Bioimaging Wiki

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<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>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>Software</b>: LAS X<br>
<b>Software version</b>: 3.5.7<br>
<b>Year</b>: 2017 <br>
<b>SN</b>: 8100001758 <br>
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&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>
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&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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&rarr; <a href="/facility/bioimaging/doku.php?id=leica_sp8_mp_usage"><img src="/facility/bioimaging/lib/exe/fetch.php?media=chart_line.png">  Leica SP8 MP Usage Statistics</a>
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===== 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 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

{{: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  ^
|  InSight DS+ Dual (multi-photon)  |  680 - 1300 nm  |
| ::: |  1040 nm  |
|  SS OBIS 488-20 (confocal)  |  488 nm  |

==== 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

----
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