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leica_sp8_mp [2020/01/16 18:36]
bioimaging [Microscope overview]
leica_sp8_mp [2020/07/09 03:39] (current)
bioimaging
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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>:​ 3.5.6<br>
 <​b>​Year</​b>:​ 2017 <br> <​b>​Year</​b>:​ 2017 <br>
 <​b>​SN</​b>:​ 8100001758 <br> <​b>​SN</​b>:​ 8100001758 <br>
 &nbsp <br> &nbsp <br>
 &nbsp <br> &nbsp <br>
-&rarr; <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>​+&rarr; <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> ​
 &rarr; <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>​ &rarr; <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 [[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  |  ok  |
 +| ::: |  1040 nm  |  1.5 W  |  ok  |
 +|  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  |  2.50  |  15506374 ​ |
 +|  63x  |  HC PL APO Glyc CORR CS2  |  Glycerol ​ |  1.30  |  0.30  |  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'​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 in the flexible supply unit
 +{{::​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'​s 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 Resources page]]
leica_sp8_mp.1579196217.txt.gz ยท Last modified: 2020/01/16 18:36 by bioimaging