File formats for microscopy

TIFF, JPG . . .

Always use TIFF. TIFF is a lossless format and all data is preserved. JPG, PNG etc are formats for making the files smaller while keeping the images looking pretty much the same. They involve the loss of at least a little bit of information - fine for pictures of your holiday, not so good for microscopy. TIFF stands for Tagged Image File Format. The tagged bit is very useful for microscopy as the systems often populate the tags with useful information like the objective used, exposure, binning etc.

Lossless compression (eg LZW) attempts to compress exactly the same image into a smaller file. In general, it is quite difficult to compress complex images in this way so it tends not to save much drive space and I wouldn't bother with it.

Bit depths

This is how many grey levels there are in the image (or in each colour channel of the image). The detectors used in microscopy produce very accurate measurements of intensity so often we need a few extra grey levels. Here are some relevant types

8-bit	28 =	256
12-bit	212 =	4096
16-bit	216 =	65 536

256 grey levels are enough for most things so that is the basis of most standard formats. A standard colour image is a 24-bit RGB - 8bit RED, 8bit GREEN and 8bit BLUE.

How should I save images on a metamorph system?

I recommend saving each individual original image as a TIFF. This is the raw data, without loss, and this is the file you should use for any kind of quantification (especially anything with intensities). Images from the Axio Imager's fluorescence camera have 4096 grey levels. TIFFs only really come in 8bit and 16bit varieties, so the 12bit data is put into a 16bit file (but only the first 12 bit levels are populated).

This image won't open in programs like Powerpoint, Windows Picture Viewer, Mac Preview etc. You can look at the original images on the metamorph workstations, use imageJ or photoshop can open the files if you adjust the levels (be careful!).

You may find it useful to have a more easily viewed form of the file in addition to the original. If you have more than one channel, produce an overlay and then save this as a TIFF. This will be standard 24bit RGB format and work with nearly any program. If you just have one channel, you can save an 8bit copy from the short cut on the taskbar. Remember these formats involve the loss of some information - the image is put into the 8bit copy or the overlay as displayed on the screen and some intensity information is lost for all the pixels outside the scaling triangles.

. . .and the confocals?

The software on the Zeiss and Leica confocals uses proprietary formats. It is generally easier to use the software (free versions are available) to view images etc. You can export the data as TIFF for publications etc. Confocals in general produce 8bit data so the bit depth is not usually something to worry about.

Movie format

Quicktime is a good format for exporting timeseries for display in talks etc. It compresses the images so the files are of a more manageable size and free viewers are available for most platforms.