invisible labels with optimal sensitivity.

Mouse intestine tissue section
Mouse intestine tissue section stained for Substance P (cyan, Alexa 488) labeling the presynaptic contacts in the enteric nervous system, HuC/D (yellow, Alexa 568) labeling the enteric neurons, and neuronal Nitric Oxide Synthase (nNOS, red, Alexa 750) labeling a sub-population of enteric neurons. Sample Courtesy of Pieter Vanden Berghe, LENS & CIC, University of Leuven, Belgium.

Multiphoton microscopy can be combined with 3D Tiling and Stitching in order to image large samples, such as this example of mouse cerebellum. Airyscan 2 imaging in Superresolution mode can be used to acquire superresolution images of specific areas of interest and can be seamlessly combined with two-photon imaging. ZEN Connect can bring all the information from your different experiments together, allowing you to map the high-resolution images on the larger structure, maintaining the context and simplifying your file organization.

Mouse brain cerebellum labelled with anti-calbinding (Alexa-568) and anti-GFAP (Alexa-488).
Mouse brain cerebellum labelled with anti-calbinding (Alexa-568) and anti-GFAP (Alexa-488).

Mouse brain cerebellum labelled with anti-calbinding (Alexa-568) and anti-GFAP (Alexa-488). The fluorophores were both excited with the two-photon laser at 780 nm and the emission spectra were simultaneously collected by the BIG.2 detector. 3D Tilling and Stitching were used to cover whole structure, and an orthogonal projection was created in ZEN Blue. Specific areas of interest were imaged with the Airyscan 2 detector in order to acquire high resolution images of the Purkinje cells. The Airyscan 2 datasets were processed and orthogonal projections were created with ZEN Blue. The individual superresolution images were aligned with the cerebellum using ZEN Connect. Sample courtesy of L. Cortes, University of Coimbra, Portugal.

Downloads

ZEISS LSM 980 with Airyscan 2

Your Next Generation Confocal for Fast and Gentle Multiplex Imaging

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The Basic Principle of Airyscanning

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ZEISS LSM 9 Family with Airyscan 2

Multiplex Mode for Fast and Gentle ConfocalSuperresolution in Large Volumes

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Beam Path of ZEISS LSM 980

Poster

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ZEISS LSM 980 ve Airyscan 2

Hızlı ve hassas multiplex görüntüleme için yeni nesil konfokal mikroskobunuz

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