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How To …

Here are a few things which might be of help in analysing your images. It's impossible to anticipate everything people might want to do, so if you want to know something which isn't listed here it's worth asking to see if there's a way to do it - then it will appear here for other people to use.

Keep in mind that some images, often confocal or other fluorescent ones, have good contrast between what you want to measure - and what you don't. These images are usually easy to analyse. When there is little contrast between what's hot and what's not, it can often be difficult to impossible to do what you require. There's also no single answer to how to analyse an image, don't take anything you read here as the final word, take the initiative to find, or develop, solutions of your own. Being objective is however a universal requirement.

See the Image Analysis page for links to download ImageJ and Fiji - and other open source imaging programs.

Click on the pdf icons to download the instruction file(s) you require.
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Quantifying Fibre Thickness - Fiji

How to spatially calibrate, make a binary image and calculate fibre thickness using Fiji. The term 'fibre' can apply to many weird and wonderful things, so even if your sample isn't described as a fibre, it may still be applicable to this method.
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Removing Uncorrected Background Images for Montage Files - Fiji

The montage collection software 'Volocity' saves background corrected and uncorrected images as paired files. This procedure tells you how to discard the uncorrected files and then merge them into a single montage using Fiji.
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Analysing Matching Regions of Interest - Fiji / ImageJ

If you have many small regions of interest in an image, this method allows you to measure intensity related parameters in paired images. One of two fluorescence (usually) channels is thresholded for the structures of interest. These are converted into regions of interest (ROI) and can be duplicated on the image of the second fluorescent channel. This means the relative relationship between two markers can be analysed across a large number of discrete points.

The parameters you measure can be set in the Analyse / Set Measurements menu.

If you are unsure on how to use thresholding or the ROI Manager, download the 'Getting Started' pdf at the bottom of this page.
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Joining Image Arrays - Fiji

This is one method to automatically join together images which have a degree of overlap between them to form a single montaged image. Generally, a 10% overlap works well, but may vary depending upon how many features can be identified. Large featureless areas of a single colour may cause the method to fail, so some trial and error is sometimes necessary.
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Spatial Calibration of Images - ImageJ / Fiji

To obtain 'real' dimensions from your images, they usually have to be calibrated against a known scale. This describes the process and is used in many of the other procedures described here. In other words, download this file too.
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Measuring a Ringed Region of Interest - ImageJ / Fiji

Remove an inner region and surrounding areas to leave a ringed region of interest (ROI) which can be measured.
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Automatic Alignment and Overlay of Images - Fiji

Uses rotation and lateral shift to align images taken on different sections. Colours regions of interest and then overlays two images to show interaction between two different regions of interest, ie. one per image.
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Measuring Particle Characteristics - ImageJ / Fiji

How to measure particle sizes using thresholding to select them from the rest of the image.
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Measuring Surface Roughness from Confocal Scanning - ImageJ / Fiji

How to process a data file obtained from scanning a surface using a confocal laser scanning microscope.
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Thresholding - ImageJ / Fiji

Also known as segmentation, it selects pixels within a certain intensity range to either remove them from an image, or limit analysis to only the region of interest (ROI) required.
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Measuring Area - ImageJ / Fiji

A common request, so here's a way to measure an area in tooth sample, but it could apply to just about anything.
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Getting Started - ImageJ / Fiji

The basics of using ImageJ, how to open files, calibrate images, threshold and where to find the program. Used as part of a workshop, this is biased towards confocal images and µCT. The techniques are however applicable generally.