Leica SP8 Setup guide

Training Guide for Leica SP8 Confocal/Multiphoton Microscope
LAS AF v3.3
Optical Imaging & Vital Microscopy Core
Baylor College of Medicine (2017)

2 - Leica SP8 Training Guide
Power ON Routine
1
Turn ON power switch for
epifluorescence light source.
This lamp will be used to visualize
fluorescence in the microscope
eyepieces.
2
Turn ON Laser Power, Scanner
Power & PC Microscope green
switches then turn Laser Emission key
to ON state.
3
Power ON MP-EOM Power Strip.
Wait for Windows PC to boot and
Login to User Account.

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Power ON Routine – for NLO Only
A
Turn Chameleon TiS laser key from
‘Standby’ to ‘ON’.
B
Turn ON power strip for APT.
C
Toggle Laser selector to position A -
‘Leica’.

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Power ON Routine – for NLO Only
D
Select USB hub to Position 1.
E
Once logged in, verify that the USB
connection is active for the APT.
Green is active.
F
Start ‘APTUser’ application from
desktop.

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Power ON Routine – for NLO Only
G
Click ‘Home/Zero’ to home the
device.
H
Once homing is complete, click the
current position display.
I
Enter ‘36’ into position for Ch1 and
press OK.
Minimize APT software.

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Start LAS Software
4
Double-click LAS AF icon.
5
Choose configuration and
microscope (With Stage and
DM6000 respectively).
Choose toggle for Resonant
scanner.
Note: If you do not require high
speed scanning – leave Resonant set
to OFF. If you turn it on you will not
be able to use slower speeds.
Click OK to initialize system.
6
When prompted to Initialize Stage
select YES.
Note: Ensure that no samples are
mounted or condenser does not
interfere with stage travel movement.

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Getting Started with LAS
The LAS AF software is separated into four distinct areas.
Image Acquisition & Control Settings
These functions control the static acquisition settings for image collection. Here you can find settings such as Frame Size,
Scan Speed, Averaging, Zoom, etc.
Beam Path Configuration
This area is where we set our beam path configuration and control our detector settings to optimize our fluorescence
signal.
Image Display
This area is where the image will be displayed. Settings here allow the user to control how the image is viewed on screen.
Scan Action Functions
These functions start/stop scanning and initiate our experiment acquisition.

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Getting Started with LAS

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Powering On Lasers
Before we configure the beam path for the particular fluorophores being imaged we need to turn on the individual lasers we
require.
1. Click on the + icon for the Visible Laser dialog.
2. Toggle on each laser required.
Note: For optimal conditions set the Argon laser power to 10% output power.
3. Close Available Lasers dialog.
4. To enable Visible Lasers for use – toggle the ON/OFF switch above + to ON state.

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Mounting Sample on Microscope
Once the system has been powered on, we can mount our specimen on the stage below the microscope objective.
1. Raise stage to appropriate height and mount sample.
2. Switch microscope TFT to ‘FLUO’ to activate the incident path
from default ‘CS’ in Combi.
3. Choose filter (I3=Green, N21=Red).
4. Open/Close reflected light shutter with ‘IL-Shutter’.
5. Via the microscope eyepieces, visualize your sample and adjust
the focus (Z) and stage position (X,Y) accordingly
6. When sample is in focus and positioned correctly – switch
microscope back to CS – Combi to begin imaging with the
confocal.

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Beam Path Configuration
Once the lasers are powered on we want to configure the beam path to collect a fluorescence signal. In this example, we will
design a beam path for an Alexa 568 sample.
1. From the Visible Laser dialog, choose excitation
wavelength that matches your fluorophore. In this
example, we will use the 561nm laser.
Note: Set the transmission % to a reasonable starting
value (this may be changed dynamically depending
on signal strength). A starting value between 1-5% is
recommended.
2. Choose primary dichroic mirror that matches the laser
line you wish to use.
Note: If you will use sequential scanning choose a
beam splitter that matches every line you wish to use.
3. Turn on PMT for detection and select LUT (look up
table or color) for that particular channel.
4. Define upper and lower emission band thresholds
based on the spectrum for your fluorophore.

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Beam Path Configuration
Note: PMT selection will be based on where your fluorophore(s) fall in the linear range of the emission spectrum. Meaning that
PMT 1 corresponds to the lowest wavelength and PMT 4 corresponds to the longest wavelength fluorophore. In our example, we
will be using a sequential scan with Alexa 568 and Alexa 488, so PMT 1 will be used for the Alexa 488 signal and PMT 2 will collect
the Alexa 568.
Sequential Imaging for more than one color:
1. Once setup for initial single color is complete (in this example, Alexa 568),
ensure the ‘Seq’ button is selected in your Acquisition control settings (see
Acquisition Setup, Step 2, page 14). This will activate the Sequential Scan
dialog.
By default, your previously designed single beam path will be Seq 1.
Note: The default selection of ‘Between Lines’ is recommended so the system will
switch between acquisition tracks every line to minimize any potential sample drift.
2. Once these options are selected you can click + to add ‘Seq. 2’ and repeat steps 1-
4 on the previous page to change the beam path settings to match your second
fluorophore.
3. Repeat process for total number of channels required.

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Beam Path Configuration
Note: We always recommend that you collect your longest wavelength fluorophores first in order to ensure minimal photo
bleaching of lower wavelength fluorophores.
Example Beam path for Seq. 1 – Alexa 568
Example Beam path for Seq. 2 – Alexa 488

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Acquisition Setup
The Acquisition Mode parameters located on the left-hand side of the software display contain all the settings available for image
optimization and collection.
1. From the dropdown, choose the Acquisition Mode. For example, if you want to
collect 3d image stacks, select ‘xyz’ from the dropdown.
The remainder of the settings in the acquisition mode control dialog can remain at their
defaults to start. Depending on sample morphology and signal quality these settings will
need to be adjusted later.
Format
– image size in pixels. Default is 512x512 however this can be optimized
based on zoom and objective settings. Ideal Nyquist sampling frequency can be
quickly selected with ‘Optimize XY Format’.
Speed
– overall speed of scan during acquisition. Generally, try to run this at the
default 400Hz to start and change depending on noise levels.
Zoom Factor
– change magnification without changing objective lens.
Line Average
– selection >1 will scan each X-line number of times selected then
average result. This will be the most effective method for reducing noise in the
resulting image.
Pinhole
– this controls the optical section thickness. To start – select ‘Airy 1’ button
to set optimal pinhole diameter. This can be increased or decreased to adjust
image quality.

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Scanning an Image
Once you have completed the beam path configuration for your fluorophore(s) it is time to begin scanning to optimize the
image quality.
1. Press ‘LIVE’ to begin scanning.
2. Image will appear in Image Display area (Page 8, Area 3).
3. From the Acquisition Mode dialog, set the Pinhole to ‘Airy 1’.
Note: Setting the pinhole to 1 AU is a compromise between axial resolution and signal level. When you start with an
open pinhole, you are at a point where your signal is the highest but your axial resolution is at its lowest. As you reduce
the aperture diameter you are increasing your Z resolution but reducing your signal level. This is a reasonable trade off
until you reach 1 AU. Reducing the pinhole lower than 1 AU will still linearly increase your Z resolution but now signal will
start to drop exponentially.
4. Beginning with one channel, adjust ‘Gain’ in under PMT for selected channel until you can see
a reasonable image on screen.
Note: Increasing Gain will effectively increase PMT sensitivity to signal, making your image
brighter. This will also increase noise which can be reduced with scan speed/averaging
adjustments.
5. Repeat for remaining channels.
6. Stop scan and move to ‘Fine Tuning Image Quality’ section.

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Fine Tuning Image Quality
In order to find the optimal gain settings for a particular condition, you must first be able to identify the thresholds of the signal
level in the image.
1. While scanning a live image, click Quick LUT and toggle to Glow mode (over &
under) in the Image Display.
Note: Glow mode assigns a green color to underexposed areas in the image with a
grey value of 0 (or less). It assigns a blue color to overexposed areas in the image
with grey values >255 (for 8 bit).
2. Switch ‘Visible’ lasers toggle to OFF.
3. Adjust ‘Offset [%]’ for PMT to the minimum possible value so the image is just
above the display of any green pixels.
4. Switch ‘Visible’ lasers back to ON state.
5. Adjust the ‘Gain [V]’ so that your brightest areas fall just below the overexposed
limit (blue pixels).
6. Click ‘Capture Image’ to collect final scan.
Note: Capture Image will scan with same scan speed and frame size as LIVE, and in
addition it will apply any Line or Frame Averaging you have set to control image noise in
the Scan Control settings.

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Fine Tuning Image Quality
Ideally, you do not want an area in the final image to contain blue or green pixels with the Glow mode LUT. For maximum
contrast, adjust the Gain so the brightest areas fall just below overexposure (blue) and the Offset so the background areas fall just
above underexposure limit (green). In a properly balanced image you should see no blue or green pixels with this LUT.
Example of Overexposure (
blue) and
Underexposure (
green)
Solution? Reduce Gain and increase
Offset
Example of Good Exposure

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Understanding Image Quality
The result of the final scan above may or may not produce acceptable image quality. Therefore, it is important to have an
understanding of the three key factors that play a role in image quality.
Pinhole (Confocal Aperture)
The confocal aperture controls
both axial resolution and signal
level. Opening the aperture
reduces axial (Z) resolution but
increases signal level reported to
the detector. Closing this aperture
will reduce the signal level but
increase axial resolution.
Laser Intensity
The intensity of the laser
illumination source has obvious
effects on the signal level.
Increasing laser power will increase
signal levels but may also introduce
nonlinear effects such as
phototoxicity and photobleaching.
Detector Sensitivity
The detector sensitivity is regulated
by the high voltage gain applied to
the detector. Increasing the gain
directly increases detected signal
but also amplifies the inherent noise
in the system.

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Understanding Image Quality
Pinhole (Confocal Aperture)
Ideally, the confocal aperture should be set to the size of the structure(s)
you are trying to resolve. However, for example, some sub-cellular
structures of interest may be beyond Abbe’s diffraction limit and
therefore beyond the microscope’s capability to resolve. The confocal
aperture has some practical limits that can be used to guide the usage
of this setting.
Start by closing down the confocal aperture to ‘1 Airy’. You can certainly
close the confocal aperture below this value and continue to improve
resolution, just understand that below ‘1 Airy’ you will lose signal level at
an exponential rate.
Conversely, you can increase the confocal aperture diameter to improve
the detected signal level if you can sacrifice axial resolution.
In some cases, the signal level may be so low that increasing the aperture diameter is the only way to lower the gain enough to
get a usable image.
In other cases, if the gain is too high (causing excess noise) and the laser power cannot be increased due to photo effects then
increasing the confocal aperture is the only option to improve image quality.

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Understanding Image Quality
Laser Intensity
The overall power of the laser will directly affect image quality but most
importantly it will have the greatest impact on the health of your sample
and fluorophore(s). Increasing laser power will yield more signal but it
will also induce negative photo effects such as phototoxicity and
photobleaching that can harm the sample. Lasers also generate
considerable heat when used at higher powers and that may have
unforeseen effects on the sample.
For most lasers on this microscope, we recommend laser power
settings between 1% and 5% to start. It is recommended to start as low
as you can possibly go and still get an image. Then it can be increased
as necessary to help balance image quality.
To discover your fluorophore(s) saturation level (i.e. how much laser power you can use before your fluorophore stops absorbing
additional photons) you can start imaging at a low laser power and gradually increase the power slider until you reach a point
where your image stops getting brighter. Continual increases in laser power will stop yielding more signal. That point is the
practical laser power limit.