Point scanning confocal

Technik

Vergrößern

der Anregungsstrahl eines Laserscanning Confocal Mikroskop fokussiert das anregungslicht auf einen beugungsbegrenzten Punkt in der Probe. FLuoreszenzlicht, das von diesem Punkt abgestrahlt wird räumlich durch eine Lochblende gefiltert. Als Konsequenz wird nur Licht eines kleinen 3D Volumens (Voxel), das bis zu Größen von 250nmx250nmx500nm(x,y,z) reichen kann, vom Detektor erreicht wird. Durch Abtasten der optischen Anordnung über diese Probe können 2D optische Schnitte aufgenommen werden.

Nutzung

Goldstandard um optische Schnitte aufzunehmen, 3D Bilder mit hoher auflösung und Kolokalisationsstudien. Lebendproben tolerieren of die hohe Photonenbelastung nicht. Geringere Geschwindigkeit der Bildaufnahme aufgrund des Point Scanning Prinzip.

Konfiguration

Stellaris I (3OG)

Manufacturer:
Leica Microsystems
Model: Stellaris 5 (Point Scanning Confocal Microscope)

Applications:
• fixed samples (fluorescently labelled)
• live-cell imaging

Body:
• Leica DMi8 platform
• Inverted
• LED light source - filter cubes:
- DAPI
- GFP
- RHOD
• Brightfield light source
• Transmitted light detector
• Motorized xyz scanning stage
- Universal flexible specimen frame. - Live-cell imaging chambers (temperature, CO2 and humidity control)
Plate
3 cm dish

Lasers:
• Tunable White Light Laser Stellaris 5 (485 – 685 nm)
• 405nm laser

Detectors:
5 Power Hybrid HyD S detectors
• Analog, Reflection and Photon Counting detection mode
• Tau Sense capabilities: TauGating, TauContrast, TauSeparation

Objectives:

• 10X HC PL FLUOTAR (NA 0.3; DRY; WD 11 mm)
• 20X HC PL APO (NA 0.75; IMM Water, Gly, Oil; WD 0.66 mm; CS2)
• 40X HC PL APO (NA 1.1; WATER; WD 0.65 mm; CS2)
• 63X HC PL APO (NA 1.4; OIL; WD 0.14 mm; CS2)

Scanner, Speed, Resolution:

• 8 kHz Tandem Scanner – switchable between:
o Non-resonant mode:
• confocal point scanner for highest axial resolution (adjustable speed 1-2600 Hz)
• up to 10 fps 512 x 512 px
• up to 5200 lines/second
• max resolution 8192 x 8192 px
• zoom 0.75X – 48X
• scan field: 22 mm diagonal

o Resonant mode:
• adjustable speed up to 8 000 Hz

• up to 28 fps at 512 x 512 px
• max resolution 2496 x 2496 px with up to 3 channels simultaneously
• zoom 1.25X – 48X • scanfield: 13 mm diagonal

Okolab incubation chamber:

• Temperature control
• Humidity control
• CO2 control

*The system is equipped with active liquid cooling and mounted on an air table.

Software:
• LAS X
• Image Compass: routine multichannel/Z-stack acquisition
• Navigator basic package: tile scan, merge
• LAS X 3D Visualization
• TauSense capabilities: TauGating, TauContrast, TauScan
• Dynamic Signal Enhancement
• LIGHTNING basic detection package
• LAS X Live Data Mode
• LAS X Assay Editor
• LAS X Dye Finder

Konfiguration

Stellaris II (UG)

Manufacturer:
Leica Microsystems
Model: Stellaris 5 (Point Scanning Confocal Microscope)

Applications:
• fixed and unfixed samples (immunofluorescence or fluorescence reporters)
• Microscope live-cell imaging

Body:
• Leica DMi8 CS Premium platform on pressured air table
• Inverted
• LED light source - filter cubes:
- CFP
- GFP
- YFP
- DsRed
• Brightfield light source
• Transmitted light detector
• Motorized xyz scanning stage
- Universal flexible specimen frame

Lasers:
• STELLARIS 5 extended pulsed White light laser: 485nm - 790nm
• diode lasers 405nm and 448nm

Detectors:
5 Power Hybrid HyD S detectors
• Analog, Reflection and Photon Counting detection mode
• Tau Sense capabilities: TauGating, TauContrast, TauSeparation

Objectives:
• 10X HC PL FLUOTAR (NA 0.3; DRY; WD 11 mm)
• 20X HC PL APO (NA 0.75; IMM Water, Gly, Oil; WD 0.62 mm; CS2)
• 25X HC FLOUTAR (NA 0.95; IMM Water, WD 2.5 mm; W)
• 40X HC PL APO (NA 1.1; Water; WD 0.62 mm; CS2)
• 63X HC PL APO (NA 1.2; Water/SALT Water; WD 0.3 mm; CS2)
• 63X HC PL APO (NA 1.3; Gly; W/ 0.3 mm; CS2)
• 100X HC PL APO (NA 1.4; OIL; WD 0.13 mm; CS2)

Scanner, Speed, Resolution:

• 8 kHz Tandem Scanner – switchable between:
o Non-resonant mode:
• confocal point scanner for highest axial resolution (adjustable speed 1-2600 Hz)
• up to 10 fps 512 x 512 px
• up to 131 fps 512 x 16 px
• up to 5200 lines/second
• max resolution 8192 x 8192 px
• zoom 0.75X – 48X
• scan field: 22 mm diagonal

o Resonant mode:
• Line frequency 16000 Hz (bidirectional)
• up to 28 fps at 512 x 512 px
• up to 290 fps at 512 x 16 px
• max resolution 2496 x 2496 px with up to 3 channels simultaneously
• zoom 1.25X – 48X

Software:
• LAS X
• Image Compass: routine multichannel/Z-stack acquisition
• Navigator basic package: tile scan, merge
• LAS X 3D Visualization
• TauSense capabilities: TauGating, TauContrast, TauScan
• Dynamic Signal Enhancement
• LIGHTNING basic detection package
• LAS X Live Data Mode
• LAS X Assay Editor
• LAS X Dye Finder
• LAS X MicroLab (for FRAP and FRET experiments)

Aivia Go
• Semi-automated analysis and editing tools
• 3D objects generated
• Pixel and object colocalization analysis
• Render multi-channel 2 to 4D images of up to 3 TB
• Virtual reality integration for interactively navigating