PrerequisitesBefore starting this lesson, you should be familiar with:
Learning ObjectivesAfter completing this lesson, learners should be able to:
Understand that images have a data type which limits the values that the pixels in the image can have.
Understand common data types such as 8-bit and 16-bit unsigned integer.
Images contain numerical values that must be somehow stored on the hard disc or within the computer memory. To do so, for each pixel a certain amount of space (memory) must be allocated (usually measure in bits). Generally, the more bits you allocate, the bigger are the numbers that you can store, however, you also need more space. Thus choosing the right data type usually is a balance between what you can represent and how much space you want to afford for this. Especially, for large image data such as volume EM and light-sheet data, the choice of the data type can have quite some impact on your purse. In addition, certain operations on images can yield results with values outside of the original data type; this is a serious and frequently occurring source of mistakes when handling image data and thus must be well understood!
Open the following images and discuss their data type and whether there are any signs of intensity clipping.
- Appreciate that this image has no major issues.
- This image has saturation issues (many pixels of value 255).
- This is problematic as one cannot compare the intensity of some of the nuclei.
- This image clips intensities at low gray scale levels (many zeros in the image).
- This is problematic as the data could be seen as if there is no DNA within some places of the nuclei, which most likely would be the wrong biophysical interpretation.
- Even though this is an unsigend 8-bit integer image there are only two of the possible 256 gray values used. Thus, in practice this probably represents a binary image.
- This is an unsigned integer 16-bit image.
- Not all 65535 gray values are used.
- In fact, the highest value in the image is 1200 which is less than 4095, thus, maybe this image was actually acquired with a 12-bit camera or some other bit-depth less than 16.
- In general, working with bit-depths between 8 and 16 is a bit problematic because there is not much support in terms of file formats and analysis software. Thus in the process one may loose track of the original bit-depth of the image.
- This image is stored as an unsigned integer 16-bit image.
- The range of the data is from 32963 to 36863
- In fact, this image is a 12-bit image that has an offset of 2^15.
2^15 = 32768(minimal possible value, which does not occur in this particular image because there is some background)
2^15 + 2^12 - 1 = 36863(maximal value possible in this image)
- This example, which is produced by a real commercial microscope, again demonstrates that interpreting 16 bit images can be tricky, as they may actually be of a lower bit depth.
Show activity for:
- For each image mentioned in the activity perform the below tasks.
- Open the image in Fiji.
- Use various ways to inspect the image and verify the comments that are given below the respective image in the activity.
- To this end, useful tools are:
Image › Show Info...
- Inspect pixel values by hovering over the image with the mouse.
Analyze › Histogram
Analyze › Plot Profile
Observe that for some software the datatype of the loaded image does not match the datatype given in the metadata.
The reason is that some software only support data types where the bit depth is a multiple of 8. For example, unsigned integer 12-bit data may not be supported.
This is very important as you may misinterpret whether your image contains saturated pixels or not.
Show activity for:
- Download any of the above images
- Open the image using
Plugins > Bio-Formats > Bio-Formats Importer
[X] Display OME-XML metadata
[ OK ]
- Check whether the information in
Image > Typeis the same as the one mentioned in the displayed metadata (look for
- Also check the maximum value in the image, e.g. using
Analyze > Histogram
- How does this maximum value compare to the image datatype?
- For example, you may find a value of 4095, which is the maximum of an unsigned integer 12-bit image, which may be the datatype mentioned in the image metadata, however ImageJ may represent this image as a 16-bit image. Appreciate that this can be confusing!
- If you find the maximum of the image to be identical to maximum that the datatype of the image can represent you may have an issue with saturation! Check this
- by hovering with the mouse over bright regions
- using the
HiLoLUT with appropriate contrast settings, i.e. the maximum should be the maximum of your datatype!
True or false? Discuss with your neighbor!
- Changing pixel data type never changes pixel values.
- Converting from 16-bit unsigned integer to 32-bit floating point never changes the pixel values.
- Changing from 32-bit floating point to 16-bit unsigned integer never changes the pixel values.
- There is only one correct way to convert from 16-bit to 8-bit.
- If the highest value in an image is 255, one can conclude that it is an 8-bit unsigned integer image.
- If the highest value in an image is 1034, one can conclude that it is not an 8-bit unsigned integer image.
- If the bit-depth is 16 and there are a lot of neighboring pixels with the value 4095 and no pixels with a higher value, most likely this image was acquired with 12-bit camera.
Image data types
The pixels in an image have a certain data type. The data type limits the values that pixels can take.
For example, unsigned N-bit integer images can represent values from 0 to 2^N -1, e.g.
- 8-bit unsigned integer: 0 - 255
- 12-bit unsigned integer: 0 - 4095
- 16-bit unsigned integer: 0 - 65535
Intensity clipping (saturation)
If the value of a pixel in an N-bit unsigned integer image is equal to either 0 or 2^N - 1, you cannot know for sure whether you lost information at some point during the image acquisition or image storage. For example, if there is a pixel with the value 255 in an unsigned integer 8-bit image, it may be that the actual intensity “was higher”, e.g. would have corresponded to a gray value of 302. One speaks of “saturation” or “intensity clipping” in such cases. It is important to realise that there can be also clipping at the lower end of the range (some microscopes have an unfortunate “offset” slider that can be set to negative values, which can cause this).
Recommended follow-up modules: