Leica Sp5 WLL STED Operation instructions

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 SP5WLL.

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)

3
System Start up (step 1)
On the STED Tower, Switch “ ON ” the button

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.

5
System Start up (step 3)
Turn the key from the depletion laser 592-nm in
vertical position.
The SHG led keep green.

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”).

7
Turn “on” the XCite Light.
System Start up (step 5)

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

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

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