Microscopy Technical Notes

These image files can be opened as Foreign Format files.

On the Macintosh:
Define a Foreign Format with the following settings:

Yes, IPLab for Windows is completely compatible with Windows XP and Windows 2000.

The IPLab installer automatically installs the Sentinel NT driver required to make the key/dongle work.

If you need to install them yourself, you will find the Sentinel NT installer on the IPLab distribution CD. Please read the simple installation directions that come with the installer.

IPLab calculates an object's area by measuring the number of piixels within the object.

If you tell IPLab the size of a pixel, then IPLab will automatically convert the area from pixels into the units you defined. For example, if you tell IPLab that a pixel is 2 µm tall and 2 µm wide, then IPLab will automatically report an object with a 10 pixel area as having an area of 40 µm2. You only need to define the pixel-to-units conversion factor once, but of course you can define multiple units for different objectives and imaging systems.

IPLab does measure the actual area of objects within the digital image. However, it is important to remember that CCD cameras and scanners always convert images into rectangular arrays of square pixels. Regardless, the measured area is the real area so long as the object is significantly larger than the size of a single pixel.

Multiply the image's width in pixels, its height in pixels, and the bit depth of the image.

Bit Depth:

• Byte, or 8 bit, images obviously have 1 byte for each pixel.
• Short Integer and Unsigned 16 images both have 2 bytes. Common digital cameras acquire 12 bits of data but store it in one of these 2 byte data types.
• Color 24 images have 3 bytes per pixel.
• Color 48 images have 6 bytes per pixel.

File size = (image width in pixels) x (image height in pixels) x (bytes per pixel)

Example #1:
Your monochrome digital camera acquires an image 1280 x 1024 pixels in size. The camera acquires 12 bit data, so the images are stored in Short Int or Unsigned 16 images, with 2 bytes per pixel.
Example File Size #1 = (1280 pixels) x (1024 pixels) x (2 bytes/pixel)
= 2,621,440 bytes
= ~2.6 MB

Example #2:
Your color video camera acquires an image 640 x 480 pixels in size. The camera and its frame grabber produce a color 24 image, with 3 bytes per pixel.
Example File Size #2 = (640 pixels) x (480 pixels) x (4 bytes/pixel)
= 921,600 bytes
= ~921 KB

1. First, you select the area you want to measure.
2. Next, you can choose which measurement to perform.
3. Finally, you measure!

All of the commands you need are in the Analyze menu, unless stated otherwise.

You're done! You may want to read the Analyze section of the IPLab User's Guide to learn how to perform more complex measurements.

By using a USB-to-serial-port adapter or a serial port PCI card.

Now that RS-232 serial ports are no longer included on the newest Macintosh computers, BD Biosciences Bioimaging has tested various third party devices that provide these necessary ports. The serial port PCI cards also work with older Macintoshes, in case you need more than the two built-in ports.

Remember to install the software supplied with the hardware.

PCI Solutions:

• Keyspan SX4 Pro Serial Card
• Creative Solutions Lightning PCI
• Megawolf Remus / 2

USB Solutions:

• Keyspan Twin Serial Adapter (USA28X) - this is an adapter that plugs into your USB port and provides two serial ports.

Within IPLab, use the Point Function command from the Math menu. "Point Function" means that the function you select will be applied to each pixel in the image.

1. In order to subtract a constant, use the first function, (ax + b)/c. The variable x represents the intensity value of the pixel.
2. In the bottom left corner of the Point Function dialog box, set a = 1 and c = 1.
3. Set b equal to the constant you want to subtract (i.e. 120).

IPLab can measure the mean intensity and the area using different units. If you set the units of distance with the Set XY Units command in IPLab, then the mena will not equal (measured sum / measured area) because the units will be different.

IPLab measures the mean as intensity units per pixel. It measures the area in the units of distance assigned by the Set XY Units command (for example, microns or millimeters).

You can define new distance units with the Define XY Units command, and you can apply the units to images by using the Set XY Units command. Both commands are in the Analysis menu.

IPLab will open only uncompressed TIFF files. If you wish to open a TIFF file with IPLab, please make sure that you turn off all compression options when the image within another application.

The image-sensing CCD can only capture a small area of the image that the microscope projects, so on the monitor you will see a smaller area at higher magnification.

If you need to widen your field on the computer, you will need a camera mount that has a reducing factor, typically 0.6x, 0.5x or 0.45x. BD Biosciences Bioimaging can help you select an appropriate mount. In addition, some microscope manufacturers produce variable magnification or zoom mounts. These let you customize the magnification and field size on your monitor.

Installing camera controls and extensions will add menus of commands to IPLab's menu bar. If IPLab cannot find these extensions, the menu will disappear. To find the extensions and replace the menu, answer these questions:

• Did you drag IPLab to the desktop?
   
  If so, you need to put it back in its folder and put an alias of it on the desktop instead.
• Is there more than one copy of IPLab on your computer?
   
  If so, one copy of IPLab might not have extensions installed. You might be running that copy, and that is why your menus are missing. It is best to have only one copy of IPLab on your disk. Otherwise you need to keep track of which copy of IPLab is running when you open an image.
• Have you just installed a new version of IPLab?
   
  Then you also need to install any other extensions that you own into the new IPLab folder.
• Have you read the manual?
   
  New versions of some extensions are put into different menus than in the past. For example, you will find the new Ratio Plus for Windows commands in their own Ratio menu. We no longer use the Ext. menu for anything.

You have to restart the computer, even though the installer didn't say that you needed to. This can happen when you install a camera that you previously used with the Evaluation version of IPLab.

The program is telling you to allocate more memory to it. Macintosh OS 9 and earlier allocates memory to the program when you launch the program. If you open too many images, the program will run out of memory and give you the "Insufficient Contiguous Memory" error.

Please do the following:

1. Quit the program if it is running.
2. From the Macintosh Finder, find the program. (For example, find IPLab in the "IPLab" folder).
3. Click once on the program's icon once to select it.
4. Choose the Get Info: Memory command from the File menu. If you only see Get Info, choose that.
5. Increase the Preferred Size value in the "Memory Requirements" section of the dialog.
   The amount to set depends on the amount of RAM in your machine, and the amount of memory used by other programs that you want to run at the same time.

(This solution is the same for both IPLab and ONE-Dscan/Mac.)

It is possible that the program does not have enough memory available to grab the image. When this happens, it tries to put up an alert to notify you of this situation. Sometimes there is not enough memory left to display the alert, so all you hear is a beep. Try closing some windows in the program, or assign more memory to the program.

Here's how to assign more memory to IPLab or ONE-Dscan/Mac:

1. Quit the program.
2. Open its folder.
3. Single-click on the program's icon to highlight it.
4. Select Get Info from the File menu.
5. Increase the Preferred Size of IPLab.

Error 65 is a timeout error. This means that the Snapper frame grabber board is waiting for data from the camera. Usually this means that the cables are not connected correctly.

If you are using version 1.2 (or higher) of the Orca acquire module, or any version of the Orca II acquire module, make sure that the BNC cable labeled 'O' at the end of the data cable is connected to the EXT. TRIG. port on the Orca controller.

The VirtualCam is a camera control which simulates a camera in software. You can use it to write scripts or demonstrate the software when you don't have a real camera available.