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leica_sp8_mp [2020/01/17 12:12] bioimaging [Microscope overview] |
leica_sp8_mp [2022/08/30 12:27] bioimaging [LASERs] |
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<a href="/facility/bioimaging/lib/exe/fetch.php?media=leica_sp8_mp_wiki_new.png"><img src="/facility/bioimaging/lib/exe/fetch.php?media=leica_sp8_mp_wiki_new.png" width=300></a> | <a href="/facility/bioimaging/lib/exe/fetch.php?media=leica_sp8_mp_wiki_new.png"><img src="/facility/bioimaging/lib/exe/fetch.php?media=leica_sp8_mp_wiki_new.png" width=300></a> | ||
<td style="border:0px solid white;"> <p style="line-height:1.8"> | <td style="border:0px solid white;"> <p style="line-height:1.8"> | ||
- | <b>Location</b>: <a href="/facility/bioimaging/lib/exe/fetch.php?media=leica_sp8_mp_map_new.png">Room P2-B-42</a> (<img src="/facility/bioimaging/lib/exe/fetch.php?media=phone_neg.png" width=15> 47246) <br> | + | <b>Location</b>: <a href="/facility/bioimaging/lib/exe/fetch.php?media=leica_sp8_mp_map_new.png">Room P2-B-42</a> (<img src="/facility/bioimaging/lib/exe/fetch.php?media=phone_neg.png" width=15> 47236) <br> |
<b>Manufacturer</b>: <a href="https://www.leica-microsystems.com/" target="_blank">Leica Microsystems</a><br> | <b>Manufacturer</b>: <a href="https://www.leica-microsystems.com/" target="_blank">Leica Microsystems</a><br> | ||
<b>Model</b>: <a href="https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-mp/" target="_blank">TCS SP8 MP</a><br> | <b>Model</b>: <a href="https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-mp/" target="_blank">TCS SP8 MP</a><br> | ||
<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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===== 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 [[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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{{: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 ^ | ||
+ | | InSight DS+ Dual (multi-photon) | 680 - 1300 nm | 1.3 W | | ||
+ | | ::: | 1040 nm | 1.5 W | | ||
+ | | SS OBIS 488-20 (confocal) | 488 nm | 20 mW | | ||
+ | |||
+ | ==== 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|}} | ||
+ | * Log in to Windows (Bioimaging User) | ||
+ | * 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 Resources page]] | + | [[resources|Back to the Equipment page]] |