Leica DM 5500 tutorial.pdf - EPFL

Welcome to the Leica DM 5500
Microscope tutorial
• How to start up the system (p.3)
• How to use the stand DMI 5500 (p.4)
• Which soft to use (p.12)
LAS (for color images)
• How to use the stand from the soft (p.13)
• The Differential Interference Contrast (DIC) (p.15)
• How to acquire a Bright Field image and its different adjustments (p.16)
• How to focus easier (p.22)
• The Mark & Find positions option (1) (p.23)
• The Multi-step acquisition mode ( Mark & Find (2), Mosaic) (p.24)
• How to create a shading reference (p.30)
• How to see, to save your images and their information (p.31)
• How to annotate your image (p.32)
LAS AF (for black and white images)
• Create your experiment (p.34)
• How to acquire a fluorescence image and its different adjustements (p.35)
• How to acquire several channels and overlay them (p.38)
• The Relative Focus Correction (p.40)
• How to acquire a Z-stack (p.41)
• The Mark & Find positions option (p.45)
• The Tile scan option (p.46)
• How to shut down the system (p.47)
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Leica DM 5500 tutorial
Before using the
Leica DM 5500
You need to
reserve the
system =
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/index.phpo
ption=com_vi
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w&id=52
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System Start up
• Turn on the Fluorescence lamp
• Turn on the Microscope button
You have to turn on the light
only if it is cold (approximately
30 minutes after its last use)
• Turn on the PC
• Introduce your Username
• Introduce your password
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Microscope (left side)
• Intensity of halogen or
fluorescence lamp (+/-) (INT)
• Condenser Aperture for the
contrast (+/-) (AP)
• Field diaphragm for the Köhler
adjustment (+/-) (FD)
• Toggle between reflected and
transmitted light
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Microscope (front part)
• On the downer front part of the
microscope, you’ll find a tactile
screen
• On its left side, you will see some
tabs, to open the one you need to
setup more options, you have to
touch it
You only need to use two tabs:
• The Eyes-piece-camera position
selection
• The stage position
For the other options (Settings
summary/Illumination/Magnification
), you can change them from the
software
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Tactile screen (step1)Eyes-pieces/camera
• When you’ve touched and opened the
tab of eyes-piece - camera position
selection,
you can change between eyes-pieces
and camera position (or a fifty-fifty
mode)
You will also find both of the shutter.
• For fluorescence
• For brightfield
To open or close them, you have to
touch the shutter button, and you
will see
• The LCD off window allow you to
switch off the tactile screen. To
switch on again, just touch it
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Tactile screen (step2)About the stage
• When you’ve touched and opened the
tab of stage controller,
You’ll see two news tabs:
• The first one named Focus and care
about the Z-focus-Axis
• The second one named Stage and
care about the XY-Stage position:
you will see the X, Y positions in mm
and you can change the step of X/Y
stage controller between fast and
precise
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Tactile screen (step3)About the stage
Under the window FOCUS, you can
setup the Z Axis Stage
• By choosing a fine or coarse focus
wheel step
• By doing go down and up the stage in
some positions
• Neared focus position (
Z=0)
you can modify it with the
set button and delete it
with the clear button
• a down position, to put your
sample or your immersion oil
In both cases, you have to click on the
GO TO button to activate your
chosen position.
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Microscope (upper part) Cameras
• On the right up side of the
microscope, you will find The camera
button selection.
Indeed this microscope has two
cameras:
• A color camera (the upper one)
• A black and white camera ( the right
one)
When you’re opened your soft, you have
to choose the right camera:
• With the LAS soft select the color
camera (pull the camera button)
• With the LAS AF select the black
and white one (push the camera
button)
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Microscope (right side)
• Magnifying lenses ( 1x/1.25/1.6x), to change
the lens, you have to turn this wheel
Choose your objective manually:
• HCX PL FLUOTAR 5x/0.15
• HC PL FLUOTAR 10x/0.30
• HC PL FLUOTAR 20x/0.50
• PL FLUOTAR/ OIL 40x/1.00-0.5
• HCX PL APO/ OIL 63x/1.40-0.60
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Next to the microscope, you will find a
Joystick.
This Joystick allows you to manage the
stage:
The Joystick
Turning the upper button, you will
manage the Y-axis direction
Turning the downer button, you will
manage the X-axis direction
Turning the backwards button you will
manage the Z-axis direction
On each side of the joystick you will
find two small black buttons which
allows you to choose
Between a coarse or fine moving in
Z direction
Or
Between a fast or precise X-Y
moving direction
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Which soft to use
When you arrive on your
computer session, you
will see that this
computer has two softs,
you have to open the
right one.
• If you work with color
camera, you have to open
the LAS soft
• If you work with black
and white camera, you
have to open the LAS AF
soft
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LAS Soft Microscope on the soft(MIC1)
Under Acquire and Mic1,
• you can see the objective that you’re
using
• You can setup the aperture and field
diaphragm
• You can setup the intensity of the
transmitted light and use the
shutter
• You can select the eye piece or
camera position
• For Acquiring your image after all
settings done
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Microscope on the soft, step 2 (MIC2)
Under Acquire and
Mic2, you can
first
• setup the focus
wheel
(fine or coarse)
• setup the X/Y
stage manager
(fast/precise)
You can also Mark
some positions
(6) that you
could find later.
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Differential interference contrast (DIC)
Under Acquire and MIC1, select
DIC contrast
• To change and choose the right
contrast , turn the DIC prism
wheel
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Acquire a transmitted light image
• Under Acquire and Camera,
you can adjust
• the exposure time
• The contrast (gamma)
As you’re working with a color
camera, you have to apply a
white balance before
acquire a picture (see next
slide)
If you have no other option to
setup
You can click on Acquire an
image
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White Balance
As you’re acquiring a Bright filed
picture with a color camera, you
need to do a white balance
You have to
• select an area of your live image
which has to be white
• Create around this area a
selection with the left button of
your mouse and Click on White
balance
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Histogram and saturation options
Under Acquire and Camera and
Histogram,
• You can see the grey scale
• You can tick Show Over/Under
Exposure, and you will see the
over exposure in red and the
under exposure in blue, you
shouldn’t have some red dots on
your picture
• You can also reduce the grey
scale
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Input option
Under Acquire and camera and input
option, you can choose
• if you want a greyscale picture
• The bit depth (8/12 bits)
• The right format (2088x1550 HQ)
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The binning
Under Acquire and Camera and
Input option,
• You can choose a binning, it will
increase the camera sensitivity,
reduce the exposure time
But you will lose some resolution
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Scale Bar
• Under Camera and
scale bar,
You can choose to show
and change the scale
bar, if you click on
show
Be careful, with this
option the scale bar
won’t stay on your
picture. if you really
want a scale bar on
your image you have
to do it under process
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How to focus easier
Under Acquire and Camera and
Region of interest,
• If you select zoom focus, it will
zoom the region you want so you
can easily find the right focus.
When it’s done click on off, and
the right focus will appear on all
the picture
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Under Acquire and Mic2,
Mark and Find options (1)
You can Mark some positions (maximum 6) that
you could find later.
• To mark a position you have to click on the
dark tab, to see all positions and select one
of them and click on store
When you’ve clicked on store, your position will
be saved.
You will see the coordinates of your chosen
position next to its name and on the right
part of your screen, a circle surround the
number of the position
When all positions you want have been marked,
to find them you have to
Select the position you want to find and
Click on GoTo
Or
Dobble clicked on the right part of the
screen on the right number surrounded
position.
To clear position you have to select it and click
on CLEAR
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As the DM 5500 owns a
motorized stage,
under multi-step you
will find some
applications linked to
the stage, the two
most important:
Multi step options, step1
• A Mark and Find
option
• A multi-step option,
which allows you to
create a mosaic
picture with several
single images
To accede to the options
you have to select
the MULTI STEP
acquisition mode
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Multi step options, step 2
When you’re opened the multi-step
acquisition mode, the “S” tab will
appear and open,
under Methode choose the Mark and Find
options
• To mark a position, you have
to choose the position you
want and click on Create
Do that for each position
you want to mark
• To see your saved positions,
Click on them
• To delete your positions click
on Clear. If you want to remove
only one position, select it and
click on remove
To acquire each position which was
marked, click on Acquire Multi-Step
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Multi step options, step3(mosaic mode)
With the Multi step
option, you can scan
a “large” part of
your sample in
several images
which be put
together,
By default, you will use
the bi-directional
scan option
Then you have to
select the start
point of your
mosaic and click on
create
Afterwards you have
to choose the end
point and click on
expand
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Multi step options, step4 (mosaic mode)
You can now see your mosaic.
• if you’re area is too small, you can zoom it
• To remove it, you have to click on clear
Before acquiring our picture, under option you
can
• Save or load configurations
• Choose to create a Mosaic image or/and subimages
• Choose what you want to see during the
acquisition (Mosaic or singles images)
• Change the reduction factor of your mosaic
When all your settings have been done
you can click on Acquire MultiStep
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Multi step options, step5(mosaic mode)
During acquisition you can
see the advance of your
mosaic
When your acquisition is finished,
it will open under Browse,
you can again
• Choose to see single images or
a Multi Step image
• Change the factor reduction of the
Multi Step image
If you want to attenuate boarders between
Single images, you have to apply
a shading reference (see next slide)
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Create a shading reference
You will find the shading reference
under Acquire, camera and
processing.
You can first choose one alreadyexisted
shading reference , clicking
here
To create a shading reference,
you have to
• Find an empty area of your sample or
better a new empty slide
• Setup illumination until our picture
becomes totally white
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Create a shading reference
• Go under Acquire, camera and
processing and click on Create a
shading reference (the downer
selection you can do)
• Name it
If the illumination setup doesn’t
be alright, the soft will tell you
• Finally click on Apply to live
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See your images and their information
• Under Browse and file
information, you can see the
information linked to the image
• Under Browse and File
directory, you can see in which
file is saved your image
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How to annotate your image
• Under Process and Annotate,
you can choose to note some
information about the image
• The name , description , date
and time
• The scale bar
• Lines, measurements (ex.
distance line)
• To save the picture with the
information, you have to select
Merge.
Be careful, if you do it, you
can’t go back. So if you need
the original picture, copy it
before you annotate it
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LAS AF soft
When you’re opened the LAS AF
soft, during the configuration
time, you will see this window
appears:
If you want to use the
motorized stage to acquire some
tile scan or marking some
positions, you have to initialized
the stage
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Under Acquire and
Experiment
How to acquire a fluorescence image:
Create your experiment
When the software is open,
experiment name is
create, you can change it
You can also open some
saved experiment
And if you want more than
one experiment, you can
create them with the
New button
When you experiment is
finished you have to save
it
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How acquire a fluorescence image(step1)
Under Light Path Settings,
you have to set up your
channel
• Choose the name
• Choose the color
• Select the FLUO
contrast method
• Choose the filter
Under load/save single
settings, you can save or
load this settings
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How acquire a fluorescence
image(step2)
You have to click on Live to
see your live image
Under Acquire and
Acquisition, you can
adjust
• the exposure time
• Change the intensity
For acquiring your image
after all settings done,
you can click on Single
image ; Capture image;
Start
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Bit depth
Under
Configurations,
and camera
You can choose
between a 8 or
12 bits depth
image
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Histogram
If you click on this button,
You will see
• The histogram of your
image
For seeing over or under
exposure you have to go
on the right screen.
Normally your image has to be
in color, then you have to
click on this button to
change into Over-under
exposure mode
(Over-exposure would be in
blue and under exposure
in green)
or Greyscale mode
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How acquire several channels
Under the Light Path Settings,
• you can add or delete some new
channels, clicking on the plus or
minus
For each channel you have to
• Select the channel
Under the Light Path Settings,
• Choose a name, a color, the FLUO
contrast method and the filter
Under Acquire and Acquisition,
• Setup the exposure time and the
intensity
You can also save or load some
configuration
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How to overlay several channels
On your right screen, on the right
side you will see each channel
you’re acquiring.
If you want to see only one channel,
you have to select the channel
which interested you
If you want to see all your channels,
select all your channels
Finally if you want to see an overlay
of channels, select channels you
want to overlay and select the
overlay mode
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Relative Focus Correction
• The Focus changing option, permit
you to acquire a several channels
image , when several channels
have different focus point
between them
• To open the focus changing option
, click on this button
Relative Focus Correction (RFC)
Then you have to
• Select the channel
• Find the focus
• Click on Store Z Position
For each channel you want
Finally you have to click on apply
Relative Correction Focus
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How to acquire a Z-stack (step1)
First, under Acquire and setup,
you have to select the
Lambda then Z Z-movement
Under Acquire and Acquisition,
you have to
• open the Z-stack mode
• Go to the Z-stack dialogue
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How to acquire a Z-stack (step2)
• Activate live mode
• 1) Find the first section by
moving the z position until the
objective is positioned on the
top of your sample
• 2) select Begin
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How to acquire a Z-Stack (step3)
• Continue in Live Mode
• 3) Draw and setup the last
section by moving the z position
until the objective is positioned
below to your sample = move the
red plane into the z-stack
dialogue
• 4) Select End
• 5) Stop live mode
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How Acquire a Z-stack (step4)
• 6) Introduce the step size into
the calculator
• Do enter
Or select the system optimized
mode
• 7) Select Start
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How to mark some positions
With the mark and find option,
you can choose to save the
position of several points on
your sample and it will be
able to find them later
You have to
• Select the Mark and Find
option
• Select a new position
• Choose on the sample the
position you want and click
on this button
You can check the X-Y position
Then you have to click on Start
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With the tile scan option, you can scan a
“large” part of your sample in several
images which be put together
You have to
• Open the tile scan option
• Select the start point of the scanning
on your sample and click on this button
• Select the end point of the scanning on
your sample and click on this button
On the tile scan dialogue, you can see,
which surface will be scan and also in
how many tiles it will be done
If you want to delete your mosaic to
create a new one, you have to stop the
Live mode
You can also see the positions of your point
Then you have to click on Start
Tile scan option
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How to shut down the system
• If you was using oil/glycerol objective, clean the objective with
ethanol/water
Before shutting down the system, you have to look on the reservation
web-page if there is somebody using this microscope after you. In this
case you haven’t to shut it down but you have to log off
your session
If nobody comes after you, you have to
• Exit the software
• Shut down the PC
• Turn off the microscope
• Turn off the light
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