Using Micro-Manager/ImageJ
1. ImageJ is an image analysis program. Micro-manager is a plug-in to ImageJ that
allows control of a microscope, camera or other equipment. We are using it to control
the light sources and the camera. We also have a program called Fiji which is
different version of ImageJ that is set up specifically for analysis. I recommend you
download Fiji to your personal computer so you can use it outside of the lab. It is
freeware and has a huge development and support community to which anyone can
subscribe. On the two sites below is also documentation of each of the programs if
you need help. These are also bookmarked on the lab computers.
a. Micro-manager: http://www.micro-manager.org/index.php
b. Fiji: http://pacific.mpi-cbg.de/wiki/index.php/Main_Page
2. The microscopes are manual except for the illumination system. There are 3 LED’s
to provide light; White, Blue and Green. Blue and green are for fluorescence
illumination which we will use in a few weeks. Today we will only use White (on top
of the microscope). There are three small cubes mounted on a plate next to the
microscope. These control the 3 LED’s. Each has a switch on top labeled
On/Off/Pulse. When switched to ON, the LED is on all the time. When Off, it is
always off. In Pulse mode, the LED is switched off and on by Micro-manager. Most of
the time, we will work in Pulse mode.
3. In Pulse and On mode, the dial on top of the microscope controls the brightness of
the LED. If the image is all white, it means the light is too bright. If it is all black, it
means no light is getting to the camera. Either the light is not on (check by looking at
the sample and you should see light shining on it) or the camera/eye knob on the
microscope is set to eye (if so you should see light when you look through the
eyepieces).
4. To use the system; turn on the outlet strip that powers the LED’s. The switch will light
up when turned on. When you are done, turn this switch off! Now switch the white
cube to pulse. We have a small problem that in some cases, the light is too bright
even at its lowest setting. I will show you tricks to overcome this problem
5. Start Micro-manager by clicking once on the icon in the dock. You will be asked to OK
a dialog box to initialize the camera control. When the program opens you will see
the ImageJ toolbar and the Micro-manager control window (see images)
ImageJ Toolbar
Micro-Manager Control Window
6. If you click on the Micro-manager control window, the menu’s on the top of the page
will be the Micro-manager menus. If you click on an image or the ImageJ toolbar, the
menus will switch to ImageJ.
7. In the Micro-Manager window, choose White from the Channel popup. If you click
Live in Micro-manager, the White light will come on and the camera will start sending
images to the computer.
8. We will talk about all the settings in a future lecture.
In Pulse and On mode, the dial on top of the microscope controls the brightness of the
LED. If the image is all white, it means the light is too bright. If it is all black, it means no
light is getting to the camera. Either the light is not on (check by looking at the sample
and you should see light shining on it) or the camera/eye knob on the microscope is set
to eye (if so you should see light when you look through the eyepieces).
9. To use the system; turn on the outlet strip that powers the LED’s. The switch will light
up when turned on. When you are done, turn this switch off! Now switch the white
cube to pulse. We have a small problem that in some cases, the light is too bright
even at its lowest setting. I will show you tricks to overcome this problem
10.
When you start Micro-manager it will open with a control window. There is a tool
bar associated with ImageJ. When you snap an image, you see a new window open
with the image. If you click on the image, the menu’s on the top of the page will
switch to ImageJ. If you click on the micro-manager control window, the menus will
switch to the Micro-manager menus.
11.
Start Micro-manager by clicking once on the icon in the dock. You will be asked
to OK a dialog box to initialize the camera control. When the program opens you will
see the ImageJ toolbar and the Micro-manager control windowl