Location: Room P2-B-42 ( 47236)
Manufacturer: Leica Microsystems
Model: TCS SP8 MP
Nickname: "SP8"
Software: LAS X
Software version: 3.5.7
Year: 2017
SN: 8100001758
 
Data will be deleted after: 3 months  
 
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===== Microscope overview ===== {{ :confocal_vs_2photon.png?0x300}} The Leica SP8 MP is a both a confocal and //multi-photon microscope// able to generate high-resolution three-dimensional images of thick specimens. Multi-photon excitation (most commonly two photon excitation) is particularly advantageous for imaging thicker samples. Rather than exciting the fluorophore with one photon, multi-photon excitation is produced by two or more lower energy photons which can penetrate deeper in the sample. Moreover, contrarily to confocal, the multi-photon excitation light will only achieve sufficient intensity to cause fluorescence in a specific region. Because of this, no pinhole is needed in multi-photon microscopy to exclude the light from out-of-focus planes and achieve optical sectioning. The Leica SP8 MP is an upright microscope equipped with water and glycerol immersion objectives especially suitable for intra-vital and thick samples immersed in water or glycerol imaging. In confocal mode, its scanning unit includes a spectral detector PMT to be used with a 488 nm laser for excitation. In multi-photon mode, its Insight DS+ Dual pulsed laser can be tuned from 680 to 1300 nm and has a second laser line emitting at 1041 nm, to be used with four non-descanned detectors with specific filtersets: two PMTS and two HyD detectors (hybrid detectors with 45% QE compared to ~25% QE for conventional PMT). {{:info.png?25|}} Click on the image on the right to see it in higher detail {{:warning.png?25|}} Data files older than **3 months** will be automatically deleted on this system, please copy your data to the iMM server using the desktop link. ===== System components ===== ==== LASERs ==== ^ Laser Unit ^ Wavelength ^ | InSight DS+ Dual (multi-photon) | 680 - 1300 nm | | ::: | 1040 nm | | SS OBIS 488-20 (confocal) | 488 nm | ==== Objectives ==== ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ Reference ^ | 25x | HC FLUOTAR VISIR | Water | 0.95 | 2.50 | [[https://www.leica-microsystems.com/objectivefinder/detail/objective/506374/|15506374]] | | 63x | HC PL APO Glyc CORR CS2 | Glycerol | 1.30 | 0.30 | [[https://www.leica-microsystems.com/objectivefinder/detail/objective/506353/|15506353]] | ==== Filtersets (Ocular) ==== ^ Filterset ^ Reference ^ Excitation ^ Dichroic ^ Emission ^ Fluorochromes ^ | Blue | I3 | 450-490 nm | 510 nm | > 515 nm | GFP, FITC, Alexa488 | ==== Emission Filters (multi-photon) ==== {{::default_configuration.png |}} \\ \\ \\ ^ Detector ^ Filter ^ Transmission ^ Fluorochromes ^ | HyD-RLD 1 | Red | 570-640 nm | mCherry, Alexa568, Cy3 | | HyD-RLD 2 | Green | 500-550 nm | GFP, Alexa488, FITC | | PMT-RLD 3 | Far Red | 662 - 737 nm | Alexa647, Cy5 | | PMR-RLD 4 | Red | 570-640 nm | mCherry, Alexa568, Cy3 | \\ \\ \\ \\ \\ **Upon request:** \\ (requires changing filters and the dichroic beamsplitter) {{::alternative_configuration.png |}} \\ \\ \\ ^ Detector ^ Filter ^ Transmission ^ Fluorochromes ^ | HyD-RLD 1 | Green | 500-550 nm | GFP, Alexa488, FITC | | HyD-RLD 2 | Blue | 415-485 nm | DAPI, Hoescht, Alexa350 | | PMT-RLD 3 | Far Red | 662 - 737 nm | Alexa647, Cy5 | | PMR-RLD 4 | Red | 570-640 nm | mCherry, Alexa568, Cy3 | \\ \\ \\ ===== System Turn On Procedures ===== * Check that the cooling and power supply for the infrared laser are ON (''InSight DeepSee READY'') {{::0_infrared_laser.png?100|}} * Turn on the fluorescence lamp {{1_metal_halide.png?100|}} * Turn on the HyD (Hybrid detectors) supply unit {{::2_hyd_supply.png?100|}} * Turn on the computer * Turn on the microscope electronics box {{::3_electronics_box.png?100|}} * Turn on the microscope control panel {{::4_control_panel.png?150|}} * Switch on the scanner power, laser power and turn the key switch to on {{::5_scanners_laser.png?120|}} * Login in to Windows with your Agendo credentials * Start the **LAS X** software ===== Microscope Turn Off procedures ===== If there is another user for this microscope in the __next hour__: * Leave the fluorescent lamp and lasers on * Clean up immersion objectives Else: * Clean up immersion objectives * Set the infrared laser to ''hibernate'' (deactivate the infrared laser in the Currently Available Lasers window) * Close the **LAS X** software * Turn of the 488 nm laser with the key switch in the flexible supply unit * Switch off the HyD supply unit * Switch off the microscope control panel * Shutdown the computer * Switch off the laser power and scanner power in the flexible supply unit * Turn off the microscope electronics box * Turn off the fluorescent lamp ---- [[resources|Back to the Equipment page]]