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.

3 - Leica SP8 Training Guide
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’.

4 - Leica SP8 Training Guide
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.

5 - Leica SP8 Training Guide
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.

6 - Leica SP8 Training Guide
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.

7 - Leica SP8 Training Guide
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.

8 - Leica SP8 Training Guide
Getting Started with LAS

9 - Leica SP8 Training Guide
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.

10 - Leica SP8 Training Guide
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.