Adjusting Brightness and Contrast

Introduction

It is sometimes essential to alter the brightness and contrast of an image to make it better for publishing or highlighting an area of interest. Adjusting the balance of an image needs to be done carefully so as not to remove or add data that was not in the original image. To this end it is usually safe to adjust linear parameters such as brightness and contrast (histogram levels) as long as under and over saturation is avoided. Adjusting non-linear parameters such as gamma and curves is generally discouraged as it will change the relationship of the data in a given image. For example, adjusting gamma can result in the dark values of an image not changing very much but the bright values being greatly increased.

This section uses the DAB 01.tif image found in Demo Images\Widefield Images\DAB\ and the FITC.tif image found in Demo Images\Widefield Images\DIC and Fluorescence\

Adjusting Brightness and Contrast – Colour Images

1. Open DAB 01.tif from Demo Images\Widefield Images\DAB\

2. Go to Image 🡪 Adjust 🡪 Brightness/Contrast 

3. In the resulting dialog window there is a histogram of the image data at the top with the range of intensities in the image below (in this example 0-255, 8bit). 

Several options exist for adjusting the brightness and contrast - if at any point you do something weird just press the Reset button and everything will return to the original: 

a) You can move the Minimum and Maximum sliders. This should only be done to the limit of the data and not beyond it. So, in this example the maximum value should not be changed and the minimum value can be adjusted to about 80 without affecting the output.

b) You can press the Auto button and a histogram stretch will be calculated with a target of 0.35% of pixels being saturated. The result of this can sometimes leave you with a very strange looking image.

c) You can press the Set button and enter in your own values directly

d) You can move the Brightness and Contrast sliders. The same rules apply to theses as to adjusting the Maximum and Minimum sliders.

4. Once you are happy with your settings press the Apply button. NOTE: Once the Apply button is pressed the original values in the image are altered to the new ones and the original data is lost. For this reason, it is best to save the result as a different file so as not to lose the original data.

Adjusting the Colour Balance – Colour Images

It is possible to adjust the colour balance of an image to remove subtle tints that may occur due to staining or imaging artefacts. This should be done with caution as it can result in misrepresentation of data. Images that have been colour balance adjusted should not be used for further analysis.

1. Open DAB 01.tif from Demo Images\Widefield Images\DAB\

2. Go to Image 🡪 Adjust 🡪 Colour Balance 

3. The resulting window contains a histogram like before. The default view is to have the red channel selected. You can select different colour channels from the pull-down menu and see the resulting histograms for each 

The minimum, maximum and brightness values can be adjusted for each colour channel to change the balance in the image.

To lock in a change to a colour channel you need to press Apply before selecting another colour channel. Once Apply is pressed the data is changed as before with brightness and contract adjustment.

NOTE: Selecting All from the list will let you change all colour channels equally. This is what is being done in the brightness and contrast adjustment from before. 

Adjusting the Brightness and Contrast – Monochrome Images

Adjusting monochrome images follows the same principals as shown above for adjusting colour images. The one major difference is the dynamic range of the images tends to be larger as they are captured on higher bit depth cameras.

Open FITC.tif from Demo Images\Widefield Images\DIC and Fluorescence\ 

2. Go to Image 🡪 Adjust 🡪 Brightness/Contrast 

3. Notice this time the range on the bottom of the histogram is from 0 – 65535 because this is a 16-bit image. You will also notice that the bulk of the image data is in the bottom part of the histogram. 

4. Very little adjustment can be made to the Minimum value before data is lost but there is some range in the Maximum for adjustment. If you press the auto button, you will notice the result is too saturated in the higher end. 

5. Setting the maximum value to around 45000 will give a well-balanced image without over-saturating too many pixels. As before the Apply button needs to be pressed before the values are applied (permanently) to the image.