1. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 1 of 29 Slides BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” Lecture 3: Image file formats Steve Kelley Department of Basic Medical Sciences, School of Veterinary Medicine Weldon School of Biomedical Engineering Purdue University J. Paul Robinson, Ph.D. SVM Professor of Cytomics Professor of Immunopharmacology & Biomedical Engineering Director, Purdue University Cytometry Laboratories, Purdue University This lecture was last updated in January, 2007 You may download this PowerPoint lecture at http://tinyurl.com/2dr5p Find other PUCL Educational Materials at http://www.cyto.purdue.edu/class These slides are intended for use in a lecture series. Copies of the slides are distributed and students encouraged to take their notes on these graphics. All material copyright J.Paul Robinson unless otherwise stated. No reproduction of this material is permitted without the written permission of J. Paul Robinson. Except that our materials may be used in not- for-profit educational institutions ith appropriate acknowledgement.

2. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 2 of 29 Slides Data files are a representation of an original image, which is itself a representation of reality. The chain of digital image processing includes both creation of digital data from an image, and recreation of an image from the digital data. Data file formats are created in order to make specific operations more convenient. The most convenient format may differ with the particular application. What are data file Formats?

3. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 3 of 29 Slides Image process - subject to output Analogue digital

4. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 4 of 29 Slides How it works in real life

5. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 5 of 29 Slides For most purposes, a one-to-one mapping of pixels to data values is most useful, but the internal representation of the data values may be different for different file formats. Files can be either compressed, or not, and compression can be either lossy or not. For scientific analysis lossy compression is unacceptable; it may be useful for overview presentations. Image manipulation can take place before image acquisition, during image acquisition, on the digital data, or during recreation of an output image. What is image manipulation?

6. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 6 of 29 Slides 8 bits provide 256 possible gray levels

7. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 7 of 29 Slides How does this translate from an image into an image map?

8. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 8 of 29 Slides How does this translate from an image into an image map? 512

9. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 9 of 29 Slides How does this translate from an image into an image map? 512

10. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 10 of 29 Slides

11. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 11 of 29 Slides 512 255 0

12. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 12 of 29 Slides Image size The higher the resolution of the image, the more data points there are Very high resolution files need to be reduced in size to store the data We need to employ compression algorithms to reduce the file size But the goal is to maintain the quality of the image

13. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 13 of 29 Slides Run Length Encoding – count the number of identical values, replace the values with a count followed by the value A kind of compression algorithm which replaces sequences ("runs") of consecutive repeated characters Compression

14. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 14 of 29 Slides 0% Compression50% Compression 75% Compression80% Compression 76k 42k 28k26k

15. Copyright J. Paul Robinson, Steve Kelley Purdue University 1996-2005 15 of 29 Slides Brightness and contrast variation are controlled by a system input-output curve. Spatial kernel filtering and median filtering use information local to a particular area of an image to modify that area. Digital image analysis is “Data Analysis”. What is image analysis?

16. Copyright J. Paul Robinson, Steve Kelley Purdue University 1996-2005 16 of 29 Slides Input-Output Curve

17. Copyright J. Paul Robinson, Steve Kelley Purdue University 1996-2005 17 of 29 Slides Input-Output Curve

18. Copyright J. Paul Robinson, Steve Kelley Purdue University 1996-2005 18 of 29 Slides Input-Output Curve

19. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 19 of 29 Slides Mechanics of kernel convolution

20. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 20 of 29 Slides 121 242 121 127129123121124130 139134133134137 127130133134132131 123122131129 125 118122 124123122 124121117116114118 127121114110109114 136124122117111109 Divisor=1+2+1+2+4+2+1+2+1=16 Gaussian kernel filter Section of image file

21. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 21 of 29 Slides Smoothing

22. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 22 of 29 Slides Smoothing Filter 111

23. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 23 of 29 Slides Smoothing original3 x 3 Smooth 9 x 9 Smooth

24. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 24 of 29 Slides Sharpening original3 x 3 Sharpen 9 x 9 Sharpen

25. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 25 of 29 Slides Gaussian Filter 121 242 121

26. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 26 of 29 Slides Sobel Filter 121 000 -1-2-1

27. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 27 of 29 Slides Application of a median filter

28. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 28 of 29 Slides

29. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 29 of 29 Slides

30. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 30 of 29 Slides

31. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 31 of 29 Slides

32. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 32 of 29 Slides Lossy Compression Most filters will result in lossy compression This means once you compress the image, you can never return the image back to its original self One needs to be careful with biological data to ensure that you preserve the raw files Lossless: GIF, TIF Lossy: jpg, png

33. Copyright J. Paul Robinson, Steve Kelley Purdue University Cytometry Laboratories 1995-2007 33 of 29 Slides Summary Structure of data files Image manipulation Kernels Filters