Leica DMi8 User manual

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NeuroTechnology Studio
Leica DMi8
User Manual
For questions or concerns contact:
Lai Ding: [phone] [phone] [email protected]rvard.edu

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Table of Contents
3…. Start-up
5…. Microscope Display
6…. Channel Set-up
8… Apply Previous Settings
9…. Display Window
10…. Acquisition Window
11…. Navigator
12…. Objective Resolution Info
13…. Camera Binning Format Info/ Shutdown

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DO NOT TOUCH ANY BUTTON/SWITCH UNLESS IT IS SPECIFIED BELOW
Start-up
1) Turn on the power strip, It is located on the wall above the power bench.
2) Wait until the display screen at the front of the microscope stand finishes startup
3) If Computer is not ON, turn on the computer.
4) Log in to your lab account on the computer:
Login to the computer using the lab account and password you were given during your training.
Remember to log off when you are finished with your session.
5) Next, open the software “LAS X”

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A dialog window will pop up. Make sure the selection is the same as picture below, click the
button: OK.
As the hardware configures, you will hear the microscope parts starting to activate. This is
normal. If any error message shows up, take a picture and notice Lai Ding or Teri Zgoda
immediately.
Select your camera: click “Preferences” under camera section.
Then choose the camera under “Set Camera to” section. “Leica DFC9000GT LEFT” is for
fluorescence imaging. “DMC5400 RIGHT” is for bright field color imaging
You are now ready to image! Please save the files to E: drive “User Images” folder and the
corresponding lab folder. DO NOT SAVE IMAGES UNDER C: or D: DRIVE!

5
Microscope Display
Almost all these features can also be changed using the LAS X software.
Status of current selections. Can also change from TL to IL and open/close shutter
Change light type and filter cube
Change the objectives and light path (directed towards eyepiece, left camera port,
or right camera port)
Press to bring the objective all the way down, and to set the focus

6
Channel Set-up
•To add a channel, press the PLUS button in the middle window. To delete a channel press
the MINUS button
•To change the false color of the channel, double click on the gradient rectangle
•Determine what wavelengths you need. Select the filter cube, which will be outlined in
red when active.
•Turn on the LED source. You may have to change the wavelength it is emitting at using
the dropdown below the slider. If it can’t use that wavelength with that filter, the box will
be grayed out with a line through it.
Make sure that the wavelengths in these dropdown boxes are the same throughout all
the channels. This quickens the time it takes to take a photograph.
•Use the slider to increase/ decrease the light intensity
•Press the LIVE button in the lower left-hand corner to view your subject

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•Use the knob circled in red to adjust Z
•Use the knobs bellow to adjust X and Y. There is a small toggle switch circled in red below
that changes between fine and coarse movement.
•Adjust exposure time and laser intensity for each channel.
•Under PROJECT SETTINGS dropdown, uncheck SEQUENCER MODE
•Press SINGLE IMAGE to photograph one single channel.
•Press CAPTURE IMAGE to photograph all channels.
•Press START to begin a z-stack or time series.

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Apply Previous Settings
•Open a previous project using button circled in yellow
•Go to “Configuration” tab and “Param Recall” section, click “Load” button, under the
pop-up file dialog window, select “setting.xml” file
•Go back to “Acquire” tab, press the button circled in red to apply the settings

9
Display Window
d
Annotation: Add shapes or scale bars to image. Measure distance between points.
Select what
channels you want
to view.
This button is to
view a composite of
all the channels.
Open 3D viewer
window. Useful only
if you’ve taken a Z-
stack.
•Change Look Up
Table (color,
intensity, or black
and white)
•Autoscale all
channels
•Stack autoscale
•Channel auto scale
•Reset
•Use background LUT
to scale
•Open Histogram
Look up table and contrast. Moving the two circles closer together will increases contrast.

10
Acquisition Window
Select Zfor Z-stack and Tfor time series.
Mark and Find
Tile Scan
FIM: fluorescence intensity manager, higher % more light
IL –FLD: field stop, should always be at 6
Camera %: (light is directed towards eyepiece) or 100 (light is
directed to camera)
Change objective here
Keep “Magnification Changer” as “ 1 “
Binning: select if you want a faster, quicker image with less
resolution exposure here.
Longer exposure = more light = higher chance of photobleaching
Binning: increases exposure but decreases resolution
Scroll through the Z to find when your subject just begins to dim and
go out of focus, then press BEGIN. Go through the focus to the next
point the subject starts to go out of focus and click END.
The system will provide number of steps with “optimized” Z-step
size if “System Optimized” box is checked. User can assign step size
or number of steps with corresponding box checked.

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Navigator
•If doing a Tile Scan, click on Link Shading to open up a new window. Check the ACTIVATE
boxes on the new window, then close. This will smooth out some of the dark edges you
would see on the final tiled image.
Pressing the Navigator button will open up a new window. Press the X to close the
window and get back to the main acquisition page.
•Click LIVE to find the center of your subject and bring it in focus
•Click SPIRAL to begin a spiral overview scan. It will scan in the channel selected at the top,
in this screenshot the red channel is highlighted.
oThe time it would take to scan the entire channel is displayed in the lower right
corner
oThe shorter the exposure time, the faster the spiral will be
•Press STOP to end the scan

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•Select what area you want to photograph using the polygonal tools in the bottom
window
•Click blue to put manual focus points on the scan area. Green is autofocus.
•Click FOCUS MAP to bring up a window displaying the points. Press live, then double click
on the first point to bring it into the field of view. Focus and press SET Z, then next to
bring it into the next focus point.
•Hit START to begin the tile scan.
•Once the scan is finished, click on the MOSAIC MERGE tab at the top of the window.
•Select SMOOTH BLENDING on the drop down menu, and click merge tiles.
Leica DMi8 Objective/Camera Info
Objective
FOV: Field Of View FL: Fluorescence Camera (LEFT) CC: Color Camera (RIGHT)
Objective
5X
10X
20X
40X*
63X (oil)*
100X (oil)*
N.A.
0.12
0.45
0.4
0.95
1.4
1.4
FOV_FL (mm)
2.67 x
2.67
1.34 x
1.34
0.67 x
0.67
0.34 x
0.34
0.21 x 0.21
0.13 x 0.13
FOV_CC (mm)
2.64 x
1.76
1.32 x
0.88
0.66 x
0.44
0.33 x
0.22
0.21 x 0.14
0.13 x 0.09
Working Distance
14000um
2800um
6900um
170um
140um
90um
Resolution XY @
550nm
2.80um
0.75um
0.84um
0.35um
0.24um
0.24um
Resolution Z @
550nm
38.2um
2.72um
3.44um
0.61um
0.43um
0.43um
*: not suitable for plastic bottom dish or multi-well plate

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Camera binning/format
Pixel Scale FL : Fluorescence Camera (LEFT DFC9000GT) Camera Pixel Size : 6.5um
Bin \\
Objective
5X
10X
20X
40X
63X (oil)
100X (oil)
No binning
1.3um*
0.65um*
0.325um*
0.163um*
0.103um*
65nm
2x2
2.6um
1.3um
0.65um
0.325um
0.206um
130nm*
3x3
3.9um
1.9um
0.975um
0.489um
0.310um
195nm
4x4
5.2um
2.6um
1.3um
0.65um
0.41um
260nm
8x8
10.4um
5.2um
2.6um
1.3um
0.82um
520nm
*: recommended binning setting
-----------------------------------------------------------------------------------------------------------------------------
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Pixel Scale CC : Color Camera (RIGHT DMC5400) Camera Pixel Size : 2.4um
Format \\
Objective
5X
10X
20X
40X
63X (oil)
100X (oil)
20MP
480nm
240nm
120nm
60nm
38nm
24nm
5MP
960nm
480nm*
240nm
120nm*
76nm
48nm
2.2MP
1450nm*
720nm
360nm*
180nm
114nm *
72nm*
4K
480nm
240nm
120nm
60nm
38nm
24nm
*: recommended format setting
Shutdown
•Make sure you save all of your files and transfer electronically. If using an external
hard drive, make sure it is clean!
•Delete any sensitive data off the server
•Check to see if there is anybody else using the microscope on the schedule and log
off your user account
•Press the switch on the power strip to turn off the microscope
•Cover the microscope