(Database for the nematode branch of Assembling the Tree Of Life)

Imaging

Switch Olympus IX2-UCB (microscope, stages) but the X-Y of the stage will not start working until you switch the Proscan (Software) control. At this level you can use the microscope for simple study without image capturing. Should you want to proceed to image capturing you need to connect the microscope to the computer and switch the camera. You may do the following to connect start imaging.

1. Insert the IPLab software key to the computer slot.
2. Switch the Q-Imaging Retiga Camera on the left side of the microscope (light is green when on)
3. Start your computer (Mac) OS 9.2 interface.
4. The computer will check connection with the microscope, the motorized stage and the camera. When connection is well with all three, a list of function keys appear on the right side of the screen.
5. Most image capturing can be done using the Single Z-Stack option. This function enables you to take Z-stacks at one defined position and offers the flexibility to move to another position manually, or do the collection of Z-stacks on the same position using a different objective, totally different setting or different light set up.
6. Click Single Z-Stack
7. It will ask you to select thickness of Z-stacks or slices. There are two choices given at this level, o.5µm and 1µm. One can chose any at this level and proceed, but it is always to change to any thickness at a later stage before the actual capturing.
8. Then a box with the message please select to of sample then press OK, click OK. Two windows will appear at this level. One will appear on the left side of your screen with the image you see under the microscope and a smaller one (Exposure window) will appear that will allow you to change the light exposure of your image.
9. Indicate the desired upper position of the image by moving the objective-tuning knob towards the direction indicated close to the knob (on the right side). This window has Set Top on it to remind you at what stage you are working on.
10. If the quality of the image is acceptable, you may click Grab on the lower right side of Exposure window. (see below if image quality is unacceptable).
11. A window will appear that says Please select bottom of sample then press OK. The window will have Set Bottom to remind you the at what stage you are working on. You may do the same as in setting the upper limit, except you have to move the focus-tuning knob to the opposite direction. When you click Grab, a small window appear that asks you to enter Exposure time and Step size.
12. This step will decide what light exposure should be used in capturing the image. One has to try and find what is the best for every image, but one would develop a sort of feel for quality at this level based on the image quality observed at the earlier stages. Also it gives the opportunity to change the size of each slice in case one needs to change this. Then you can click OK. The computer will immediately start capturing slices of images starting from the defined upper level to the defined lower level. Here it will give you the opportunity to view every slice as being captured. In case the light is too dark or too bright, you may modify light intensity by closing the diaphragm slightly, though it is not advisable to start modifying image quality at this level.
13. In case image quality is not acceptable either it is too dark, too bright or lacks good contrast, then click cancel at the end of the image capturing and when the savings window appears. Discard the window and click Single Z-Stack again. Follow the procedure indicated above until you arrive at the stage where you set the upper level of the image. Here using the second window Exposure window, you can change settings for the light. There are many ways of changing the light of an image. One can do it at the microscope level or at the computer level. In both ways keeping the quality in mind and the purposed of each clip will dictate choice. At the microscope level there are many ways of modifying the light atmosphere of an image. Again the structure being observed may determine the amount of light and how much contrast is needed, apart from personal preference.
14. At the computer level you can use the Exposure menu to change the time (msec) of exposure. This simply increases the time as you increase time, the image becomes brighter. The second option is to use Gain button to modify the amount of light the whole background gets. Typically one needs to modify a combination of both the exposure and gain values to get a good quality image. Repeated trial of various combinations may be needed in some cases to get a good contrast and light intensity and ultimately a good image. The default setting for the exposure is 2msec and the value for the gain is 9.

Saving

Once good quality Z-stacks are acquired, the saving window will appears asking where to save. You can indicate the destination file and it will deposit the stacks as IPLab files.

Naming of files:

To make searching and data management smooth, each Z-stack and the video clip that result from it are to be named in a standard format following the one provided below:
Name of body part + magnification of objective used + any magnification lens employed + additional specifications

Note: The way body part names are to be written is provided in the form of a list in the website (http://nematol.unh.edu/how_to_use.php#body ).

Example to give a name for a Z-stack of the tail tip, taken at 60x objective with additional 1.6x magnification lens and DIC (Differential Interference Contrast) would be:
tail_tip60x1.6xDIC

But the nematode from which the image was taken would be given a unique number i.e. a Nematode ID.

The way the Nematode ID is a combination of the date the image was taken and the person who took it. Each name will have six numbers followed by two letters and one or two numbers. The first two numbers are the month, the following two numbers are the date and the last two numbers are the year, followed by two letters from the preferred name of the person and the last number is an indicator of which individual it was from those imaged the same day.

Example: the tenth nematode (10) whose image was collected on, lets say, January 20, 2003 (012003) by a person named Eyualem (Ey) would be: 012003Ey10 This number will be unique to that single worm and will not be given to any other worm in the database. If the same person images another worm after the same day, it would be given the unique Nematode ID of 012003Ey11.

Exporting images as movie clips

1. Start IPLab software. You don't need to get connected to the microscope to do this, but you need the software key to use the program.
2. Open the IPLab file you intend to export as movie.
3. Under File menu click Export, it will open an extension window sequence to movie. Click it and a window Export Sequence to Movie will appear.
4. Click on the downward arrowhead to indicate the source file and click the file in use.
5. Click on the Save in. Here you can chose the folder where the movie clip will be deposited and the filename to be used. Click Save when you finish selecting the destination. Now you have selected what to export and where to export, what remains is to define the conditions of the process of exporting the file.
6. The middle lower left side of the window gives you the opportunity to chose either all the slices or you may also select a defined range of slices for export.
7. The middle lower right gives you the option to chose different speeds, i.e. seconds per frame, for the export the image. After trying different settings for the export the best suited for exporting is the 0.05second per frame setting. But one can change this setting if a faster or a slower speed is preferred.
8. We do not use the other options indicated on the lowest part of the window such as compression etc. But one can use them if need be.
9. Click OK and your image will be exported from an IPLab Z-stack format to a QuickTime movie and will be deposited in the folder specified.

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Copyright © 2002-2009  UNH HCGS and the University of New Hampshire Database design, development and management: Fangning Liu

Funded by NSF