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Scanning Electron Microscopy


from other physical data, such as characteristic X-ray emission or cathodoluminescence spectrometry. Color has also been added by mixing the signals from multiple electron detectors, each detector coded for a diff erent color [ 1 ]. But the question is: how does one go about colorizing SEM images with only the information contained within the SEM images themselves? Pseudocolor . T e numbers representing each pixel intensity can be arbitrarily matched to a color via a lookup table. T is is known as pseudocolor or “false color.” Lookup tables may be based on the colors of the rainbow (blue-green-yellow-orange- red-white), the colors of the thermal scale (black, red, orange, white), or some arbitrary color scale [ 2 ]. Using this approach


Figure 2 : Image of calcifi ed particles in cardiac tissue. (a) Secondary electron (SE) SEM image, (b) BSE image, (c) DDC-SEM image obtained by superimposing the two previous images and adding color to the BSE image via a lookup table. This colorization technique helps to reveal both the composition and texture of the sample. Original images courtesy of Sergio Bertazzo.


obviously doesn’t add any additional information to the image, but it can allow better visualization of image detail (or material phases) in a sample. T is is the case when color is applied to SEM backscattered electron (BSE) images in which image brightness increases with increasing atom number in the specimen. Figure 1a shows a raw BSE image displayed in grayscale. T is image exhibits non-uniform brightness, the right side being visibly darker than the leſt . In Figure 1b , false color has been added by arbitrarily matching a color to each gray level; however, because the gray levels were not uniform across the fi eld, it is not possible to distin- guish the phases confi dently at this stage. Figure 1c shows the result of applying mathematical correction of the gray levels. T is type of image processing consists of subtracting the 2nd degree polynomial that best fi ts (least square method) the homogeneous (low variance) areas of the gray-level image. In the corrected image, each color represents a diff erent mineral phase that can yield a quanti- tative volume fraction. Image superposition . A BSE image with strong compositional contrast also can be superimposed onto a secondary electron (SE) image. T e result is a composite or mixed image in which the texture and composition of a sample are both visible: composition being represented by false color diff erences, while topographical diff erences show up in the details of the shadowing ( Figure 2 ). T is is known as a


Figure 3 : Image of erythrocytes (red blood cells). Images from left to right show a sequence of stages in the colorization process using MountainsMap ® SEM software. From one step to the next required only a single mouse click. Original image courtesy of Thierry Thomasset, Université Technologique de Compiègne.


2018 May • www.microscopy-today.com 13


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