Leica Stellaris 5 Confocal Laser Scanning Microscope
The Leica STELLARIS 5 is an inverted point-scanning confocal microscope for high-resolution multichannel fluorescence imaging of fixed and living samples. The system combines tunable white-light laser excitation, five sensitive spectral Power HyD S detectors, conventional galvanometric and fast resonant scanning, and a fast SuperZ galvo stage for 3D and time-resolved imaging.
Capabilities
- Point-scanning confocal fluorescence microscopy on an inverted Leica DMi8 platform
- Pulsed white-light laser (WLL), tunable from 485–790 nm in 1 nm steps, plus 405 nm diode laser
- Five fully tunable Power HyD S spectral detection channels covering 410–850 nm
- Conventional galvanometric scanning and high-speed 8 kHz resonant tandem scanning
- Multichannel imaging, optical sectioning, Z-stacks, time series, sequential scanning and spectral imaging
- Fast Z-stacking with a SuperZ galvo stage
- TauSense lifetime-based contrast, gating and fluorophore separation
- LIGHTNING computational image restoration / super-resolution
- Navigator-based large-area acquisition, stitching and multi-position imaging
- Optional temperature- and CO₂-controlled stage-top incubation for live-cell and time-lapse imaging
Objectives
| Objective | NA | Application |
|---|---|---|
| 5× HC PL FLUOTAR | 0.15 | Low-magnification overview / large-area imaging |
| 10× HC PL FLUOTAR | 0.30 | Overview and large-area confocal imaging |
| 16× HC FLUOTAR L IMM CORR VISIR | 0.60 | Long-working-distance multi-immersion imaging; selected cleared samples |
| 20× HC PL APO IMM CORR CS2 | 0.75 | Multi-immersion confocal imaging of tissue and cellular samples |
| 40× HC PL APO GLYC CORR CS2 | 1.25 | High-NA glycerol-immersion confocal imaging |
| 63× HC PL APO OIL CS2 | 1.40 | Maximum-resolution confocal imaging |
Confocal fluorescence imaging
- Excitation is provided by a 405 nm diode laser and a pulsed white-light laser (WLL) tunable from 485–790 nm in 1 nm steps, allowing excitation wavelengths to be matched closely to the fluorophores used.
- Fluorescence is detected by five spectral Power HyD S detectors with freely adjustable detection windows across 410–850 nm; transmitted-light detection is also available.
- The 8 kHz tandem scanner can be switched between conventional galvanometric and resonant scanning. Resonant scanning is particularly suited to rapid imaging of dynamic or light-sensitive samples.
- TauSense provides fluorescence-lifetime-based contrast, gating and signal separation; spectral excitation, emission and excitation–emission scans are supported.
Excitation and detection settings should be selected according to the fluorophores and experimental requirements; CFM staff can assist with acquisition strategy for complex multicolor experiments.
Confocal live-cell imaging
- A Tokai Hit STXG-GSI2X-SET stage-top incubator can be installed on the SuperZ stage for long-term live-cell and tissue-culture imaging.
- Temperature control: ambient temperature to 50 °C; integrated digital gas mixer for CO₂ control from 5–20%; objective heater included.
- Compatible sample holders include 35 mm and 50/60 mm dishes and slide chambers.
Live-cell experiments should be discussed with CFM staff in advance so that incubation, sample mounting and acquisition settings can be planned appropriately.
Software
- LAS X STELLARIS Control Software for microscope control, image acquisition, processing, quantification and data export.
- Available acquisition/processing functionality includes TauSense, Dynamic Signal Enhancement, LIGHTNING Base, Navigator/Stitching/Mark & Find, Lambda and Lambda-Lambda scans, and Z-intensity compensation.
- LAS X 3D Visualisation, Dye Finder, LAS X Offline Software and LAS X Lightning Process are available for downstream processing and visualization.
- LIF viewing software: A limited free-of-charge viewer version of LAS X is available from the manufacturer website upon registration.
Typical applications
- Multichannel confocal fluorescence imaging of fixed samples or live-cell/time-lapse
- High-resolution 3D Z-stacks
- Fast resonant scanning of dynamic and light-sensitive samples
- Spectral or fluorescence-lifetime-based imaging for separation of overlapping fluorescence signals
- Large-area confocal tile scans and multi-position experiments
- LIGHTNING deconvolution
- 3D imaging of optically cleared samples
Publication requirement
Important:
The Leica Stellaris 5 was originally funded by means of a DFG Major Research Instrumentation Grant. Therefore, publications featuring data acquired at it must state this funding in the acknowledgments, including the DFG project number 533293533.
Suggested acknowledgement text for Leica STELLARIS 5 use:
“Imaging was performed at the Core Facility Microscopy (CFM), Central Institute of Mental Health (ZI/CIMH), using a Leica STELLARIS 5 confocal microscope, funded by the Deutsche Forschungsgemeinschaft (DFG/German Research Foundation) – project number 533293533.”
Furthermore, a PDF of the publication must be sent to the CFM management.
Sample Methods Section
“Confocal fluorescence images were acquired using a Leica STELLARIS 5 point-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with an inverted DMi8 stand, a pulsed white-light laser tunable from 485–790 nm and a 405-nm diode laser, and Power HyD S spectral detectors. Images were acquired using a [5x/0.15 / 10x/0.30 / 16x/0.60 / 20x/0.75/multi-immersion / 40x/1.25/glycerol / 63x/1.40/oil] objective and LAS X acquisition software v4.9.2.31016. [Single optical sections / Z-stacks with a z-step size of … µm / time series] were acquired using excitation wavelength(s) of […] nm and emission detection window(s) of […] nm. Images were acquired using [galvanometric / 8-kHz resonant] scanning at [scan speed / frame rate], with a pinhole diameter of […] Airy units, a pixel size of […] µm and [line/frame] averaging of […]. Where required, adjacent image fields were acquired and stitched using LAS X Navigator. [For live-cell experiments, samples were maintained at … °C and …% CO₂ using a Tokai Hit stage-top incubator.]”
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