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leica_sp8_mp [2020/06/01 19:23] bioimaging |
leica_sp8_mp [2021/08/06 19:17] bioimaging [System Turn On Procedures] |
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<b>Nickname</b>: "Multiphoton"<br> | <b>Nickname</b>: "Multiphoton"<br> | ||
<b>Software</b>: LAS X<br> | <b>Software</b>: LAS X<br> | ||
- | <b>Software version</b>: 3.5.5.19976<br> | + | <b>Software version</b>: 3.5.7<br> |
<b>Year</b>: 2017 <br> | <b>Year</b>: 2017 <br> | ||
<b>SN</b>: 8100001758 <br> | <b>SN</b>: 8100001758 <br> | ||
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{{ :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]]. | {{ :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]]. | ||
+ | |||
+ | [[https://www.drbio.cornell.edu/cross_sections.html|Two-photon action cross sections - Cornell University]] | ||
{{: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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^ Laser Unit ^ Wavelength ^ Maximum Power ^ Current Status ^ | ^ Laser Unit ^ Wavelength ^ Maximum Power ^ Current Status ^ | ||
| InSight DS+ Dual (multi-photon) | 680 - 1300 nm | 1.3 W | ok | | | InSight DS+ Dual (multi-photon) | 680 - 1300 nm | 1.3 W | ok | | ||
- | | ::: | 1040 nm | 1.5 W | **not working** | | + | | ::: | 1040 nm | 1.5 W | ok | |
| SS OBIS 488-20 (confocal) | 488 nm | 20 mW | ok | | | SS OBIS 488-20 (confocal) | 488 nm | 20 mW | ok | | ||
==== Objectives ==== | ==== Objectives ==== | ||
^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ Reference ^ | ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ Reference ^ | ||
- | | 25x | HC FLUOTAR VISIR | Water | 0.95 | 2.50 | 15506374 | | + | | 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 | 15506353 | | + | | 63x | HC PL APO Glyc CORR CS2 | Glycerol | 1.30 | 0.30 | [[https://www.leica-microsystems.com/objectivefinder/detail/objective/506353/|15506353]] | |
==== Filtersets (Ocular) ==== | ==== Filtersets (Ocular) ==== | ||
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* Turn on the microscope electronics box | * Turn on the microscope electronics box | ||
{{::3_electronics_box.png?100|}} | {{::3_electronics_box.png?100|}} | ||
- | * Turn on the microscope's control panel | + | * Turn on the microscope control panel |
{{::4_control_panel.png?150|}} | {{::4_control_panel.png?150|}} | ||
* Switch on the scanner power, laser power and activate the key switch to turn on the 488 nm laser in the flexible supply unit | * Switch on the scanner power, laser power and activate the key switch to turn on the 488 nm laser in the flexible supply unit | ||
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- | [[resources|Back to the Resources page]] | + | [[resources|Back to the Equipment page]] |