Bioimaging Wiki

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<b>Location</b>: [Oeiras] 0B06 
<br><b>Manufacturer</b>: Prairie (now Bruker!)<br>
<b>Model</b>: Prairie Ultima <br>
<b>Nickname</b>: "Prairie"<br>
<b>Software</b>:  PrairieView 4.5 <br>
<b>Year</b>: 2008 <br>
<b>SN</b>: N/A <br>
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Data older than 3 months is automatically deleted!</b>
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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> XXX 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">  XXX Usage Statistics</a>
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===== Microscope overview =====

{{  :confocal_vs_2photon.png?0x300}}
A multi-photon, or 2-photon, microscope uses modules similar to those of a laser scanning confocal microscope: A laser source for sample excitation, a scanhead with galvo mirrors to scan the excitation beam, and photo-multiplier tubes to detect fluorescence signals. 2-photon imaging has advantages over confocal: the use of a near-infrared laser allows deeper penetration into tissues, the radiation is less energetic and potentially less phototoxic, and because the excitation effect is confined to the focal plane, de-scanning and pinholes are not necessary, so there are less light looses on the detection side.
This Prairie Ultima system is integrated with a custom modified fixed stage ready for intra-vital imaging, and 2photon is ideal for studies in thick tissue slices, in-vivo, or imaging of small organisms.

{{: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 institutional server using the desktop link.

===== System components =====

==== LASERs ====
^  Laser Unit  ^  Wavelength  ^
|  Coherent - Chameleon  |  650 - 1050 nm  |

==== Objectives ====
^  Magnification   ^  Model  ^  Immersion  ^  NA  ^  WD (mm)  ^  Reference  ^
|  20x  |  Olympus (now Evident!) XLUM FL Plan  |  Water (dipping)  |  1.00  |  2.00  |  [[https://www.thorlabs.com/thorproduct.cfm?partnumber=N20X-PFH]]  |


==== Filtersets (Ocular) ====

^  Filterset  ^  Reference  ^  Excitation  ^  Dichroic  ^  Emission  ^  Fluorochromes  ^
|  2p (pos1)  |  -  |  mirror  |  -  |  -  |  all  |
|  Green (pos3)  |  -  |  blue  |  -  |  ~525 nm  |  GFP, FITC, Alexa488  |
|  Orange (pos4)  |  -  |  green  |  -  |  ~580 nm  |  TRITC, Alexa568  |
|  Blue (pos5)  |  -  |  violet  |  -  |  ~460 nm  |  DAPI  |

==== Emission Filters (multi-photon) ====

{{::xxfiltersetDiagram.png  |}}
\\
^  Detector  ^  Filter  ^  Transmission  ^  Fluorochromes  ^
|  CH1 (PMT)  |  Red  |  640/680 nm  |  Alexa647, Cy5  |
|  Ch2  (GaAsP) |  Green  |  570-620 nm  |  mCherry, Alexa568, Cy3  |
|  Ch3  (GaAsP)  |  Far Red  |  500-550 nm  |  GFP, Alexa488, FITC  |
|  Ch4 (PMT)*  |  Blue  |  435-485 nm  |  DAPI*, CFP or SHG  |

*Ch4 can be used either in fluorescence mode, or alternatively as a BF ("DODT") channel or for SHG - ask staff

\\
**Upon request:**
\\ (requires changing filters and the dichroic beamsplitter)

UNDER CONSTRUCTION!!!

===== System Turn On Procedures =====

  * Check the cooler. The temperature should be 20 Cº. {{::img.png?100|}}
  * Turn on the fluorescence lamp {{1_img.png?100|}}
  * Switch the laser from "stand by" to "on" by turning the key clockwise (unit under the table)
  * Make sure the workstation is ON
  * Turn on the pre-amps for the detectors you will use (Ch2 and Ch3 pre-amp unit in shelf above monitor, and Ch1 and Ch4 below the keyboard. Note that any switching of the Ch1&2 pre-amps should be preceded by "override" for protetion).
  * Ask staff to change the Ch4 for DODT (BF) detector or fluorescence/SHG

===== System turn Off procedures =====

  * Transfer your data to the institutional server (data is deleted every 3 months)
  * Clean up immersion objective(s)
  * Remove all your samples and accessories and leave the working area ready for the next user

If there is another user for this microscope in the __next hour__:
  * Leave the fluorescent lamp and LASER "ON"
  * Inform staff you are leaving the system "ON"
 
Else:
  * Set the LASER to "Standby" (LASER key to the left)
  * Turn "OFF" all pre-amps (make sure to protect 1st for Ch1&2!
  * Turn "OFF" the fluorescence lamp
  * Turn "OFF" monitor, lights, and close the microscope curtains
  * Make sure you turn "OFF" any life support unit used (heating, CO2, O2...)

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