leica_sp8_mp
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leica_sp8_mp [2020/01/16 18:36] bioimaging [Microscope overview] |
leica_sp8_mp [2020/01/20 16:44] bioimaging [System Turn On Procedures] |
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| ===== Microscope overview ===== | ===== 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 1040 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). With this system you can perform optical sectioning high resolution imaging of fluorescent samples that are two thick for confocal microscopes such as the [[zeiss_lsm_880|Zeiss LSM 880]] or the [[zeiss_lsm_710|Zeiss LSM 710]], albeit with a slightly lower resolution. If you do not need optical sectioning and your sample is thin, check out a widefield system instead, such as the [[zeiss_cell_observer|Cell Observer]] or the [[nikon_eclipse_ti|Nikon Eclipse Ti]. If your personal computer cannot handle all the data you collected, check out the [[big_guy|Big Guy]] or [[colossus|Colossus]]. | + | {{ :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). With this system you can perform optical sectioning high resolution imaging of fluorescent samples that are two thick for confocal microscopes such as the [[zeiss_lsm_880|Zeiss LSM 880]] or the [[zeiss_lsm_710|Zeiss LSM 710]], albeit with a slightly lower resolution. If you do not need optical sectioning and your sample is thin, check out a widefield system instead, such as the [[zeiss_cell_observer|Cell Observer]] or the [[nikon_eclipse_ti|Nikon Eclipse Ti]]. If your personal computer cannot handle all the data you collected, check out the [[big_guy|Big Guy]] or [[colossus|Colossus]]. |
| {{:info.png?25|}} Click on the image on the right to see it in higher detail | {{:info.png?25|}} Click on the image on the right to see it in higher detail | ||
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| {{: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. | {{: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 ^ Maximum Power ^ Current Status ^ | ||
| + | | InSight DS+ Dual (multi-photon) | 680 - 1300 nm | 1.3 W | **not working** | | ||
| + | | ::: | 1041 nm | 1.5 W | **not working** | | ||
| + | | SS OBIS 488-20 (confocal) | 488 nm | 20 mW | ok | | ||
| + | |||
| + | ==== Objectives ==== | ||
| + | ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ Reference ^ | ||
| + | | 25x | HC FLUOTAR VISIR | Water | 0.95 | 2.5 | 15506374 | | ||
| + | | 63x | HC PL APO Glyc CORR CS2 | Glycerol | 1.30 | 0.30 | 15506353 | | ||
| + | |||
| + | ==== Filtersets (Ocular) ==== | ||
| + | |||
| + | ^ Filterset ^ Reference ^ Excitation ^ Emission ^ Fluorochromes ^ | ||
| + | | Blue | I3 | 450-490 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's control panel | ||
| + | {{::4_control_panel.png?150|}} | ||
| + | * Switch on the scanner power, laser power and activate the key switch to turn on the 488 nm laser | ||
| + | {{::5_scanners_laser.png?120|}} | ||
| + | * Log in to Windows (Bioimaging User) | ||
| + | * Start the **LAS X** software | ||
| ---- | ---- | ||
| [[resources|Back to the Resources page]] | [[resources|Back to the Resources page]] | ||
leica_sp8_mp.txt ยท Last modified: 2024/01/28 12:05 by bioimaging
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