Point scanning confocal
Technique
© Carolin Bleese
The excitation beam path of a laser scanning confocal microscope focuses the excitation light to one diffratction limited spot in the specimen. Fluorescence light that is emitted from this spot is spatially filtered by a pinhole. In consequence, only light from a small 3D volume (voxel) that can be as small as 250nmx250nmx500nm (x,y,z) reaches the detector. By scanning this optical arrangement over the specimen, 2D optical sections can be aquired. Aquiring several 2D sections spaced in z allows to image 3D objects.
Use
Gold standard for imaging of optical sections, high-resolution 3D images and colocalisation studies. Living specimens often do not tolerate the high photon exposure. Reduced speed of image acquisition due to the point scanning principle.
Configuration
Stellaris I (3 OG)
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
For using the Stellaris UG, a prior training is required. The dates for the next introductions can be found in the following TeamUp calendar. Please use Sign Up to register for your preferred date. A TeamUp account is not required. For further questions regarding the introductions, booking, or usage, please contact Susanne Mühlbauer.
Configuration
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