Leica Sp5 WLL STED Tutorial Manual Download

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Wellcome on the STED Tutorial

Before using the Sp5 WLL STED microscope ,

Version 1 27.08.2013



You will need to put down your name on the

reservation system

https://sv-bbs.epfl.ch/ptbiop/

STED, on the SP5WLL.

Summary of Contents for Sp5 WLL STED

Page 1: ...Wellcome on the STED Tutorial Before using the Sp5 WLL STED microscope Version 1 27 08 2013 You will need to put down your name on the reservation system https sv bbs epfl ch ptbiop STED on the SP5WL...

Page 2: ...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: ...3 System Start up step 1 On the STED Tower Switch ON the button...

Page 4: ...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: ...5 System Start up step 3 Turn the key from the depletion laser 592 nm in vertical position The SHG led keep green...

Page 6: ...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: ...7 Turn on the XCite Light System Start up step 5...

Page 8: ...8 System Start up step 6 When the computer is on Introduce your Username Introduce your Password Introduce your login Start the LASAF software Under Configuration select Machine STED Do OK...

Page 9: ...9 Choosing the objective Under Acquire and 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: ...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: ...11 Under Configuration select 2times the 592 depletion laser Be sure the Laser Enabled led appear white on the STED tower Laser Switch 1...

Page 12: ...ration and 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 Increase the Argon l...

Page 13: ...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 Wait until the calibration is finish And do OK Laser calib...

Page 14: ...se to work Set the WLL laser line that you need to use 10 to 30 Select the Hyd 2 detector and set it at 100 Introduce to the Hyd a bandwidth that is 10nm fahter to the WLL line that you set and maximu...

Page 15: ...o the Hyd a bandwidth that is 10nm fahter to the WLL line that you set and maximum 10nm lower than the wavelength of the laser depletion 582nm Open the Gate Setting set the Wll value which is equal to...

Page 16: ...0HRZ Use a zoom factor from 4 to 6 Very important control that the pixel size is 20nm or less Use 3 4 Accu in frame mode Use 0 4 average in line mode Ex if you use 200 HRZ use 2 3 accu without average...

Page 17: ...ntionel 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 Use Start and not live to acquire the image STE...

Page 18: ...18 System Shut Down step 1 Before you exit from the software PLEASE clean the objective Turn off the 3 lasers...

Page 19: ...19 System Shut Down step 2 Exit from the LASAF software Go to Start and shut down the computer...

Page 20: ...20 Turn on the XCite Light System Shut Down step 3...

Page 21: ...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: ...22 System Shut Down step 5 On the STED Tower turn the Key vertical position Switch off the button...