Bioimaging Wiki

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<b>Location</b>: [Oeiras] Room 0B06<br> 

<b>Manufacturer</b>: <a href="http://www.zeiss.de/micro" target="_blank">ZEISS Microscopy</a><br>

<b>Model</b>: <a href="https://www.zeiss.com/microscopy/en/products/light-microscopes/confocal-microscopes/lsm-980-with-airyscan-2.html">LSM 980 with Airyscan 2</a><br>
<b>Nickname</b>: "980"<br>

<b>Software</b>: ZEN 3.3 <br>
<b>Year</b>: 2019<br>
<b>SN</b>:  <br>
&nbsp <br>
Data will be deleted after: <b>1 month</b>
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&nbsp <br>
&rarr; <a href="/facility/bioimaging/doku.php?id=oeiras_lsm_980_quality"><img src="/facility/bioimaging/lib/exe/fetch.php?media=list-icon.png" width=18> ZEISS LSM 980 with Airyscan 2 Quality Control</a>
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&rarr; <a href="/facility/bioimaging/doku.php?id=oeiras_lsm_980_usage"><img src="/facility/bioimaging/lib/exe/fetch.php?media=chart_line.png">  ZEISS LSM 980 with Airyscan 2 Usage Statistics</a>
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===== Microscope overview =====

{{  :confocal_pinhole.png?340}} The Zeiss LSM 980 with Airyscan 2 is a [[https://www.youtube.com/watch?v=rTkjQ6Q0p5I|point scanning confocal microscope]] is an inverted microscope equipped with a laser scanning head with a total of 6 PMT/GaAsp + 1 Airyscan detectors. The Airyscan can achieve non-diffraction limited optical sectioning with resolutions down to 140nm laterally and ~400nm axially. It also allows acquisition in “Multiplexing” mode(s), which accelerate the acquisition speed (4/8x faster) or in sensitivity mode that enhances the signal-to-noise acquisition.

The microscope is also equipped with a dark heating and atmospheric control chamber to allow live imaging of mammalian cells (O2 control is also possible, upon request). We have available an objective inverter for some intra-vital or open-dish applications (upon request only, requires preparation). 

  * **Microscope**: [[https://www.zeiss.com/microscopy/en/products/light-microscopes/widefield-microscopes/axio-observer-for-life-science-research.html|Zeiss Axio Observer]]
  * **LED Illumination**: [[https://www.zeiss.com/microscopy/en/products/accessories/colibri.html|Zeiss Colibri 7]]
  * **Features**
    * |  [[https://www.imdik.pan.pl/images/Us%C5%82ugi_dokumenty/Przydatne_informacje/2d_Definite_Focus_information.pdf|Definite Focus.3]]
    * | [[https://www.youtube.com/watch?v=tUiEa7vcggQ|Airyscan 2 Multiplex]]
    * | [[https://www.zeiss.com/microscopy/en/about-us/newsroom/press-releases/2021/zeiss-lsm-plus-and-zeiss-airyscan-joint-deconvolution.html|LSM Plus]]
    * | [[https://www.zeiss.com/microscopy/en/about-us/newsroom/press-releases/2021/zeiss-lsm-plus-and-zeiss-airyscan-joint-deconvolution.html|Joint Deconvolution]]

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<p align="center"><iframe width="420" height="236" src="https://www.youtube.com/embed/Ji_PVld77Gs" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture" allowfullscreen></iframe></p>
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  * [[https://www.youtube.com/watch?v=zA4xqq-EL9g|ZEISS Webinar: LSM 980 Airyscan 2 Multiplex mode]]

{{:warning.png?25|}} Data files older than **1 month** will be automatically deleted on this system, please copy your data to the GIMM server using the desktop link.

===== Booking Rules =====
  - **Users** can book at maximum **8 hours per week**
  - This usage restriction does not apply for **weekends** and for working days **before 9:00** and **after 21:00**
  - Exceptions to these rules require **approval from [[joserino@medicina.ulisboa.pt|José Rino]]**.

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

==== Lasers =====

^  Laser ^  Excitation line  ^  Power  ^  Description (use)   ^   
|  <fc #ff00ff>405 nm (violet)</fc>  |  405 nm  |  15 mW  |  <fc #00ffff>DAPI, Hoechst</fc>   |  
|  <fc #6495ed>488 nm (blue)</fc>  |  488 nm  |  13 mW  |  <fc #00ff00>GFP, FITC, Alexa488</fc>   |  
|  <fc #00ff00>561 nm (green)</fc>  |  561 nm  |  13 mW  |  <fc #ffa500>RFP, TRITC, Cy3, Dylight Orange</fc>   |  
|  <fc #ff0000>639 nm (red)</fc>  |  639 nm  |  10 mW  |  <fc #cd5c5c>Cy5, APC, Alexa/ATTO633-647</fc>   |  

If you require more laser lines (eg, 445nm - CFP/Cerulean, 514nm - YFP, or 594nm mCherry/AF594), we suggest as an alternative the LSM980-32 in Lisbon:
https://imm.medicina.ulisboa.pt/facility/bioimaging/doku.php?id=zeiss_lsm_980


==== Objectives ====
^  Magnification   ^  Model  ^  Immersion  ^  NA  ^  WD (mm)  ^  Reference  ^
|  2.5x  |    |  -  |  0.085  |  8.8  |  [[https://www.micro-shop.zeiss.com/en/us/shop/objectives/420320-9902-000/Objective-EC-Plan-Neofluar-2.5x-0.085-M27|420320-9902-000]]  |
|  10x  |  EC Plan-Neofluar  |  Air  |  0.45  |  2.0  |  [[https://www.micro-shop.zeiss.com/en/ee/shop/objectives/420640-9900-000/Objective-Plan-Apochromat-10x-0.45-M27|420640-9900-000]]  |
|  20x  |  Plan-Apochromat  |  Air  |  0.75  |  0.55  |  [[https://www.micro-shop.zeiss.com/en/ee/shop/objectives/420150-9900-000/Objective-Fluar-20x-0.75-M27|420150-9900-000]]  |
|  25x  |    |  W/Sil/Gly/Oil*  |  0.80  |  0.57  |   [[https://www.micro-shop.zeiss.com/en/ee/shop/objectives/420852-9870-790/Objective-LD-LCI-Plan-Apochromat-25x-0.8-Imm-autocorr-DIC-M27|420852-9870-790]]  |  
|  40x  |  LD C-Apochromat Corr  |  Water  |  1.10  |  0.62  |  [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/421867-9970-000/Objective-LD-C-Apochromat-40x-1.1-W-Corr-M27|421867-9970-000]] |  
|  63x  |  Plan-Apochromat DIC  |  Oil  |  1.40  |  0.19  |  [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420782-9900-799/Objective-Plan-Apochromat-63x-1.4-Oil-DIC-M27|420782-9900-799]]  |

*ask staff for help setting up the proper immersion medium setting


==== Detector(s) ====

1x blue-green multialkali PMT (15-20% QE)

4x GaAsP PMT spectral detectors (tunable between 450-750 nm) (~45% QE)

1x green-red multialkali PMT  (10-25% QE)

1x T-PMT detector (brighfield)

1x Airyscan2 detector (~45% QE after processing Airyscan data)

==== Airyscan2 emission filters =====
^  Position  ^  Emission  ^
|  1  |    |
|  2  |    |
|  3  |    |
|  4  |    |
|  5  |    |
|  6  |    |
|  7  |    |
|  8  |    |

==== Eyepiece filterset(s) (COLIBRI LED illumination) =====
^  Position  ^  Transmission  ^  Description (use)  ^  Excitation   ^
|  DAPI  |  445/50 nm  |  <fc #00ffff>DAPI, CFP or Hoechst filter</fc>  |  385nm   |
|  GFP   |  525/50 nm  |  <fc #00ff00>RFP or TRITC (LongPass)</fc>  |   493nm  |
|  RFP   |  LP590 nm  |  <fc #ffa500>GFP or FITC filter</fc>  |  555nm  |
|  Mirror   |  -  |  Mirror for LSM  |  -  |  


===== Turning ON =====

1. Verify if the fluorescent lamp is not hot (touch it), otherwise wait until it’s cold (10-15min). Afterwards, turn on the switch. NOTE. If Zen or the microscope are started with the fluorescence illuminator "OFF" they will not see it and need to be restarted afterwards. 

2. Turn on the microscope and the Definite focus control box.

3. Turn on laser key (turn laser key clockwise (or counter-clockwise?) to downward position) in the unit below the table.

4. Login into your aGina account on the computer. Start ZEN Blue 3.3.

===== Turning OFF =====

DO NOT TURN OFF THE COMPONENTS SWITCH, AND THE STAGE AND PIEZO CONTROLLERS (UNDER THE TABLE)

1. Remove the sample and Gently wipe with optic cleaning paper and EtOH paper the immersion objective(s) used. If you suspect the objective needs deep cleaning please inform the staff.

2. Transfer your data to the institutional server (never connect USB sticks or disks on this workstation).

3. Check that the incubator(s) are off, unless they are needed by the next user (ask staff). 

4. Close ZEN and logout from windows (if there’s an user after you, you can now leave the microscope).
If there is no one in the next 2h, turn off the fluorescence lamp (behind the computer screen) and also exit Zen.
If you are the last user of the day (check bookings in Agendo): Turn off Laser key (to horizontal position) in the large unit under the table and turn off the microscope and definite focus control box.

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