Leica SP8 Setup guide

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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.