leica_sp8_mp
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leica_sp8_mp [2020/01/20 16:50] bioimaging [System Turn On Procedures] |
leica_sp8_mp [2021/04/13 18:27] bioimaging [Objectives] |
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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> | ||
|   <br> |   <br> | ||
|   <br> |   <br> | ||
| - | → <a href="https://imm.medicina.ulisboa.pt/intranet/booking/146"><img src="/facility/bioimaging/lib/exe/fetch.php?media=date.png"> Leica SP8 MP Booking</a> | + | → <a href="https://my.agendo.science/calendar/?id=MzRjYWxlbmRhcnM="><img src="/facility/bioimaging/lib/exe/fetch.php?media=date.png"> Leica SP8 MP Booking</a> |
| <br> | <br> | ||
| → <a href="/facility/bioimaging/doku.php?id=leica_sp8_mp_quality"><img src="/facility/bioimaging/lib/exe/fetch.php?media=list-icon.png" width=18> Leica SP8 MP Quality Control</a> | → <a href="/facility/bioimaging/doku.php?id=leica_sp8_mp_quality"><img src="/facility/bioimaging/lib/exe/fetch.php?media=list-icon.png" width=18> Leica SP8 MP Quality Control</a> | ||
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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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| ==== LASERs ==== | ==== LASERs ==== | ||
| ^ 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 | **not working** | | + | | InSight DS+ Dual (multi-photon) | 680 - 1300 nm | 1.3 W | ok | |
| - | | ::: | 1041 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 | 2.5 | 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 | | + | |
| + | **Upon request:** | ||
| + | ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ Reference ^ | ||
| + | | 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) ==== | ||
| - | ^ Filterset ^ Reference ^ Excitation ^ Emission ^ Fluorochromes ^ | + | ^ Filterset ^ Reference ^ Excitation ^ Dichroic ^ Emission ^ Fluorochromes ^ |
| - | | Blue | I3 | 450-490 nm | > 515 nm | GFP, FITC, Alexa488 | | + | | Blue | I3 | 450-490 nm | 510 nm | > 515 nm | GFP, FITC, Alexa488 | |
| ==== Emission Filters (multi-photon) ==== | ==== Emission Filters (multi-photon) ==== | ||
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| Else: | Else: | ||
| * Clean up immersion objectives | * Clean up immersion objectives | ||
| - | * Set the infrared laser to ''standby'' (deactivate the infrared laser in the Currently Available Lasers window) | + | * Set the infrared laser to ''hibernate'' (deactivate the infrared laser in the Currently Available Lasers window) |
| * Close the **LAS X** software | * Close the **LAS X** software | ||
| * Turn of the 488 nm laser with the key switch in the flexible supply unit | * Turn of the 488 nm laser with the key switch in the flexible supply unit | ||
| Line 120: | Line 125: | ||
| ---- | ---- | ||
| - | [[resources|Back to the Resources page]] | + | [[resources|Back to the Equipment page]] |
leica_sp8_mp.txt · Last modified: 2024/01/28 12:05 by bioimaging
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