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1

NeuroTechnology Studio

Leica DMi8

User Manual

For questions or concerns contact:

Lai Ding: (617)264-5946 (781)901-6116 [email protected]rvard.edu

2

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

3

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”

4

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

7

•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.

8

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.