Zeiss LSM 980 with Airyscan 2

Confocal · Zeiss

Our most advanced confocal system. Combines super-resolution Airyscan 2 detection, AI-assisted navigation, multiphoton excitation, fluorescence lifetime imaging, and spectral unmixing — all with live cell environmental control.

Instrument Features

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Airyscan 2 Detector

32-element GaAsP detector array delivers ~1.7× lateral resolution improvement over conventional confocal. Multiplex mode enables fast simultaneous multi-channel Airyscan imaging with full sensitivity.

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AI Sample Finder

AI-powered automated detection of cells or regions of interest across the slide. Dramatically reduces setup time by automatically positioning the system over relevant areas without manual scanning.

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2-Photon Laser

Tunable near-infrared Ti:Sapphire laser for two-photon excitation. Enables deep tissue imaging (up to several hundred microns) with reduced phototoxicity and improved signal-to-background in thick specimens.

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B&H FLIM System

Becker & Hickl Time-Correlated Single Photon Counting (TCSPC) module for fluorescence lifetime imaging. Enables FRET measurements, environmental sensing (pH, ion concentration), and separation of spectrally overlapping fluorophores by lifetime.

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Spectral Detector

34-channel spectral detector for full emission spectrum acquisition. Enables linear unmixing of multiple fluorophores with overlapping spectra and separation of autofluorescence from specific signal.

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Live Cell Incubation

Full environmental control including temperature (37°C), CO₂ (5%), and humidity. Suitable for long-term time-lapse imaging, drug treatment experiments, and live cell dynamics studies.

Startup Guide

1

Power On the System & locat sample

  1. Live imaging only: turn on the incubation system first. It must be on before the software starts so that ZEN recognises it.
  2. Press the single on/off switch on the microscope.
  3. Open ZEN Blue. No special login is needed.
  4. During start up the stage may ask you to calibrate for the AI Sample Finder. Skip it. Calibration is performed by Adeela only.
  5. ZEN Blue starts in “Locate” tab
  6. Find & focus sample with low power objective (5x or 10x) and then move to final objective, add immersion as needed.
    1. NOTE if you plan on using AI sample finder you can skip this step
2

Set up imaging parameters

  1. Go to the Acquisition tab.
  2. Reuse (recommended): load the saved configuration made with Adeela, or open a previous image and click Reuse
  3. Smart Setup: click Smart Setup and choose LSM Confocal or Airyscan. Add each dye with Dyes and Contrast Methods, then click OK. The Smartest option reduces bleed through and hardware changes between tracks, and the software may switch to multitrack if the dyes are too similar. In Airyscan mode you will select between “speed and resolution”, as Adeela for recommendations
  4. In Channels, select one track at a time, click Live, and turn on the Range Indicator so red pixels show saturation. Reset the display before judging saturation
  5. Set laser power as low as gives a usable image. Values of 0.1 to 5% are suggested to limit bleaching.
  6. Set detector gain. Ideally in the low 700 range for Airyscan. Adjust gain to up to 800 before laser power, and keep digital gain at 1.0
  7. Set the pinhole to 1 AU for confocal, and keep pinholes identical across channels if you compare them, because section thickness differs otherwise. For Airyscan SR/MPLX the pinhole is not adjusted
  8. Set image size and pixel size. Confocal sampling follows Nyquist, and scan speed of 6 or 7 is average. Use 16 bit depth.
  9. Bidirectional scanning halves the scan time but can show a phase shift. Use Auto or the Correction X slider to fix it if that occurs.
  10. Keep averaging low, below 4 for confocal and 2 for Airyscan, since it slows acquisition.
  11. Press Continuous to check all ticked tracks and to make sure “Airyscan” detector is aligned. The “AIryscan” icon on the bottom will turn green when ready to image. NOTE the DAPI doesn’t always align fully, just make sure other channels are aligned before beginning
3

Process Airyscan data in ZEN Blue either on LSM 980 computer or ZEN Blue Airyscan workstation

Airyscan images need processing before they reach full resolution. Do it before stitching, orthogonal projections or unmixing.

  1. Open the raw Airyscan image and go to the Processing tab.
  2. Choose Airyscan Processing. Use 2D SR for single planes, or the 3D option for z stacks, with Auto Filter ticked.
  3. Select your image and click Apply. The result opens as a new image.
  4. For manual control, filter strength 1 gives pixel reassignment with weak filtering, about 1.4x the confocal resolution. Auto applies conservative deconvolution, up to about 2x. Too strong a setting creates structured noise [3].
  5. Process each channel’s data the same way when comparing channels.
  6. Tiled data: process with Airyscan first, then stitch. In Processing choose Stitching, tick New Output and Fuse Tiles, and use the channel with the brightest, most extensive signal as the reference.
  7. Save the processed image as CZI and keep the raw file.

Choose the Airyscan mode

ModeUse it for
SRMaximum resolution and signal to noise, about 120 x 120 x 350 nm at 512 x 512
SR-4Y (Multiplex)Faster super resolution, about 140 x 140 x 450 nm at 25 fps. This is the recommended Multiplex setting
SR-8Y (Multiplex, LSM 980 only)Fastest, about 120 x 160 x 450 nm at 47.5 fps [3]. Available on the LSM 980, but 4Y is the optimal setup
CO (confocal Airyscan)Improved signal to noise at about 1x Nyquist, NOT super resolution
4

Extra LSM 980 features

These features exist only on the LSM 980. Speak to Adeela before using any of them.

  • Incubation. Turn the incubation system on before starting the system so that ZEN recognises it.
  • Spectral detector. The LSM 980 has a spectral detector for linear unmixing. This is separate from the Airyscan detector. See Adeela if you want to perform linear unmixing.
  • FLIM. The system has a Becker and Hickl FLIM system. See Adeela if you want to perform FLIM.
  • Two photon laser. The LSM 980 has a two photon laser. It works best in confocal mode, but it can also be used in Airyscan mode.
  • SR-8Y Multiplex. Available on this system, but 4Y is the optimal setup.
5

References

  1. Max Perutz Labs, LSM 980 quick guide (Zeiss)
  2. HKU CPOS, LSM 900 with Airyscan 2 SOP (April 2026)
  3. Queensland Brain Institute, LSM 980 guide (2025)
6

Shutting down

  1. Clean oil off objectives and save data for processing
  2. Exit out of ZEN
  3. Turn off incubation if used
  4. Check iLabs schedule in and if anyone is coming in within an hour leave system on
  5. If no one is coming in for more than an hour turn “on/off” switch to off position and cover microscope

⚠️ For FLIM experiments, contact Adeela before your session for additional setup steps specific to the B&H FLIM module.

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