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Summary of Contents for Leica Sp5 WLL STED
- Page 1 STED, on the SP5WLL. Wellcome on the STED Tutorial Before using the Sp5 WLL STED microscope , You will need to put down your name on the reservation system https://sv-bbs.epfl.ch/ptbiop/ Version 1 27.08.2013...
- Page 2 Welcome to the Leica Sp5 WLL Confocal Invert tutorial System start Up. (Page 3-8) Laser switch and calibration (page 11-13) Sted configuration(page 14-17) System shut down. (page 18-22)
- Page 3 System Start up (step 1) On the STED Tower, Switch “ ” the button ...
- Page 4 System Start up (step 2) The SHG led turn green for 3sec, then it’s turn orange for 3 minutes, then it’s turn green again after 1 minutes. ...
- Page 5 System Start up (step 3) Turn the key from the depletion laser 592-nm vertical position. The SHG led keep green. ...
- Page 6 System Start up (step 4) Turn on the PC/Microscope button Turn on the Scanner Power button. Turn on the Laser Power button (White laser is “on”). Turn the Key on the right position (Argon laser is “on”).
- Page 7 System Start up (step 5) Turn “on” the XCite Light. ...
- Page 8 System Start up (step 6) When the computer is Introduce your Username Introduce your Password Introduce your login Start the LASAF software. Under “Configuration” select Machine STED. ...
- Page 9 Choosing the objective Under Acquire Acquisition Choose and select your objective in the list. To acquire a STED image you need to use only the HCX PL APO CS 100x 1.40 OIL...
- Page 10 Choosing the Filter You need to introduice a Notch filter to stop the laser 592nm Select “Analyzer Filter” Deselect “ Auto Select” Select “NF STED ” filter ...
- Page 11 Laser Switch 1 Under “ Configuration” select 2times the 592 depletion laser Be sure the “Laser Enabled” led appear white (on the STED tower) . ...
- Page 12 Laser Switch 2 Under “Hardware Configuration ” “Laser” select the WLL and Argon Laser. Increase the power of the 592 Depletion at 80% ( minimum) 100% possible. Increase the wll laser at 70% ( good value = the laser is very stable). ...
- Page 13 Laser calibration/ alignement Every time that you want to use the STED , you need to calibrate the 592 laser vs WLL laser. Go to the “ STED Setting “ dialogue and press the “ Align Beams “ fonction. ...
- Page 14 STED configuration ( step1) By using the depletion laser 592nm, the samples can be bleach very quickly, so first scan the sample in standard Confocal mode to find the focus and prepare the STED configuration close to work. Set the WLL laser line that you need to use (10 to 30 ...
- Page 15 STED configuration ( step2) Select the STED mode. Set the WLL laser line that you need to use ( for ex. 488nm 30 % to 50% transmission Select the Hyd 2 detector and set it at 100%. Introduce ...
- Page 16 STED configuration (step3) Use a format like 1024x1024 to 2048x2048. Use a scan speed like 200 to 400HRZ Use a zoom factor from 4 to 6. Very important ,control that the pixel size is 20nm or less. ...
- Page 17 STED configuration (step4) Select” Switch Conventionel Laser”. Select the ” STED ” . Set the Depletion 592nm laser line at ” 100% ” You can use less than 100%, but you may lost some resolution, and win some intesity. ”...
- Page 18 System Shut Down (step 1) Before you exit from the software …….PLEASE clean the objective. Turn off the 3 lasers ...
- Page 19 System Shut Down (step 2) Exit from the LASAF software. Go to Start and shut down the computer.
- Page 20 System Shut Down (step 3) Turn “on” the XCite Light. ...
- Page 21 System Shut Down (step 4) Turn the Key on the left position (Argon laser is “off”). Turn off the Laser Power button (White laser is “off”). Turn off the Scanner Power button Turn off the PC/Microscope button ...
- Page 22 System Shut Down (step 5) On the STED Tower, turn the Key vertical position Switch “ ” the button ...
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