Goldberg Lab
USING LEICA AS LASER MICRODISSECTION (LMD6000)
MICROSCOPE
Written By Jungim Hur
Digital Video Camera
Eyepieces
Laser
module
Laser
safety UV
shield
Specimen
holder
Smart move
control
LEICA CTR6500
electronics box
1. Mains switch
2. Pilot indicator
Front side of the laser box
1. Key-operated switch
2. Switch for external trigger
3. Status LED: Operating status of laser
4. Push-button: Laser on/off
5. Status LED: Laser active/inactive
6. Service plug
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Goldberg Lab
6. Description and assembly of the components
• Place the specimen holder (fig. 8) onto the
stage you are using and fasten it with the two
Leica LMD6000
Operation Protocol
screws
6.2 Condenser
You can use either a motorized or a manual
condenser. Both are equipped with condenser
head S28.
1) Clean the LMD station including slide holder, PCR tube tray sub-stage holder, the 3-D
• Screw the condenser head into the
10 Collection
device
with caps damped with DEPC treated H2O (500 µl
SatelliteFig.controls
and Fig.
keyboard
with
Kimwipes
condenser.
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Removable specimen holder
1 ml
ForH
motor
stage
a 3-fold holder forin
scanning
stage
DEPC solution
500
2O).
b holder for motor stage
2 For scanning stage • Using the condenser height adjuster (10.4),
turn the condenser holder (10.1) completelyThe name of
downwards.
corresponds
•
a
1
•
the specimen (“Specimen ID“
to the name in the “Change
Specimen“ dialog which is opened with the
Unscrew the clamping screw for the condenser (10.3) far enough so that the“Unload“ key after a change of specimen. A
condenser can be inserted from the front.
data sheet is created in the "LMDDB" archive for
From the front, insert the condenser into theeach specimen name.
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condenser holder as far as it will go. On the
underside of the condenser, there is an orientation pin, which must be located in the
guiding notch (9.1).
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2) Turn on power to:
• Pull the condenser’s clamping screw (10.3) so
that the condenser
in place.self diagnostic tests, stage
a. CTR-MIC control box (microscope
will gois locked
through
will move; requires about 30 seconds).
b. CPU/monitor
the usual way by the on button on the computer.
b
c. Laser (with key). During warm-up, the laser power box will display one green
Fig. 10 Condenser holder
light (left). When the second
green
1 Condenser
holderlight (right) comes on, the laser is ready.
2 Condenser centering
Touch
the
red
button
between
the
two
green lights on the laser power supply to
Fig. 9
Condenser holder
3 Clamping screw for condenser
1activate
Guiding notch the laser. Now there4 will
Condenser
adjuster
beheight
one
green light (left) and one red light
(right). The laser is ready to fire. Requires about 5 minutes. If you want to take
a break more than 5 minute, then press red button and the laser box will
display two green lights. It will be
1 in the “stand-by” mode and will be reactivated by 1pressing the red button between the two green lights.
3) Allow the microscope to fully initialize the stage2 before
starting
the LMD application
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software.
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4) Check to be sure the Laser Safety Shield is properly in place. Laser will not fire
without the laser safety shield in place.
5) Activate the LMD software from the Desktop by double clicking on the LMD icon. Click
on the “LOAD” slide (left) button in the software menu. Slide holder will move forward;
pull the slide holder forward and out of the microscope stage. Insert the special foil
membrane slides with mounted tissue sections into the slide holder (be sure to have the
proper orientation of the specimen facing down in the microscope). Return the slide
holder back into the microscope stage by sliding the holder along the slide rails until it
snaps into place. Click the “Continue” button on the dialog box to take the slide holder
into the microscope.
Create Specimen Overview images by clicking “Options/Specimen
Overview“. Use lower magnification (4X-10X) and define scanning
are. Then, save scanned overview image.
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Goldberg Lab
6) Click the “Unload” Collector (right) button in the menu bar to unload the collection
device (moves forward), Load PCR tubes with caps into the collection device.
Inserting the cap into the holder
1. Lid of the cap
2. Holder
For RNA isolation from LCM samples, place Extraction buffer (20-30 µl) from
PicoPure RNA Isolation kit into the caps (Avoid bubbles when you place buffer into the
caps). Return the collector to the sub-stage holder and click OK to return the collector
into the sub-stage of the microscope. Select a cap to collect samples by clicking the
corresponding circular red marking at the bottom left of the work window. The selected
cap turns green.
While collecting the cell types that you are interested, the buffer added into the
tube cap might be dried. You may add 10 µl of H2O into the tube cap after 1-1.5 hour
of cutting if the light is getting dimmer because the buffer is dried and should not
collect samples more than 2.5 hours into sample cap.
7) The 3-D Satellite controls all basic remote functions of the microscope.
a. These include motorized X and Y stage movements and Z microscope focus
functions.
b. The buttons on the right side switch between coarse or fine focus and stage
motion.
c. The buttons on the left side control the up and down magnification by changing
the objectives on the nosepiece, one position at a time.
Smartmove control
1. Movement in x direction
2. Movement in y direction
3. Setting the focus
4. Individual knob height position
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Goldberg Lab
8) Focus the specimen with a low power objective and adjust for proper illumination
levels and binocular phototube prism setting for an on screen image as necessary.
Move the stage to bring the area or cells of interest into the center of the field of view.
Switch to a higher power objective by touching your selection
on
the front touch screen of the microscope, for better detail and
identification of the cells of interest for cutting, and adjust
focus and light level if necessary.
If obtained sample image is not focused or it is hard to define the cell type that
you are interested, you may align condenser adjust. To align condenser, close the
field diaphragm all the way (control the field diaphragm on the microscope control
dialogue window) and rotate the knob on the condenser until the circle of light is crisp
and in focus. Then open up closed field diaphragm and adjust the aperture diaphragm.
Refer to Condenser Alignment Protocol provided by Leica microsystem.
9) Perform Laser calibration on set-up before cutting the region of interest.
10) Test different laser settings (power and speed) to obtain optimized condition for the
cell types that you are interested. Then, record the setting and proceed. On the
right side of the software interface, select the “Draw and Cut” mode. Use the optional
touch screen pen to drawn the region of interest (ROI) directly on the monitor or
move the mouse cursor to the image area, and click and hold the left mouse button to
draw a line or shape on the monitor around the cell or cells of interest. If the drawing is
not desirable, remove the line from the shape list. iClick on the “Start Cut” button in
the software, or alternately right click and select “Cut”.
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Goldberg Lab
It is recommended to save the image with drawn the region of interest before and
after cut.
11) If the laser does not cut properly through the specimen, optimize laser settings in
“Laser Control” by adjusting “Power”, and/or “Speed”. Sometimes, laser loses
power after few hours of use, then increase laser power and re-draw the line and
cut it again. (Refer to suggested laser settings for a “normal” histological tissue sections
by clicking “Factory Settings” in the Laser Control window. Also, you can refer to the
settings previously used to cut the cells of interest.)
12) Cutting options include: free hand figures with close line option, circle and rectangle
figures.
13) Cutting options also include: “Draw and Cut” or “Move and Cut”. If region of interest is
not released after cut, re-draw the line and cut again or increase laser power and click
“Move and Cut” button to release cut.
You can inspect the samples in the cap of collection tube by pressing the "Collector" key
in the toolbar. Pressing the "Specimen" key restores the previous cutting position.
14) The “Close Line” feature can simply be checked on or off, but area measurements are
only functional with the Close Line activated.
15) Laser Alignment and laser controls settings are specific to each objective and can be
saved under “File” and “Save Configuration File As.” By naming this file and saving it to
the folder “Profiles,” one is able to recall all laser settings saved during a cutting session.
16) To recall a user specific configuration files, go to “File” and “Restore Configuration File, ”
find and activate your specific file.
17) When you finish collecting samples, take out the tube from the collection device by
unloading collector. The samples on the tube cap should be collected into the tube
by brief spin and stored at -80°C until use. Also, unload slide by clicking on the
“LOAD” slide (left) button in the software menu. Pull the slide holder out of the
microscope stage and remove slide and store in the slide box.
Unload
collector
device
Unload
slide
holder
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Goldberg Lab
18) Make sure to record the laser setting and the information of the cell types you
have captured, the objective used to capture, the volume of extraction buffer and
H2O, the collection tube name and the date of capture.
19) Close LMD Software
20) Shut down Windows (computer)
21) Turn power off to:
a. CTR MIC controller box
b. Laser (turn key to off position)
c. Fluorescence Power Supply (if utilized during your session)
d. Monitor
Adjustment of CORR objective settings for the L20x, L40x and L63x objective
1) Depending on whether you use the Glass PEN foil slide or Metal PET Frame foil slides
or Metal POL Frame foil slides, one must properly set the correction collar of all CORR
objectives in order to achieve the best image possible. The wrong correction collar
setting will result in a poor or hazy image and a sub-optimal laser cutting. The correction
collar settings are dramatically different between the glass PEN foil slides and the metal
frame foil slides. Using the settings of one for the other type of slide will not give optimal
imaging results.
2) For Glass PEN foil slides, the correction collar setting should be approximately 0.9
while for any Metal frame foil slide, the correction collar setting should be
approximately 0.1. However, proper setting should be obtained by observing the
image and turning the correction collar until optimum contrast and clarity in the
image is achieved.
NOTE: Refer to Leica LMD6000 V6.3.1. Quick (Basic Operation) Start Up and
comprehensive online “Help” files from the LMD software or consult the LMD
printed instruction manual provided with the system.
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