Metamorph - measurements and processing

Detailed Metamorph manuals: Basic commands (3.2 MB pdf) | Drop-ins (4.0 MB pdf )

Adding a scale bar

Open the image on the metamorph workstation

Click Calibrate distances on the task bar (or from the Measure/Calibrate distances menu)

1. Make sure the correct calibration file is displayed. If you need to choose a different microscope press "load from file" and choose the correct .CAL file from C:/mm/app/mmproc/DATA.
2. Choose the objective you used
3. Press apply

Click Calibration bar on the task bar (or from display/graphics/calibration bar)

1. Adjust the bar size, orientation, xy coordinates of the position, colour etc.
2. Press Stamp
3. Save as a new file, perhaps make an 8-bit copy

Region measurements

Threshold the image - Click the threshold button on the left side of the image and choose "Auto threshold for light objects". You can adjust the threshold value by sliding the blue triangle on the image histogram.

Regions>Create regions around objects - this makes each distinct thresholded body into a region for measurement. Once the regions are made you can turn the thresold off if it makes it easier to see your image.

Measure>Region measurements - Choose the things you want to quantify from the configure tab. The results appear in the measurements tab. These can be exported to excel by pressing "open log" and selecting DDE. Once excel is open, pressing "log data" transfers the numbers to excel where they can be saved and further analysed.

Adjusting the image display

On the side of every MetaMorph single-channel raw image window are various useful buttons -

The histogram part gives you very useful information for the scaling of the image. Here the histogram part on its side, and in more detail . . .

The camera measures the intensity of each pixel and gives it a grey-level value of between 0 and 4096 (12 bit scale). Not all those greylevels will actually be populated. The histogram shows the spread and frequency of intensity values in the image. Having the black to white range of the display match the range of intensties in the image is generally a good idea. Slide the triangles to adjust.

Autoscale automatically adjusts the display so a certain percentage of pixels are under the intensity set to be black, and over the intensity set to be white.

When you have your original image displayed as you wish, you may want to:

• Make a colour overlay with other images (click "overlay images" on the taskbar, designate which image is which channel)
• Save an 8 bit copy of the file (a link is on the taskbar, this produces an image that looks the same as as the way the original is displayed, but some information is lost so save both).

Either of these options will give you a file that will open in virtually any software (eg powerpoint).

Review multidimensional data

A. Tell metamorph which images you want to build. Press "select base file" in the main review MDA window which gives you this box -

• Select directory - point to your folder with the images
• Select data set - choose the individual experiement you want to look at
• Press View

B. Select the wavelengths, stage positions, Z-slices and timepoints you want to view.

• Select the stage position (one at a time here) and channels you want (probably all of them)
• Right clicking in the boxes selects that image. Right click on the row/column heading selects the entire row/column
• You can choose multi-colour overlay (under display), projection, or best focus if wanted.
• Use the play and reverse arrows to view the timecouse etc.
• Load Images loads the select images to a single stack file which can be saved as one or exported to avi or quicktime . . (Stack/Make Movie from the top menu).

Save a QuickTime or AVI movie

• Make the stack as directed above
• From the top menu - Stack/Make movie . . .

Play each frame for: This adjusts the frame rate, 30 frames per second is standard movie rate. If each frame is played for 10 30th's of a second there will be 3 frames per second. A good way of choosing this is to decide how long you want the movie to last (eg 10 seconds and you have 100 frames, so 10 frames per second = each frame is played for 3 1/30th of a second.

Selecting the frames: If you want to use all the frames, tick all the boxes and choose "Save - Selected" (or make sure all boxes are unchecked and choose "Save - Unselected"). The buttons in the bottom left allow you to select the frames. If you have 200 frames but you only want to include every other one - Low Range=1, Step Size=2, High Range=200.

AVI or quicktime? These are different compression systems. AVI can be uncompressed (ie highest quality but the files remain very large) or a good choice for compresion is the Cinepak codec, which allows you to adjust the extent of compression. The codec options for AVI pop up when you press "Save". Quicktime doesn't give you a choice.