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 1
System start Up. (Page 3-8) Laser switch and calibration (page 11-13) Sted configuration(page 14-17) System shut down. (page 18-22) 2
System Start up (step 1) On the STED Tower, Switch ON the button 3
System Start up (step 2) The SHG led turns green for 3sec, then it s turns orange for 3 minutes, then it s turns green again after 1 minutes. 4
System Start up (step 3) Turn the key from the depletion laser 592-nm to vertical position. The SHG led turns green. 5
System Start up (step 4) Push the PC/Microscope button Push the Scanner Power button. Push the Laser Power button (White laser is on ). Turn the Key on the right position (Argon laser is on ). 6
System Start up (step 5) Turn on the XCite Light. 7
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 8
Choosing the objective Under Acquire and Acquisition Choose and select your objective in the list. To acquire a STED image you need to use the HCX PL APO CS 100x 1.40 OIL 9
Choosing the Filter You need to introduice a Notch filter to block the laser 592nm Select Analyzer Filter Deselect Auto Select Select NF STED filter 10
Laser Switch 1 Under Configuration select 2times the 592 depletion laser Be sure the Laser Enabled led appears white (on the STED tower). 11
Laser Switch 2 Under Hardware Configuration and Laser select the WLL and Argon Laser. Increase the power of the 592 Depletion laser to 75% ( minimum) 100%. Increase the wll laser to 70% ( good value = the laser is very stable). Increase the Argon laser to 30%. 12
Laser calibration/alignement Every time that you want to use the STED, you need to allign the 592 laser vs WLL laser. Go to the STED Setting dialogue and press the Align Beams fonction. Wait until the calibration is finish. Press OK. 13
Conventional configuration 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 %). Select the Hyd 2 detector and set it, to 100%. Set the Hyd bandwidth. Its start must be 10nm bigger than the WLL excitation line used and its end must not exceed 582nm. Use a format like 512x512 or 1024x1024. Use a scan speed like 400HRZ. Use a zoom factor from 4 to 6. 14
STED configuration ( step1) Select the STED mode. Set the WLL laser line that you need to use ( for ex. 488nm at 30 % to 50% transmission ). Select the Hyd 2 detector and set it to 100%. Introduce to 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 the value you use to excite the fluorochrome (for ex 488nm) Set the Gate Start from 0,5 to 2,5 1,5 a compromise Set the Gate End to 6,5 15
STED configuration (step2) 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. 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. If you use 400 HRZ use 2-3 accu with 2-4 average. 16
STED configuration (step3) 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. Use Start and not live to acquire the image. 17
System Shut Down (step 1) Before you exit from the software.please clean the objective. Turn off the 3 lasers 18
System Shut Down (step 2) Exit from the LASAF software. Go to Start and shut down the computer. 19
System Shut Down (step 3) Turn on the XCite Light. 20
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 21
System Shut Down (step 5) On the STED Tower, turn the Key vertical position Switch off the button 22