ZEISS LSM 980 with Airyscan 2

Your Unique Confocal Experience for Fast and Gentle Multiplex Imaging

To analyze life with as little disturbance as possible, you must use low labeling density for your biological models. This requires excellent imaging performance combined with low phototoxicity and high speed. LSM 980, your platform for confocal 4D imaging, is optimized for simultaneous spectral detection of multiple weak labels with the highest light efficiency.

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.

Cos-7 cells imaged with LSM Plus, including the ZEISS NIR detector in channel mode.

A Unique Confocal Experience

A light-efficient beam path with up to 36 simultaneous channels and full spectral flexibility up to the near infrared (NIR) range give you the perfect basis for multi-color experiments with living samples. On top of this, LSM Plus effortlessly improves all your experiments. The unique combination of spectral imaging with improved signal-to-noise ratio and resolution enables lower laser power for your live cell experiments.

Image with More Sensitivity

Airyscan 2 allows you to do more than any conventional LSM detector. Each of its 32 detector elements collects additional information, while all of them together gather even more light, yielding super-resolution quantitative results. By adding structural information with Joint Deconvolution (jDCV), you can push resolution even further. Or use the Multiplex modes to get super-resolution information up to 10 times faster.

Drosophila germarium. Imaged with ZEISS Airyscan 2 followed by Joint Deconvolution.

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

Increase Your Productivity

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.

A Light-efficient Beam Path

Highest Sensitivity and Spectral Flexibility for Your Experiments

LSM 980 brings a great deal of freedom to your experimental setup. The LSM 980 beam path design ensures imaging with highest sensitivity, which is key to visualizing the lowest signal and resolving all structures, as well as spectral flexibility, allowing you to freely select fluorescent labels from 380 nm to the near infrared (NIR) range.

LSM Plus

Improving the Whole Confocal Experience

LSM Plus improves literally any confocal experiment with ease, independent of detection mode or emission range. Its linear Wiener filter deconvolution needs next to no interaction while still ensuring a reliable quantitative result. Just as in our time-tested Airyscan super-resolution processing, the underlying optical property information is adapted automatically based on objective lens, refractive index, and emission range.

Drosophila egg chambers stained for F-actin (Phalloidin, magenta) and DE-Cadherin (cyan).
Courtesy of T. Jacobs, AG Luschnig, WWU Münster; with T. Zobel, Münster Imaging Network, Germany

Apply LSM Plus with no extra effort and benefit from:

Enhanced signal to noise at high acquisition speed and low laser power – particularly useful for live cell imaging with low expression levels
Improved resolution of spectral data with up to 36 channels in a single scan
More spatial information and even greater resolution enhancement for bright samples that allow to close the pinhole of the LSM
Integrated workflows to combine the advantages of LSM Plus with Airyscan super-resolution imaging

Airyscan 2

A Unique Combination of Super-resolution Imaging and High Sensitivity

Airyscan 2 is an area detector with 32 circularly arranged detection elements. Each of these acts as a small pinhole, contributing to super-resolution information, while the complete detector area collects more light than the standard confocal setting. This produces much greater light efficiency while capturing enhanced structural information.

Airyscan 2 schematic beam path: (1) Mirror, (2) Emission filters, (3) Zoom optics, (4) Airy disk, (5) Airyscan detector

32 Views Mean More Information

Powerful Deconvolution with Airyscan jDCV

Each of the 32 Airyscan detector elements has a slightly different view on the sample, providing additional spatial information that makes Joint Deconvolution possible. This reduces the distance that can be resolved between two points even further – down to 90 nm. Your super-resolution experiments will benefit from an improved separation of single or multiple labels.

Mitochondria in an Arabidopsis thaliana cell. Comparing the confocal image with Airyscan SR and Airyscan Joint Deconvolution.

Budding yeast cells with protein localized to the mitochondrial inner membrane (green) and mitochondrial matrix (magenta).
Courtesy of K. Subramanian / J. Nunnari, University of California, Davis, USA

When we imaged endoplasmic reticulum and mitochondria and saw their very fine details after applying Airyscan Joint Deconvolution, we thought this is just super cool. The new option could be integrated within our imaging protocols very easily. We were amazed at how quickly the images were processed, which helped us make decisions while we were still imaging.

Dr. Kelly Subramanian, Post-Doctoral Scholar, Department of Molecular and Cellular Biology, UC Davis

The Multiplex Modes for Airyscan 2

Large Fields of View and Whole Sample Volumes in the Shortest Time

In Multiplex modes, Airyscan detector advantages are combined with adapted illumination and readout schemes, giving you a choice of different parallelization options. Multiplex modes use knowledge of 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 larger steps when sweeping the excitation laser over the field of view, improving acquisition speed. Capturing more spatial information in the pinhole plane allows final image reconstruction with better resolution than the acquisition sampling.

Courtesy of A. Politi, J. Jakobi and P. Lenart, MPI for Biophysical Chemistry, Göttingen, Germany.

Efficient super-resolution imaging of a large field of view: HeLa cells stained for DNA (blue, Hoechst 44432), microtubules (yellow, anti-tubulin Alexa 488) and F-actin (magenta, phalloidin Abberior STAR Red).

LSM 980	Airyscan SR	Multiplex SR-4Y	Multiplex SR-8Y	Multiplex CO-8Y
Parallelization	1	4	8	8
Resolution	120/120	140/140	120/160	Confocal or better
Max. fps at max. field of view	0.2 (Zoom 1.7)	1.0 (Zoom 1)	2.0 (Zoom 1)	9.6 (Zoom 1)
Antibody labelling, fine structures	+++++	++++	+++	++
Antibody labelling, tiling	++	++++	++++	+++
Live cell imaging	++	+++	++++	+++++

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.

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.

Simultaneous Spectral Imaging

Fast and Sensitive Separation of all Fluorescent Labels

To separate even highly overlapping signals or to remove autofluorescence, you can take a Lambda Scan using the complete detection range with up to 36 detectors, keeping both illumination and time required to a minimum. Improve spectral imaging along the complete wavelength range, including Online Fingerprinting, with LSM Plus.

The example shows a murine cremaster muscle, multi-color labeled with Hoechst (blue), Prox-1 Alexa488 (green), neutrophil cells Ly-GFP, PECAM1 Dylight549 (yellow), SMA Alexa568 (orange), VEGEF-R3 Alexa594 (red), platelets Dylight 649 (magenta). Acquired with 32-channel GaAsP detector using Online Fingerprinting.

Comparison of the improved SNR before and after LSM Plus processing.
Courtesy of Dr. S. Volkery, MPI for Molecular Biomedicine, Münster, Germany

Multiphoton Microscopy with LSM 980 NLO

Non-invasive, Deep Tissue Imaging of Living or Fixed Samples

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

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 hindbrain vasculature acquired with the two-photon laser excitation at 1,000 nm, processed with LSM Plus. Color coded 238 μm z-stack.

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.

Data Beyond Imaging

More Options for Your Research

Combining laser point illumination, linear scanning, and detectors that can capture the signal in photon counting mode make the LSM 980 more than an imaging device:

Raster Image Correlation Spectroscopy (RICS)
Fluorescence Correlation Spectroscopy (FCS)
Fluorescent Cross Correlation Spectroscopy (FCCS)
Förster Resonance Energy Transfer (FRET)
Fluorescence Recovery after Photobleaching (FRAP)
Fluorescence Lifetime Imaging Microscopy (FLIM)

Fluorescence Correlation Spectroscopy (FCS) principle. Trajectory of a fluorescent particle through the detection volume

Applications

ZEISS LSM 980 at Work

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 multiple labels simultaneously, 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. The zoom-in views illustrate how LSM Plus improves SNR and resolution.

Cos-7 cells Anti-TOM20 AF750 (red), Anti-Tubulin AF700 (cyan), Actin Phalloidin-OG488 (magenta), DAPI (orange).

Separated fluorescent signals. The comparison illustrates how LSM Plus improves SNR and resolution.

Powerful Deconvolution

Cockroach brain neurons (Alexa 488: yellow, Alexa 647: magenta) and DNA (Hoechst: cyan), without (left) and with LSM Plus (right).
Sample courtesy of M. Paoli, Galizia Lab, University of Konstanz, Germany

F-actin (Phalloidin) staining of a ring canal in the Drosophila egg chamber.
Courtesy of T. Jacobs, AG Luschnig, WWU Münster; with T. Zobel, Münster Imaging Network, Germany

Multiphoton microscopy

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). 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.

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.

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

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

3D and 4D imaging

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.

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 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.

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.

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.