introduction to image analysis presented to microscopy and microscopy education 11 march 2000 new...
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Introduction to Image Analysis
Presented to Microscopy and Microscopy Education
11 March 2000New Orleans, LA
Topics to be Discussed
• Why do Image Analysis?
• Five Steps Involved
• The “Jargon”
• Putting it Together
Why do Image Analysis?
• Better Definition of Contrasting Areas
• Improved Precision/Accuracy in Measurements
• Reproducibility of Results
• Higher Throughput than Manual Methods
A Word About Our Eyes
• Eyes are very good contrast adjusters, but not good for distinguishing subtle variations in color
• Eyes can discern about 30 continuous levels of gray or color in a field of view
• Eyes are not good judges of distance
• Eyes cannot accurately reproduce measurements
The Steps Involved
• Acquiring Images
• Processing Images
• Measuring Regions of Interest
• Analysing Data
• Reporting Results
Acquiring the Image
Arguably, the most important aspect of all
• Proper setup of imaging apparatus is vital (microscope, camera, etc.)
• Obtain maximum contrast and dynamic range• Reduce “noise” and other unwanted artifacts
Processing the Image
• Maximize Brightness/Contrast
• Reduce Artifacts
• Separate Objects from Background
• Enhance Non-Visual Data (data visible to camera but not eye)
What is ‘Contrast Adjustment’
• Modifying Contrast, Brightness and Gamma Levels to Obtain Best Possible Image
Contrast- The degree of difference between the darkest
and lightest parts of the image Brightness- The overall amount of light in an image
Gamma- Improves contrast in very light or very dark areas of the image
What Filters Do
• Improve quality of image
• Remove artifacts introduced through instrumentation (noise, etc.)
• Enhance features within an image
• Remove background variations
• Enhance edges
How Filters Work
• Operate on Pixel Arrays (kernels) within Image
• Change Intensities of Pixels in Neighborhood to Suppress or Enhance Image Info
1 1 1
1 0 1
1 1 1
Example of a ‘Smoothing’ Filter
Identifying Regions of Interest
• Determine whether looking for morphometric data or measuring lengths
• Select appropriate measurements• ‘Threshold’ image to select objects, if necessary• Apply measurement parameters to image
Thresholding an Image
• Select appropriate morphometric characteristics
• Identify regions of interest to analyse
• Mask regions
Determining Lengths
• Automatic or manual length determination
• Place markers• Report data
L1
L2
L3
Summary
• Image acquisition is Critical!• Enhance brightness/contrast to reveal faint regions of interest• Apply image filters to improve image quality• Apply measurement factors and edit resulting data• Analyse results
References
Castleman, K. R. 1998. Concepts in Imaging and Microscopy: Color ImageProcessing for Microscopy. The Biological Bulletin. 194 (2): 100-107.
Russ, J.C. 1995. The Image Processing Handbook. 2nd ed. CRC Press. BocaRaton, FL.
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