Leica AF6000 LX User guide
1.      Live cell cultivation system:
l   Switch on CO2 cylinder (tank).
l   Switch on temperature, CO2 controllers (it takes approximately 2 hrs to reach set point conditions).
l   Ensure water in the flask (humidifier) with CO2 transit is sterile.
l   Ensure water bath inside the incubator contain water.
2.      Starting up:
Open the upper lid of the chamber and push the microscope arm backwards.
l   Switch on PC, optical microscope, CCD, Focus, and florescent light source.
l   Ensure microscope lens are at its lowest position.
Caution: Be careful with the chamber when initializing microscope stage.
3. Starting the software:
Click on Leica Application Suite Advanced Fluorescence (LAS AF) software  →    OK →    initialize? → yes
Caution: Be careful with the chamber when initializing microscope stage.
4.      Place your sample
5.      Select:
set up
l   Optical zoom
Adjust lens magnification
locate image
l   Experimental settings
□same intensity for same filter cubes
■ use sequencer board
■ use sequencer timestamp
■ single image mode
□ enable smartmove control during acquisition
l   Z move
□ Lamda than Z (slower in speed, Z is more accurate)
■ Z than lamda (faster in speed)
□ Apply relative focus correction (different Fcr channel allows different focus set up and select “Z wide” for Z-stacking)
l   Sutter
■ After each image
Ø   Select Fcr
1.          Select contrast mode (TL-BF, TL-PH, TL-DIC, Fluo)
If for Fluo, please select filter cube
1.      Tri – excitation filter 1 (B, G, or R)
2.      Fu 2 – excitation filter 2 (340, 380)
3.      Tx2
4.      Y5
2.          Light path settings
3.          Live preview, adjust exposure time and CCD gain value
Binning – 1x1, 2x2, 4x4 (camera resolution, exposure time and file size are adjustable.)
Exposure – set up exposure time
EM gain – adjust gain value
FIM – Adjust Fluorescence intensity manager to 5
                      Same exposure for all Fcr
                      Intensity vs. pixel (adjust gamma to lower background)
RFC – relative focus correction (different Fcr channel allows different focus set up)
(uncheck Use sequential board under Experimental settings first and then check Apply relative focus correction under Z movement)
DO NOT CHANGE Camera configuration!
Ø   Additional Fcr (allows different wavelengths setup) – repeat step 1 to 3
4.          Set z-Position for image stack
5.          Set Mark and Find / Tile Scan menu
6.          Set image with Autofocus (if there is no z-menu image settings)
Set up:       speed vs. accuracy
                Global vs. local
                Z wide
t:                first vs. every cycle
position:    first vs. every position
7.          Single image, capture
8.          Set image with Time Series. Please take note that total file size should not exceed 2G
9.          Click the Start button to start image acquisition
10.      Save your image under D drive \user\year\month\date
6.      Shutting down:
l   Close LAS AF
l   Save your images on own disc
l   Remove sample
l   Adjust lens to its lowest position. Clean lens if immersion oil is used
l   Switch off fluorescent source, temperature controller, CO2 controller, optical microscope, CCD, Focus and PC

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