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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Rolly Wiegand – CALM Leica SP5 manual 4
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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Rolly Wiegand – CALM Leica SP5 manual 8
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

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Open the ‘Configuration’ tab and click the ‘Laser’ icon to open the appropriate window (see
Fig. 11). Switch on the lasers you require, but leave the ones you do not need turned off. If
you are using the Argon laser set the output power to 30-50%. 30% usually gives enough
power and stability of the laser output for most applications. The lower setting of 30% will
help to elongate the lifetime of the laser.
Fig. 11
The following laser lines are available on the system:
Laser Excitation wavelength [nm]
405 Blue Diode 405
Argon 458, 476, 488, 496, 514
Helium Neon 1 543
Helium Neon 2 594
Helium Neon 3 633
4.2. Setting the USB Control Panel
In the configuration tab, open the window for the USB Control Panel by clicking the
appropriate icon (see Fig. 12, number 1). The only setting you have to change is the
sensitivity for the Smart Offset. Click the second knob representing the Smart Offset on the
USB control panel (Fig. 12, number 2). In the second box that opens, click 1% per turn,
which means that the offset will change by 1% when the dial is turned 360°(Fig. 12,
number 3).
Note: Please do not change any other setting in the configuration tab. If you need more
detailed information about the objective lenses, open the appropriate windows, but do not
change anything.

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Fig. 12
Configuration tab – settings for the USB control panel
4.3. Defining the ‘Beam Path Settings’
When you start to set up the beam path of the system, you should have the complete
information about the spectral properties of all fluorophores you have used in your imaging
experiment at hand (absorption and emission spectra). This is essential for setting up the
beam path for efficient spectral separation of the signal. You can either use saved pre-sets
or you can define your own settings (and save them for later re-use).
Before you start choosing your settings for a multi-channel image acquisition, you have to
decide whether to scan the channels simultaneously or in a sequential scan. The system is
equipped with 3 photo multiplier tubes (PMTs) and thus can record three different spectral
channels at the same time. This is the method that provides the shortest acquisition time
but is also prone to cross-talk, if the excitation and/or emission spectra are overlapping. It
is helpful though for quick scanning to find the right area of your specimen.
To avoid spectral cross-talk, sequential image acquisition is recommended. This allows the
sequential recording of up to 7 channels, each of them can be defined individually.
Fig. 13
1
2
3

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The example in Fig. 13 shows the excitation (dotted lines) and emission spectra (solid
lines) of 3 different fluorophores. The arrows display the 3 laser lines used for excitation.
The bars below the spectra indicate the 3 spectral bands separately collected by the
detectors. In this example, three possible scanning modes can be used:
1 – simultaneous scanning of all 3 channels – Cross-talk is a problem, because for
instance part of the signal from the second fluorophore will be recorded in channel 3
2 – sequential (all 3 channels separately) – Cross-talk is ruled out by sequential scanning,
the drawback is the scan time increases three-fold
3 – sequential/combined (channel 1 and 3 in the first and channel 2 in the second
sequential scan) – This combined method involves 2 sequential scans. During the first
scan channels 1 and 3 are recorded. Cross-talk is ruled out because both excitation and
emission spectra are far enough apart and thus do not affect the recording of the opposite
channel. In the second scan, the second fluorophore will be excited with the 543 nm line
and separately recorded. This method rules out cross-talk problems and saves some time
compared to scan method 2 (about 30% shorter scan time). It is also recommended to
combine the acquisition of the transmitted light channel with the channel that uses the 488
nm Argon laser line (see also 4.4.6.).
Section 4.5. explains how to set up a sequential scan.
A = Laser control module
B = Objective selection tool
C = Spectral view tool
D = Slit settings
E = PMT settings
F = Control for
saving/loading of settings
Fig. 14
‘Beam Path Settings’ windows
B
C
D
A
F
E

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4.4. Generating new Beam Path settings
The Beam Path window (in the acquisition tab) is a schematic representation of the major
components of the scanning system (see Fig. 14). It shows the laser modules with all
available excitation lines on the system (Fig. 14 A), the objective lens selector (B), a
spectral view help tool (C) and the module for the detector (PMT) settings (D). To create
new settings, you can start from scratch or use predefined settings and modify them (see
4.7.7. for loading predefined settings).
4.4.1. Excitation laser line selection
Switch on the required laser source (UV and visible) by clicking the appropriate grey dots
in the laser control window (Fig. 14, A). If the dot turns red, the laser source is activated. In
the same window, set the sliders of all laser lines you want to use to the percentage of
laser output you require. It is recommended to always start with as little laser power as
possible to avoid bleaching and photo-toxic effects. Typical ‘start settings’ for well labelled
specimens for the different laser lines are as follows:
405 blue diode – 20%
Argon laser – 5-10%
All Helium Neon lasers – 50%
As a visual help, the grey lines representing the different paths of the laser lines in the
‘Beam Path Settings’ window turn coloured when the appropriate laser line is activated.
Should the laser power not be sufficient, it can be adjusted any time. Try also to choose
the laser line as close as possible to the absorption maximum of the given fluorophore.
4.4.2. Spectrum view tool
In order to assist and facilitate the definition of the beam path settings, the software
provides a visual tool that shows the excitation lines as well as the emission spectra of a
large range of commonly used fluorophores. To use the tool, one emission spectrum can
be chosen for each channel by clicking the fluorophore setting in each PMT box (Fig.
14 E). This pulls out a list with fluorophores you can choose from. The emission spectrum
will then appear in the Spectrum View Tool as a dotted line, accordingly (Fig. 14 C).
Note: This is just an auxiliary tool that has no impact on the actual settings.
4.4.3. Spectroscopic separation and detection of the emitted fluorescence signal
The Leica SP5 uses a sophisticated filter-free technology to separate the emitted light from
the fluorescent sample into different spectral bands. In brief, the entire emitted signal is
spectrally dispersed by passing the light through a prism (see Fig. 15 - 1).
The spectrally dispersed signal is then passing through a lens that projects the light
parallel onto the first slit in front of the central PMT. The slits are adjustable in width and
position, and the mobile elements (sliders, see Fig. 15 – 2) that generate the slit have
highly reflective surfaces. Depending on the opening width of the slits a certain spectral
band of the signal can pass through it and is collected by one of the PMTs. The light of
shorter or longer wavelengths is reflected by the sliders onto the next set of slider
elements, which in turn can be adjusted to define different spectral bands for detection in
separate PMTs. The lack of filters in this system provides a superior technology with
respect to sharp spectral separation and guarantees high transmission properties,
something a filter-based system cannot deliver.

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Fig. 15
Spectroscopic detection of a system with 5 PMTs
Fig. 16
Slit and PMT settings control window
4.4.4. Setting the slits for the spectral separation of the emitted light
The sliders in the beam path window underneath the spectral view tool in Fig. 16 (S1 to 3)
represent the three slits of the spectroscopic detection unit. By left-clicking and moving
them, they can be shifted to shorter or longer wavelengths without changing the slit width.
By clicking and moving the left or right edge of the bar, the appropriate width of the slit can
be changed. The sliders can be used to adjust the position and width of each slit
accurately down to the nm. Right-clicking the sliders will open a little window that indicates
the settings of each slit in nm (see Fig. 16). This can also be used to set the spectral
separation.
S1 S2 S3

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Rolly Wiegand – CALM Leica SP5 manual 14
Thumb rules for adjusting the slits:
The slits should be set so they do not overlap with any of the laser lines.
The slit opening should be as close as possible to the emission maximum of the
fluorophore to be imaged.
The narrower the slit the more specific and less prone for cross-talk the setting is.
However, if the signal from a dim label is too weak to detect, the slit may be opened in
order to collect enough photons. The user has to optimise the balance between specificity
and the need to collect sufficient emitted photons.
The settings of the slits will be recorded with the image data as metadata and can be
retrieved at any time after acquisition. However, it is recommended to take notes of the
exact settings.
The settings of the slits can also be saved with the other beam path settings (see 4.4.7.
and Fig. 15 F).
Fig. 17
4.4.5. Adjusting the PMT settings
The CALM SP5 system has 3 different PMTs unlike the schematic representation in
Fig. 15. To use a PMT it must be activated by clicking the appropriate tick box (Fig. 17). By
clicking the individual ‘PMT’ buttons, a little window pulls out, which allows to set the gain
and the off-set of the individual PMT. These two parameters can also be set by using the
control console (see Fig. 18). The accurate adjustment of each PMT with regard to each
fluorophore signal is absolutely crucial for a valid image acquisition. For instance, gain
settings that lead to saturation and thus clipping of the intensity information in the image,
render the resulting data useless for accurate quantitation. These settings determine how
optimal the system uses the whole dynamic range of the digital image. The off-set defines
the intensity of the ‘darkest’ pixel in the image and should be set so that it is just above ‘0’.
Fig. 19 A shows the intensity frequency histogram of an image with too low settings for
both, the off-set and the gain, whereas panel B shows the situation with both settings too
high. Both inaccurate settings will lead to a poor use of the dynamic range and to clipping
of the intensity information in the image. Fig. 19 C on the other hand shows an intensity
frequency histogram with optimal setting of PMT off-set.
4.4.5.1. Setting the PMT off-set
The easiest way to adjust the PMT settings is to focus on a middle section of your
specimen with high intensity features. Click in the display window (right monitor) on the
panel representing the first spectral channel to activate the control panel for this channel.
The activated channel is indicated by a dotted frame around the respective image panel,
e.g. channel 1 in Fig. 20. Then change the look-up table (LUT) to glow scale (GloOU) by
clicking the button indicated by the circle and arrow in Fig. 20. By clicking this button
repeatedly, it will change through LUT-GloOU-grey scale, consecutively. In the GloOU

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Rolly Wiegand – CALM Leica SP5 manual 15
Fig. 18
Control console
Fig. 19
display mode, pixels that are close to saturation and over appear blue (see Fig. 21, right
window for channel 2), whereas pixels that turn green indicate a too low off-set (see Fig.
20, window for channel 1, left panel). Start continuous scanning by clicking the ‘Live’
button at the bottom of the main control panel. Adjust the off-set by slowly turning down
the ‘Off-set’ button on the control console (see Fig. 18) so that the percentage becomes
more negative until the first green pixels appear in the displayed, scanned image. A typical
setting would be -0.4%. Check whether this setting is accurate for optical sections
throughout your sample by slowly moving the Z-positioner on the control console up and
down (see Fig. 18), and adjust if necessary. Once you have finished optimising the PMT
gain (see 4.4.5.2.), check the off-set settings again and correct if necessary.
4.4.5.2. Setting the PMT gain
Whilst continuing to scan, adjust the ‘voltage/gain’ setting on the control console (the
range is 0 - 1250 V and the default 0 V) until the first pixels turn blue (see Fig. 21,
channel 2). Turn the gain slightly down a bit until no more blue pixels are visible in the
display panel and check this setting for other optical sections in the sample. A typical
setting would be between 600 and 1000 V. Avoid too high PMT gain settings because this
causes a significant increase in noise in your image. Once you have reached the
maximum voltage possible (1250 V), this will no further increase if you turn the knob on the
control console, and this will be indicated by a beeping acoustic signal.
If both settings are optimised, pixels across the image and different optical section should
be in the range of glow tones from dark red to bright yellow and white, but no blue and only
sporadic green pixels should be visible (see Fig. 22). Once you have adjusted the PMT
settings for one channel, proceed with adjusting all other channels in the same way.
Gain Offset
Z-position

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4.4.6. Additional transmitted light channel
In parallel to acquiring different spectral channels via PMT 1 to 3 it is possible to capture
images in transmitted light mode. Although these images lack a degree of specificity and
also contain out-of-focus light, they often give valuable spatial information such as cell
outlines or the position of the cell nucleus. To access the settings, click on ‘Additional
Channels’ in the main control panel (see Fig. 23 A) and activate the additional channel by
ticking the appropriate box (B). The gain and offset can be set in the same way as has
been described for the other PMT settings (C, and see above).
Fig. 20
Ch1 – off-set too low, Ch2 – off-set correct
Fig. 21
Ch1 – PMT gain correct, Ch2 – PMT gain too high, saturated

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The image acquisition in transmitted light mode requires the use of the 488 nm Argon laser
line and can be recorded simultaneously with the fluorescence channel (the laser line
activated and both detectors switched on). The settings of the condenser are being
checked and adjusted frequently and thus should be correct. However, if you feel that it
needs to be re-adjusted and if you are not familiar with the condenser unit, please inform
any CALM staff member.
Fig. 22
Both channels with correct settings
4.4.7. Saving and loading of Beam Path settings
Once you have finished adjusting all the settings, these can be saved to the hard disk for
later re-use. In the ‘Beam Path Settings’ screen click ‘save’ (Fig. 24 A). The system then
will prompt you for a name and a file. Please generate a unique file name you can
recognise later on and save the file in your folder on disk D:. This function saves the
activated laser lines, laser power settings, slit positions and PMT settings.
Fig. 23
Setting of the transmitted light channel
A
B
C

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If you want to reuse a certain preset at a later time, pull out the drop-down list (Fig. 25 B)
and choose from the list. This function can also be used to define single scan setups that
can be assembled into sequential scan protocols (see 4.5.2.).
4.5. Scanning mode
As briefly explained in section 4.3., the SP5 system allows multi-channel image acquisition
simultaneously or in a sequential mode, or as a combination of both. To avoid cross-talk
between spectral channels it is recommended to acquire images in the sequential mode,
i.e. channels will be recorded separately in a sequential order.
Fig. 24
4.5.1. Simultaneous channel acquisition
Since the system is equipped with 3 PMTs for fluorescence imaging, it can record 3
spectrally different channels at the same time. By activating the desired PMTs (see 4.4.5.)
and adjusting all necessary settings for the beam paths, the simultaneous scanning can be
set up easily.
4.5.2. Sequential channel acquisition
To define a scanning protocol for sequential channel acquisition click the ‘Seq’ button in
the window in the top left corner of the ‘Acquisition screen’ (see Fig. 25, circle). This will
activate the ‘Seq’ mode and open the ‘Sequential scan’ panel in the ‘Acquisition’ window
on the left hand side of the screen. The beam path and acquisition settings for each
individual channel must be defined as described above or loaded from saved presets.
If you use both, UV and visible lasers both need to be switched on for all the channels,
even though they might not be used in the individual channel. For instance, if your first
channel uses the 405 nm diode laser for excitation of fluorophore 1 and the second the
488 nm Argon laser line, the UV laser must be switched on in channel 2 although its laser
power is set to 0%.
A
B