Colour and Intensity Data Visualisation

Introduction

Histograms were touched on briefly previously and are a useful tool in looking at the distribution of pixel intensities and colours within an image. Other tools exist within Fiji that allow other methods of visualisation of this data as well. 

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

Histograms

The histogram of any open and active image can be shown by going to Analyze 🡪 Histogram or pressing Ctrl+H. 

Colour Images

The initial histogram of an RGB Colour image shows a combined histogram of all the colour channels with the values representing the average grey intensity of the combined channels. The graph represents the number of pixels that have the given intensity along the X axis.

At the bottom of the Histogram window are 5 buttons

• List – Opens the data for recreating the graph in a new window that can be saved in csv format

• Copy – Copies the data from the list to the clipboard to be pasted directly into another program

• Log – Places a lighter coloured overlay on the graph representing the data on a log scale. This is useful when there are some intensities that have low counts. 

• Live – Updates the histogram dynamically showing the histogram data of the pixels contained within and ROI drawn on the image 

• RGB – Cycles through different histrograms. 

  • Intensity (unweighted) the initial histogram showing average grey values

  • Intensity (weighted) a histogram weighted to account for the human visual sensitivity to green

  • R+G+B a histogram showing each of the colour channels added together instead of the average displayed in the intensity versions

  • Red, Green and Blue the individual histograms of each colour channel 

Colour Histogram

A histogram showing all three colour channels at once can be shown by going to Analyze 🡪 Color Histogram 

Grey Scale Images

Grey scale image histograms are essentially the same with the loss of the RGB button and the way the data may be represented. For 8-bit  images the histogram will look the same as the intensity ones above for colour images. For 16-bit images the histogram data is binned into bins representing 256 intensities each. For 32-bit images the number of bins will be noted in the histogram window. 

3D Colour Histogram

The histograms shown above do not easily show the relationship between the different channels. It also only shows the RGB colour space. 

The Color Inspector 3D plugin allows the visualisation of all the colour channels and can be run from Plugins 🡪 Color Inspector 3D 

The window that opens up will show your image on the left and the 3D histogram on the right. The 3D histogram can be interacted with the mouse and rotated around. 

If you hover the mouse pointer over the image on the left, you will see where the select pixel is within the 3D histogram.

RGB is one of many ways of representing colour. There is a selection of other colour models available in the 3D colour inspector. 

3D Surface Plot

The intensities within the image can also be shown in a 3D plot with the image shown in XY and the Z axis showing the intensities

Open FITC.tif image found in Demo Images\Widefield Images\DIC and Fluorescence\

Go to Analyze 🡪 3D Surface Plot 

The default view is showing the image in its original LUT colour (in this example grey) with the image filled in. Changing the Grid Size and Smoothing can give a better representation of the data depending on the resolution and intensity range in the original image. Pressing the Save Plot button generates a new image containing just the plot 

The colours of the plot can be changed by assigning different LUTs from the colour pull down menu 

Line Intensity Profile

Graphing the intensities along a ROI line can provide useful information very easily. 

Draw a line ROI across part of the image and go to Analyze 🡪 Plot Profile. 

NOTE: Rectangular, Segmented line and freehand line ROIs work as well.

A graph is generated showing the pixel intensities under the line. Double clicking the line ROI button and setting a thicker line thickness will average the pixels under the line giving smoother data. Pressing the Live button updates the graph in real time as other ROIs are added, moved or the line width changed.