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Introduction to Digital Image Analysis Part I: Digital Images

Introduction to Digital Image Analysis Part I: Digital Images. Kurt Thorn NIC UCSF. Image: Susanne Rafelski , Marshall lab. What is a digital Image?. Many measurements of light intensity. Bit depth and dynamic range. # of bits. range. 1. 2.

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Introduction to Digital Image Analysis Part I: Digital Images

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  1. Introduction to Digital Image AnalysisPart I: Digital Images Kurt Thorn NIC UCSF Image: Susanne Rafelski, Marshall lab

  2. What is a digital Image? Many measurements of light intensity

  3. Bit depth and dynamic range # of bits range 1 2 • 0 1 2 4 • 0 1 2 3 4 16 • 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 8 256 1-byte 12 4096 2-bytes 16 65536

  4. Bit depth and dynamic range 8 bit 4 bit 6 bit 2 bit

  5. Converting bit-depth Your monitor is an 8-bit display # of bits range 1 2 • 0 1 2 4 • 0 1 2 3 4 16 • 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 8 256 12 4096 16 65536

  6. 255 0 255 0 255 Mapping values onto display Display Intensity Digital Number

  7. Intensity scaling • Computer screens are 8-bit • Publishers also want 8-bit files 255 Final Intensity 0 0 4095 Original Intensity You lose information in this process – values 4080-4095 all end up as 255

  8. Intensity scaling Max 255 Contrast Final Intensity Brightness 0 0 4095 Original Intensity Min

  9. Brightness / Contrast adjustment

  10. Auto-Scale Brightness/contrast Be aware of how your software scales your image Full Range

  11. High B/C Brightness/contrast Be aware of how your software scales your image Full Range

  12. Gamma correction g=0.45 Output Intensity g=2.2 g=1 Input Intensity Other contrast stretching transforms….

  13. Gamma adjustment g=0.6 g=1 0.6 g=2.2

  14. What are acceptable image manipulations? • JCB has the best guidelines • http://jcb.rupress.org/misc/ifora.shtml#image_aquisition • http://jcb.rupress.org/cgi/content/full/166/1/1 • Brightness and contrast adjustments ok, so long as done over whole image and don’t obscure or eliminate background • Nonlinear adjustments (like gamma) must be disclosed • No cutting and pasting of regions within an image (e.g. individual cells) • Control and experiment must be treated identically

  15. Lookup Tables (LUTs) 0 1 2 3

  16. False coloring to bring out detail

  17. False coloring to bring out detail

  18. Lookup Tables (LUTs)

  19. Lookup Tables (LUTs)

  20. Color Images • Color images are made up of three gray scale images, one for each of red, green, and blue • Can be 8 or 16 bits per channel

  21. Color Images

  22. Color Images = Or

  23. Stacks:Sequences of images Can represent time series(movies), z-positions, or other variables Z

  24. File Formats Data sets can be big: 1392 × 1040 × 2 = 2.8MB 3-channels, 15 image z-stack, 200 time points: 2.8 × 3 × 15 × 200 = 25.2GB Compression!

  25. Compression Lossless vs. Lossy • Lossless: preserves all information in original image • Original image can be restored • Lossy: discards information not necessary for visual appearance • Original image cannot be restored • See:http://dvd-hq.info/data_compression.php

  26. File Formats • Most portable: TIFF • 8 or 16-bit, lossless, supports grayscale or RGB • Good: JPEG2000, custom formats (nd2, ids, zvi, lsm, etc.) • Lossless, supports full bitdepth • Custom formats often support multidimensional images • Not so portable • Generally Bad: Jpeg, GIF, BMP, etc. • Lossy and / or 8-bit

  27. Presentation Analysis • Matlab • ImageJ • http://rsb.info.nih.gov/ij/ • Imaris (3D visualization) • CellProfiler • http://cellprofiler.org • Photoshop • Gimp Software Tools Acquisition + Analysis • NIS-Elements • AxioVision • MetaMorph • Zen • Slidebook • Micro-Manager • http://micro-manager.org/

  28. Acknowledgements • NicoStuurman

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