NOTES: The chiller for the MaiTai multiphoton laser has FAILED therefore the MaiTai is currently out of service. The rest of the Leica SP5 2-photon microscope is working normally for now, so if your project does not require excitation at longer wavelengths (700nm+) it can continue. This includes intravital imaging without the multiphoton laser.

Leica swapped the Argon laser with the one on the SP5 STED. We recommend keeping the power level low (between standby and 20% under configurations->laser) to prolong the life of the laser.

The HyD2 detector is failing, signal detected is very low. We recommend avoiding this detector if possible.

Overview: This laser scanning confocal system features software selectable conventional or high-speed resonance scanner galvanometer system with three (chilled, high sensitivity) internal PMT detectors (spectral) and two external (NDD) PMT detectors. Six visible laser lines and a tunable NIR pulsed laser (Spectra Physics Mai Tai broadband 710-990nm) provide excitation. The system features turnkey operation and full software control in addition to the features listed below.

Location: KCBD 1250B

Training Contact: Lorraine Horwitz

Fluorophores this microscope can image:

• Blue (ex: DAPI, Alexa 405)
• Cyan (ex: CFP)
• Green (ex: GFP, Alexa 488)
• Yellow (ex: mCitrine, YFP)
• Orange (ex: Cy3, Alexa 543)
• Near Red (ex: Texas Red, Alexa 594)
• Far Red (ex: Cy5, Alexa 647)
• Near IR (possible but not ideal, ex: Alexa 700)
• 2-photon imaging of Blue, Cyan, Green, Yellow, Orange - NO LONGER IN SERVICE

Excitation Light Source(s):

• near UV laser at 405nm
• 5 line Argon laser at 458, 476, 488, 496 and 514nm
• HeNe laser at 561nm
• HeNe laser at 594nm
• HeNe laser at 633nm
• MaiTai laser tunable from 710 - 990nm - NO LONGER IN SERVICE

Emission Detection:

• Custom emission detection of any wavelength range between 410nm and 800nm
• 3 chilled photon multiplier tubes (PMTs)
• 2 hybrid GaAsP/PMT detectors (HyDs)
• 2 non-descanned detectors (NDDs)
• Transmitted light detector
• 8-, 12-, or 16-bit grayscale output

Objectives (with additional optical zoom possible):

• 20x / NA 0.8 dry
• 40x / 0.8 water
• 63x / 0.9 water
• 63x / 1.4 oil
• 100x / 1.46 oil
• 25x / NA 0.95 water (must be installed)
• Objectives listed under the SP5 STED description (must be installed)

Sample Chamber:

• Custom fit full incubation jacket (clear) for increased thermal stability during live cell experiments
• Inverted platform for imaging of slides or dishes
• LSM objective inverter device (allows system to be used as if it were an upright microscope platform)
• Automated XY stage for tiling / multipoint scanning
• "SuperZ" galvo focusing stage, 1.5 mm range. Demonstrated capability of three cell volumes per second (12 x 1-mm optical slices each)
• Chamber slide users see our chamber slide use warning before starting your cultures!

Special Features:

• FRET, deconvolution, and 3D software wizards
• Automated notch filters
• Spectra Physics Mai Tai broadband Ti:sapphire laser with 10W pump (tunable from 710 to 990 nm) (up to 2W output at 810 nm) - NO LONGER IN SERVICE
• Dual scanning galvanometer mirrors (max 16,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (4 channels). Standard scanner with beam park, bleaching, imaging to 8k x 8k (64 megapixels) per channel.
• AOTF control of visible laser lines, EOM and ND filter attenuation of NIR
• Excitation and emission scanning capability with linear unmixing
• Automatically optimized confocal pinhole apertures
• Transmitted detector with LWD and oil lenses and filters suited for SHG imaging