Leica TCS SP2 Reference guide

Brief manual how to start and close the Leica sp2 Confocal
(TCS SP2 AOBS system mounted on a DM IRE2)

A. Switching on hardware
B. Acquiring and saving images
C. Switching off the microscope
D. Good working practice
Assistance/instructions/any problem: Liesbeth Pierson, 024-3652012, Room 01.239, GI.
[e-mail]
Black = obligatory
Gray = optional settings
A. Switching on hardware
Switch red knob #1 on and press on the PC ON button to start Windows (the monitor is
switched on; the power unit for the inverted microscope is on). Wait until Windows XP is
fully loaded
Click on the icon of your institute (IMM, IWWR, DCN). Please, do not add a password!
If any entry is corrupted and Liesbeth is absent, you could use the GI Administrator entry.
Press red knob #2 on: the power box for the scan head and lasers switches on
Press red knob #3 on: the cooling is functioning (note that outside working hours, an
additional cooling should be switched on from the box on the corner just outside the room;
ask Liesbeth)
Switch the conventional 50 W HBO fluorescence lamp on by pressing once on #20a of
EBQ 100. (NEVER re-ignate the lamp soon after having switched it off. In general avoid
any unnecessary switching on and off of this lamp). #20c is to open/close the HBO lamp-
shutter, #20b to switch between filters:
1 is for UV excitation [A set: BP340-380 nm; DM 400 nm; LP425 nm]
2 green excitation [N2.1 set: BP 515-560nm; DM 580nm; LP590 nm]
3 for blue excitation [I3 set: BP 450-490 nm; DM 510 nm; LP515 nm]
4 not fluorescence filters/scanmodus
Settings are displayed at #20d. Leave the settings for the HBO lamp intact, please (#29 is
the HBO lamp diaphragm and #30 the intensity regulator).

Switch the lasers on that are required to excite the fluorochromes in the sample. For
reflection imaging the 488 nm line of the Argon laser is usually employed.
* The Argon laser (458, 476, 488, 496, 514nm; ) is switched on by pressing knob #3 and
turning the key #5 to the start position (clock at 14h00) and releasing it to position on
(clock at 12h00). Leave knob #4 on position "minimum" when the laser does not need to
be activated to save its life time, e.g. for a break. Turn it to 10h00 during imaging
sessions.
* The 594 nm HeNe laser is switched on by turning key #6 to position 1
* The 633 nm HeNe laser is switched on by turning key #7 to position 1
* The 561 nm Orange laser is switched on by turning key #8a from 0 to I.
* The 405 nm diode laser is switched on by first pressing the red #9a knob and then
turning the key #9b to position 1
Take care that the 10x objective is in the upright position
Choose the correct sample holder (slide holder for xy scans, or slide holder with
outstaying box for xz scans, or multiwells holder).
To start the Leica confocal software that commands the hardware (microscope, lasers,
AOBS and more) and contains the acquisition and imaging modules, click on the Leica
LCS icon on the screen. Choose the “Start” option.
If motorized coordinate positions of the sample are required, the stage will iterate
automatically to the left back corner to get a calibrated start position after answering
“Yes” to the question “Initialize Stage?” Remember to perform xy stage initialization only
with a 10x objective upright and to press the condenser arm backward if you use the xz
galvo sample holder, so that does not touch the condenser during the calibration move.
Click “No” to the question “Initialize Stage?” if automated xy positioning is not
applicable.
Choose the appropriate objective and immersion medium (check the type: dry, water, oil
or glycerol immersion!); objectives can be changed automatically under the Obj menu or
manually by turning the revolver, or with #26 (to suppress the automatic blocking between
dry and other objective press both #27↑ and #25 ↓ simultaneously, or automatically under
the LCS). The following objectives are available:
- HCPL fluotar Dry10x/ 0.30 Numerical Aperture; maximum field of view 1.500mm x
1.500 mm
- HC PL apo CS 20x / 0.70 D, also for Differential Interference Contrast (DIC); maximum
field of view 750 µm x 750 µm
- HCX apo Long working distance (up to 3.3 mm) Ultraviolet (VI) Water immersion
40x/0.80 DIC; maximum field of view 375 µm x 375 µm.
- HCX PL apo CS 63x/ 1.30 Glycerol immersion 21 C; maximum field of view 238.1 µm
x 238.1 µm.
- HCX Pl apo 63x/1.40-060 (oil immersion with diaphragm) lambda blue-GFP; maximum
field of view 238.1 µm x 238.1 µm.
- empty position.
Insert the specimen, coverslip facing the objective (down) on the specimen holder (Take
care that the coverslip is fixed and no inclusion medium can leak out. If nail polish is used
for sealing, make sure it is completely dried out.
Check # 21 for manual setting to VIS for visual or other ports (Side for scanning).

To view the specimen in bright field (BF), 4 things to do: turn the power wheel # 11 on to
about 4V (for white light at 3200 K around 8V, to be used with NDF) and the lollypop
shutter above the field diaphragm in down position, Knob #12 operating a mirror in the
condenser light path should be on VIS = visual and MicCtrl on Visual.
Focus on the specimen with the knob “Focus” ; step S3 is coarse, step S2 is medium, Step
S1 is fine.
Apply Koehler illumination by closing diaphragm #13a and adjusting the level of the
condenser at knob # 13b to obtain a sharp projection of the edges of this diaphragm in the
oculars. The illumination path can be centered with the two rings #13c.
Set #15 on BF. If more suitable for a certain objective 63x oil or 40/63 x).
Knob #22 should be pushed inward
Adjust diaphragm #14 (from 0 to over 8 and Ph) to have just enough contrast.
Light can be dimmed with Neutral density filter (NDF) # 16. For bright field illumination
pull polarization filter # 17 out of the light path.
Turn the ribbed disc # 18 to position BF.
Pull the (ICT P) analyzer (polarized filter) #19 out for bright field vision
Keep #22 pressed in (half pulled out is 50% ocular-50% upper camera port vision; fully
pulled out is 100% upper camera port vision).
Ring #23a (display #23b) can be set in position 1x (open connection for bright field); UV
for ultraviolet scan stand, B for phase contrast adjustment with Bertrand lens (not relevant
in this microscope) , and scan for non-UV scanning
Optional but easy is the function to set the z-focus level of the objective for later recovery.
Press #27 ↑ for 3 s, focus and press #27 ↑ again. The lowest position of the objectives can
be set in a similar way using the #25↓ switch (standard is around –150 micrometer).
Differential Interference contrast settings: Press the (ICT P) analyzer polarized filter
#19 in for eye vision and out for laser scanning for this S23 condenser, turn either of the
following prism on the #15 turret to the front: K10 for 63x gly and oil objectives, K8 for
20 x and D1 for 10x. Switch polarizer/analyzer #16 into the light path.
To watch wide-field fluorescence, choose the desired filter block with the arrows on 20d,
and open shutter 20c (close the transmission light lolly pop)
To go to the confocal imaging, choose scan from the MicCtrl icon, block the light of the
transmission lamp, close the fluorescence lamp shutter.

B. Acquiring and saving images
In the Leica Confocal Software (icon on desktop): the "Acquire" menu appears on the left
monitor. Images will be displayed on the right
monitor.
Switch on appropriate lasers and push the
control for each wavelength to the desired level
in %. Click active. For the 405 nm laser choose
% passed through Neutral density filter (--
means no neutral density filter).
Set PMTs for the desired wavelength ranges by
mouse dragging (trans active optional;
reflection is a special case). Functions of the
black knobs can be adjusted by right clicking
on the lower bar menu (choose configuration
for menu, choose sensitivity to adjust the coarsness of the knob): e.g. Smart offset, smart
gain. Use these knobs to control the offset and gain levels, pinhole opening or zoom factor
or to focus.

The switch between eye “Vision”and laser “Scan” is located under the “MicCtrl” icon in
the lower left corner.
Objectives can be automatically selected by clicking the from the “Obj.“ menu in the
lower left corner.
Choose a “Menu” for the acquisition from the Leica or User Window. For the xy sample
holder choose z-scan icon> z-wide. For the xz sample holder,
choose z-scan icon> z-galvo.
There are many ways to modulate the settings, for example
through the acquisition “Mode” in which spatial (xyz), time
(t) and wavelength (lambda) settings can be defined, in the
scan “Speed”, “Format” of images (number of pixels),
“Zoom” factor, uni or bidirectional “-> Scan” mode. To start
imaging press either “Continuous” for viewing a field that
will be refreshed after each scan, “Single” for acquiring one
view (can be combined with averaging of images by means of
lines “Li A.” or whole frames “Aver.” . The “Offset” and “Gain” of the photomultipliers
can be adjusted manually with the smart function (knobs on the table panel) or
automatically with “A.Gain”.
There is an extensive “Help” menu within the Leica Confocal software, as well as a print
of most help functions and tutorials in the confocal microscope room.
Acquired images are stored temporarily on a temp directory (can be retrieved if the
program crashes, but not if the computer crashes: therefore it is advisable to regularly save
data during an imaging session). Experiments (images and settings) SHOULD be saved as
a .lei file (like a manager file, which also keeps track of the metadata of each image) in a
folder, on your own directory under Users on the E:\Datadatadisk2 drive. The name of the
lei. experiment is automatically added to each image name as a prefix. Images/series of
images can be renamed after acquisition to a logical log name at your convenience.
Resave to refresh the renamed images in the lei. Keep folder with the tif images, lei and
txt files intact together to allow reloading in the LCS software later on. Images are
indexed color 8bits tiff format or RGB (in various channels, z levels , time series), or RGB
screenshots.

C. Switching off the microscope
Wipe immersion medium (oil, glycerol) from the 63x objectives using a fiber-free tissue
(small rectangles) for oil and glycerol, or a cleenex tissue to absorb water at the 40x
objective .
Set the 10x objective in the upright position
Save the files and exit the confocal software.
Switch lasers off:
405 nm laser by turning key #9b to position 0 and red knob #9a to 0,
561 nm by turning key #8a to 0,
594 nm #6 to min position,
633 nm # 7 to position 0,
Argon laser by turning the knob #4 to the minimum level and key #5 to off.
Switch the arc lamp out by pressing #20a.
Leave ventilator #3 on for another few minutes until cool air is blown out (listen and
wait until the sound drops).
Place the cover over the microscope, but prevent the plastic cover to touch the hot arc
lamp housing
COPY YOUR DATA TO A SERVER AFTER EACH SESSION! THE GEMINSTR
SERVER (CONTACT LIESBETH) CAN SERVE AS A TEMPORARY
STORAGE PLACE AS WELL. NO USB/HARD DISKS BECAUSE OF VIRUS
RISKS, especially since we had to remove F-secure to save space on the 10G C drive.
USERS SHOULD REMOVE THEIR OWN DATA FROM THE HARD DISK
WITHIN ONE WEEK to prevent congestion of the system.
Shut down Windows on the computer
When ready, switch #3 out
Switch #2 out
Switch #1 out
D. Good working practice
Always reserve time through bookings.science.ru.nl
Do not hesitate to contact Liesbeth for assistance/instructions, if you have any
question/doubts, if anything went wrong.
Behave in a responsible manner towards the equipment and to your colleagues. For
example, do not switch off the HBO lamp if somebody is coming to work within one hour
after you, clean up any spill, contact the next/another user if you have to cancel your booking
time, or if you are ready earlier. Before leaving, check whether you are the last person of the
day, and if so, switch off all equipment.
The general agreement is that users leave the lasers they used on for the next person
(unless there is a gap of >1 hr; but turn #4 of the Argon laser to min), but that they switch
off the laser(s) that they do not need at the beginning of their session. Of course, if you
need a warmed up laser, you are welcome to come to switch it on earlier.