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--- leica_sp8_mp [2019/12/31 02:28]
bioimaging created
+++ leica_sp8_mp [2020/12/14 12:41]
bioimaging
@@ Line 6: / Line 6: @@
 <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">
-<b>Location</b>: Room P2-B-42 (<img src="/facility/bioimaging/lib/exe/fetch.php?media=telephone_icon.gif" 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> 47246) <br>
 <b>Manufacturer</b>: <a href="https://www.leica-microsystems.com/" target="_blank">Leica Microsystems</a><br>
-<b>Model</b>: TCS SP8 MP<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>
 &nbsp <br>
 &nbsp <br>
-&nbsp <br>
+&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>
-&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>
  <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>
@@ Line 26: / Line 26: @@
 </table>
 </html>
+===== 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 [[big_guy|Big Guy]] or [[colossus|Colossus]].
+{{: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  ^  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
 ----
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