Leica SP5C User manual

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Rolly Wiegand – CALM Leica SP5 manual
CALM – Confocal and Advanced Light Microscopy Facility
Queen’s Medical Research Institute – College for Medicine and Veterinary Medicine
University of Edinburgh
Manual for the
Leica SP5C Spectral Confocal Laser Scanning Microscope
Version 1.11 – September 2008
Written and illustrated by Rolly Wiegand
We take no responsibility for the contents of this handbook.
The copyright lies with Rolly Wiegand and The University of Edinburgh, © 2008.
Reproduction of any kind without permission is prohibited.

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Rolly Wiegand – CALM Leica SP5 manual
Contents
1. Starting up the SP5................................................................................................... 1
2. Operation of the DMI6000 inverted microscope base .............................................. 3
3. Rules for the use of the mercury vapour arc lamp.................................................... 6
4. Image acquisition via the LAS AF software.............................................................. 8
4.1. Switching the lasers on............................................................................................. 8
4.2. Setting the USB control panel................................................................................... 9
4.3. Defining the beam path settings............................................................................. 10
4.4. Generating new beam path settings....................................................................... 12
4.4.1. Excitation laser line selection.................................................................................. 12
4.4.2. Spectrum view tool ................................................................................................. 12
4.4.3. Spectroscopic separation of the emitted fluorescence signal................................. 12
4.4.4. Setting the slits for the spectral separation of the emitted light .............................. 13
4.4.5. Adjusting the PMT settings..................................................................................... 14
4.4.6. Additional transmitted light channel........................................................................ 16
4.4.7. Saving and loading of beam path settings.............................................................. 17
4.5. Scanning mode....................................................................................................... 18
4.5.1. Simultaneous channel acquisition .......................................................................... 18
4.5.2. Sequential channel acquisition............................................................................... 18
4.6. Setting the acquisition mode................................................................................... 20
4.7. Setting the scanning parameters............................................................................ 20
4.7.1. Image resolution..................................................................................................... 20
4.7.2. Pinhole size ............................................................................................................ 21
4.7.3. Scan speed............................................................................................................. 21
4.7.4. Zoom....................................................................................................................... 21
4.7.5. Averaging................................................................................................................ 22
4.7.6. Accumulation.......................................................................................................... 22
4.7.7. Scan field................................................................................................................ 22
4.8. Acquisition of Z-stacks............................................................................................ 22
4.9. Using the ROI tool .................................................................................................. 24
4.10. Optimising settings via the USB control panel........................................................ 25
5. Saving and exporting data files............................................................................... 26
6. Post-acquisition data handling and analysis........................................................... 27
7. Shut-down procedure ............................................................................................. 28
8. Trouble shooting..................................................................................................... 29

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Rolly Wiegand – CALM Leica SP5 manual 1
1. Starting up the SP5
1.1. Before you switch on the system, fill in the log book (start time, lasers used, hours on
mercury lamp power supply, etc).
1.2. Switch on all three switches on the TCS control panel in the order shown in Fig. 1
(PC/Microscope, Scanner Power and Laser Power) and then turn the key 90°clockwise
(Laser Emission).
Fig. 1
1.3. Wait until the PC has finished booting up.
1.4. Log into Windows XP using the general account ‘TCS user’, which is not password
protected.
1.5. IMPORTANT: Make sure that the objective turret on the microscope stand is set to its
lowest position and that the condenser is in its normal position well clear of the stage. The
fastest method to do this is to press the ‘Z↓’ button on the right hand side of the
microscope base (see Fig. 6).
1.6. Start the LAS AF software by double clicking the icon on the desktop. The following
window will appear (see Fig. 2A). Choose ‘ok’ without going into the configuration or
microscope stand setup.
Fig. 2A

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Rolly Wiegand – CALM Leica SP5 manual 2
1.7. The software will start the initialisation of the microscope stand and the scan head,
and it cannot be used during this process. After a few minutes, the message shown in
Fig. 2B will appear, asking whether you want to initialise the motorised stage.
Fig. 2B
You should have already checked that the objectives and the condenser are out of the way
of any part of the stage (see 1.5., above). If you choose ‘Yes’, this will initialise the
motorised stage, which is essential if you want to use stage position coordinates (‘Mark
and Find’, ‘Tile Scan’). If you are restarting the software after a crash and you do not want
to loose the previous positions of your sample, click ‘No’.
Wait until the system is completely initialised.
1.8. Selection of the objective lens
The LAS AF software will open showing the ‘Acquisition’ page. Select the objective you
want to use in the ‘Beam Path’ window (see Fig. 3). IMPORTANT: Never change the
objective by manually turning the turret itself!
Fig. 3
The current objective lens is shown in the light path diagram of the window as seen in
Fig. 3. Before you start, you must check the immersion medium specifications of the lens
you want to use. This information is given in the ‘Beam Path Settings’ (Fig. 3), but you can
retrieve more detailed information about each lens from the objective selector by clicking
on the ‘Objective …’ in the configuration tab (Fig. 4, see circle). This will pull out the
following list as shown in Fig. 4 with all the lenses available on the system, including the
immersion medium specifications. Click the objective you want to use and apply the
appropriate, correct immersion medium, which could be:
DRY – air (never apply any fluid to theses lenses)
OIL – immersion oil only (use the provided Zeiss immersion oil)
WATER – water only (MQ water)

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Rolly Wiegand – CALM Leica SP5 manual 3
Important notes regarding the use of objective lenses:
It is vital for the longevity of the lenses that the correct immersion medium is used. Please
double check which lens is in place before you apply any immersion fluid. If you
accidentally made a mess, do not try to cover it up, but report it immediately to the CALM
staff.
You must clean your lens and your sample before switching to a different immersion
medium.
In case you would like to use the 63x WATER NA 1.2 lens, which is usually not on the
turret, please let the facility manager know in advance. This lens has a collar for adjusting
the lens to correct for different coverslip thickness. The black mark indicates the correction
at room temperature, whereas the red mark indicates the corrective settings for 37°C.
Fig. 4
Choose the lens you want to use by matching the refractive index (RI) of the immersion
medium with the medium embedding your sample (oil lens with a fixed sample, mounted in
a high RI medium; water lens with a live specimen in aqueous solution). Mismatch of the
RIs within your ‘optical sandwich’ can cause unwanted internal reflection and blurring. Also
bear in mind that the higher the NA of the lens, the higher the resolving power. If you have
the choice between two lenses with different magnification factors but the same NA (e.g.
63x or 100x oil, NA 1.4), the actual magnification should be adjusted with the system zoom
(see 4.7.4.) and should fulfil the Nyquist sampling criterion. This means the two lenses will
give you the same magnification and resolution, but the lens with the lower magnification
factor will allow you to image a larger field.
2. Operation of the DMI6000 inverted microscope base
2.1. The microscope should be already switched on via the TCS control panel (see above
section 1.2.).

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2.2. If required, switch on the mercury vapour arc lamp (for detailed user guide see
chapter 3. below).
Gently tilt back the arm that holds the transmitted light illumination.
2.3. Add a drop of the correct immersion medium on the chosen objective lens (see above,
section 1.8.). Never apply any fluid to dry lenses.
Fig. 5
Power supply ‘ebq 100’ for the mercury vapour arc lamp
2.4. Place your sample, coverslip downwards, into the appropriate slot of the motorised
stage. The coverslip you should be using should be of thickness 1 (130 – 150 μm mean
thickness). The configuration with the motorised stage is not particularly suitable for live
cell imaging and we recommend the Zeiss LSM510 META for live cell work.
2.5. Tilt the transmitted light illumination arm gently back towards you and make sure it is
in the right working position (if it is not, the safety switch in the arm will cut out the lasers in
the scan mode).
2.6. To select the right area of your sample and to bring the specimen into focus, you can
choose between brightfield (transmitted light) and fluorescence (reflective) mode. To
switch between the two modes press the TL/IL button on the left hand side of the
microscope stand (see Fig. 6). The system is not equipped for the use of any contrast
method (PC, DIC, etc).

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Rolly Wiegand – CALM Leica SP5 manual 5
Fig. 6
Left side of DMI6000 stand
2.7. Light intensity adjustment
In both modes the illumination intensity can be adjusted via the top two buttons designated
as INT (intensity). The upper button increases, whereas the lower button decreases the
brightness. Thumb rule is to use as little light as possible, in particular in the fluorescence
mode, to protect the sample against unnecessary bleaching. Accordingly, the size of the
field illuminated by the mercury arc lamp for fluorescence excitation can be adjusted via
the two buttons designated as FD (field diaphragm). By decreasing the opening of the field
diaphragm, the illuminated field will decrease to the area chosen. The fluorescence light
path is also fitted with a shutter that allows to block illumination of the sample and thus to
protect it (see the yellow arrow in Fig. 7).
2.8. In fluorescence mode, three filter cubes are provided by the system with the following
properties:
Filter set commonly used for Excitation filter Dichroic filter Emission filter
I3 FITC, AF488, GFP BP 450-490 510 LP 515
N2.1 AF568, Cy3, dsRed BP 515-560 580 LP 590
A DAPI, Hoechst BP 340-380 400 LP 425
Note: The system is not equipped with a camera and thus image acquisition in the
reflective fluorescence mode is not possible. This functionality is purely to find and assess
your specimen before image acquisition in the scan mode. It should also be mentioned
that the given filter cubes are prone to spectral cross-talk and thus care should be taken
regarding conclusions from the observed fluorescent signal.

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Rolly Wiegand – CALM Leica SP5 manual 6
Fig. 7
Front panel of the microscope stand
2.9. To focus on your sample, you can use the two main knobs on both sides of the
microscope stand (see Fig. 6). The focus operates in ‘coarse’ and ‘fine’ mode and you can
switch between the two modes by pressing the upper button on the right hand side
SmartMove remote control (see arrow in Fig. 8). To mark a desired Z-position of the focus,
press the two buttons ‘Z↑’ and ‘SET’ simultaneously (see Fig. 9). Do not use the ‘Z↓’
button. If you simultaneously press the buttons the first time, this will delete any previous
setting and the display at the front of the stand should display ‘--,-- μm’. If you press them
again, the display will be changing to ‘0 μm’ and this is your reference setting. The display
will give you a constant read-out f the actual Z-position (see Fig. 10) and allows you to
conveniently find your initial, marked focal plane again.
IMPORTANT: Never push the objective lens against the coverslip, because this might
damage the front lens and might require a very expensive repair.
2.10. To quickly move the objective turret to the lowest point, e.g. for making sure that the
lens is out of the way for stage initialisation or after finishing with your work, press the ‘Z↓’
button (see Fig. 9).
2.11. The SmartMove remote control allows you to conveniently control several functions
of the microscope such as the X- and Y-movement of the motorised stage and the Z-drive
mode (see Fig. 8).
3. Rules for the use of the mercury vapour arc lamp
3.1. Only turn the lamp (ebq 100, see Fig. 5) on when it is cool, i.e. when it has been
switched off for 30 minutes or longer. The lamp must not be switched on when it is still
warm from the previous use. Check the system log sheet for information about when the
lamp was turned off the last time. If in doubt, contact the previous user.
3.2. Once the lamp has been switched on, it has to be left on for a minimum of 30 minutes.
A
N2.1
I3

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Rolly Wiegand – CALM Leica SP5 manual 7
3.3. Before switching off the lamp at the end of your session, check the booking system to
find out whether another user is going to start using the system within the next hour. If so,
leave the lamp switched on.
3.4. Always log the times the lamp is switched on. The display at the front of the power
supply (see Fig. 5) shows the total usage time of the present bulb. If this reaches 220
hours, please inform the CALM facility staff by e-mail so that the bulb can be changed
within the next 30 hours.
IMPORTANT: There is a risk assessment in place for the use of this lamp, and every user
should have read, understood and signed the appropriate forms.
Fig. 8
SmartMove remote control
In Fig. 8, the arrow on the right indicates the selection button for the Z-focus mode
(‘coarse’ or ‘fine’)
X-drive
Z-drive
Fine/coarse
Y-drive
Z-drive

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Fig. 9
Right hand side of the microscope stand
All settings of the DMI6000 microscope stand are shown on the display at the front (see
Fig. 10).
Fig. 10
Display on the front of the microscope stand
You can find more details about the DMI6000 microscope stand in the Leica handbook,
and a copy of the pdf file can be found on the CALM website.
4. Image acquisition via the LAS AF software
4.1. Switching the lasers on
Z-position
Illumination intensity and
aperture setting
Lens and magnification
Imaging mode/shutter position
Focus mode