QUICK MANUAL FOR USING THE LEICA APPLICATION

SOFTWARE (LAS) INSTALLED ON THE COOLLEI COMPUTER

Leica camera DFC 420C (SN 384681109)

Room HG01.235 General Instrumentation - FNWI Radboud University
For (full) instructions to new users or other questions, please contact the
assistant
(see also Leica LAS Help menu):
Liesbeth Pierson, Tel 024-3652012, room HG 01.222,
Email e.pierson@science.ru.nl
Or if absent, Geert-Jan Janssen or Jelle Eygensteyn
Website: www.ru.nl/fnwi/gi and www.ru.nl/science/gi and then follow the menu under
light microscopy

A. GETTING STARTED more

B. CHOOSING BINOCULAR OR ORTHOPLAN SETTINGS
more
C. STORING CAPTURED IMAGES/MOVIES more
D. IMAGE OR MOVIES more
E. MAIN FUNCTIONS FOR IMAGE ACQUISITION more
F. THUMBNAILS, BROWSE, PROCESS more
G. CLOSING more

A. GETTING STARTED
1. Ask assistance to get instructions and to have the camera connected to the
desired device (binocular or microscope)
2. Start Windows (no password required)
3. To preview or capture images with the camera, open the LAS software by double

clicking on the LAS icon.

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B. CHOOSING BINOCULAR OR ORTHOPLAN SETTINGS
4. Magnification calibrations have been pre-entered for the two most used devices:
a) The Leica MZFLIII binocular. Its starting menu looks like this:

b) The Leitz Orthoplan microscope. Its starting menu looks like this:

If the device of your current choice is already the one appearing upon opening
the software, (or if another device that the other pre-registered device is required.
Note that in this case magnification settings indicated by the software will be
erroneous), proceed to item 10.

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 If the other pre-entered device is required, go to File> Exit LAS

To Are you sure you want to Exit LAS?, answer Yes

5. A new menu opens. Under Options, choose Hardware Setup, like in:

6. Please select the connected microscope from the list, either:

a) Leica MZ12.5 for the binocular, exactly like shown here (so do not choose
X/Y stage or other items) and press Save

or b) the Leica generic microscope and Orthoplan for the microscope, and press
Save.

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 DO NOT CHANGE ANY OTHER SETTINGS!

7. After having changed a device choice, go to File> Exit and confirm exit by Yes

8. Restart LAS software. Now the desired device setup should appear. (In case of
troubles, contact Liesbeth or back-up administrator Geert-Jan)
9. Always check the calibrations, for example with an objectmicrometer, because
errors might be introduced automatically. Photoshop calibration tables for the
Orthoplan and the binocular are available on the Coolei pc desktop and on the GI
server (geminstr-srv.science.ru.nl, login with your cncz name and password)
> \CLSM-&-Lightmicroscopes\A-Calibration-Magnification-
LightMicroscopes\LeicaDMIRE2-CLSM).

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C. STORING CAPTURED IMAGES/MOVIES
10. To save images, create (if not done already) with Explorer an own folder under
E:>DATA>Users>the own Department> and your name (first and last name), e.g.
E:DATA>Users>General
Instrumentation>Pietje Bel
where captured images/movies will be
temporarily stored
11. In order to record captured
images/movies in the own directory as
default path, go to Browse, click on the
desired path (DATA (E) >Your
department>Your name> own sub folders,
if you like, and then press the Set
capture location button.

The newly defined default path for storing

images is indicated in red.

You can retrieve data from this folder and

copy them to an own storage stick, server
or burn them on CD/DVD.

After a session, PLEASE, copy the

captured images to your own storage
station within one week. Check that all
data have been correctly transferred and
delete the original data from the Coollei
PC yourself to prevent clogging. There will be frequent clean-up actions for files older
than one week. If for any reason a longer storage time is required, contact Liesbeth

D. IMAGE OR MOVIES

12. For switching between image acquisition to acquiring movies or time-lapse

series (=Multi-time) go to Select Acquisition Mode:
and click the desired modus.

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E. MAIN FUNCTIONS FOR IMAGE ACQUISITION
13. Overview (details further on)

a automatic exposure
b automatic white balance
c pop-up menu camera settings
summarizing most functions
d is inactive
e Exposure Adjust is to set parameters
for acquiring an image
f Input Options resolution life/captured
images
g Histogram distribution of pixel values
h Processing shading correction,
sharpening, flip
i Regions of interest for crop, local
focus, zoom
j Calibration settings x pixels = y m
k Scale bar size and appearance
l Linking inactive
m Check color hue and saturation
corrections
n Acquire image to capture a view

Details of menu

14. ad a Automatic exposure. Click once to

set
15. ad b automatic white balance
Applies automatically white balance on full
preview image

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16. ad c pop-up menu camera settings summarizing most functions (see also
details step by step here below)

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17. ad d inactive

18. ad e Exposure: exposure time, Saturation (color intensity), gamma (linear or not),
gain (for strong/weak signal)

19. ad f Input Options Do not modify configure camera, keep cooling of the camera
on.

Recommended Live Format (preview) 864 x 648 Progressive large for bright
field (quite rapid refreshing rate and still reasonable screen resolution;
Progressive medium is more desirable for rapidly moving objects, the slower HG
options when details must be considered for the focus)
Recommended captured Format (final image after acquisition) are 1728 x 1296
Interlaced medium HQ or interlaced large HG (for some special applications, e.g.
posters where the objective is not the bottle neck).
For very weakly stained fluorescence samples (susceptible to bleaching) Color
binning in both Live and Capture mode may be an outcome (poor resolution, but
high sensitivity)
Standard for imaging is 8 bits/channel. 16 bits per channel (effective less, 10 bits,
I guess) is more suitable for semi-quantification (large dynamic range; can be
opened in Photoshop and Image J)

20. ad g Histogram shows the distribution of pixel intensities (over and under
exposures)

21. ad h Processing
includes shading
correction,
sharpening (in
general not
recommended on
the acquisition of
original scientific
images; sharpening
is a typical post-
acquisition
processing, e g to
apply with
Photoshop) and
image flipping.
Shading correction
is useful when the
background
illumination is
uneven or when a
spot of dirt on the
target of the
camera obscures
the view. First,
create a shading
reference, without
the object (possibly
slightly out of
focus), but with all characteristics for a certain objective or zoom, illumination

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 profile and orientation of the camera. Take care that no shading settings of earlier
images/session are inappropriately still active. Choose then None.

22. ad i Regions of interest crop to ROI, white balance, spot exposure spot focus
spot zoom (preview)
23. Automatical White Balance

24. How to switch on/off region of interest? (E.g. to determine the best focus)

25. ad j Calibration settings according to pre-entered calculations. Control

whether OK!

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 26. ad k Scale bars adjustments

Calibration images have been made

and compiled to provide scale bars in
a Photoshop document in separate

layers for the various settings

(see GI server under CLSM-
Light microscopy then
calibrations). However, always
check yourself that the settings are
correct, using an object
micrometer (ask assistant for it).

27. ad l Linking not active

28. ad m Check color to manually correct color by dragging the cursor. Drag a
region (can be resized) over the life image and click check color. A dot and a
cross appear. Shift the cross to adjust the color balance. The dot shows the
result. In an equally balanced RGB signal (e.g gray) the dot appears in the
center.

29. ad n Acquire image (image and encrypted calibration settings will be stored.
However, note which objective was used or what the zoom factor was for later
size calibrations)

F. THUMBNAILS, BROWSE, PROCESS

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30. To see thumbnails of already captured images activate the icon indicated in
blue here
To switch between the image size indication and the frame refresh rate,
press control + R (indicated in green)

31. The Browse menu allows on to view images under various zoom, to navigate
within a zoomed image, to view information under i.

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32. Functions of the Process menu

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G. CLOSING
Go to File Exit. (Captured images can be retrieved from the Users folder)

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