File formats for microscopy

TIFF, JPG, PNG . . .

In general, using TIFF is a safe and wise choice for saving scientific images. TIFF is by default a lossless format and all data are preserved.

JPEG compression makes the files smaller while keeping the images looking pretty much the same. This generally involves the loss of at least a little bit of information - fine for pictures of your holiday, not so good for microscopy. PNG format compression does not involve the irrecoverable loss of information and can be a good choice for figures. 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 (e.g. LZW) attempts to compress exactly the same image into a smaller file. In general, complex images can't be compressed losslessly that much so this option might not change your storage requirements much.

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 or use FIJI/imageJ.

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) or FIJI to view images etc. You can export the data as TIFF for publications etc.