1. Tytus Bernas Biological applications of image processing

2. JC Russ (1998) Image processing handbook Image acquisition system

3. JC Russ (1998) Image processing handbook Digitization…

4. Coordinate system of digital image (square pixels)... and its effects

5. Pixels T

6. Advantage of (mega)pixels

7. Microscope resolution

8. d ~ R Airy d = 0.6100 λ /NA d ~ JDD d = 0.4750 λ /NA d ~ FWHM d = 0.5015 λ /NA Microscope resolution

9. frequency 1x 2x 3x Nyquist criterion theoretical: 2x practical: 3x Perfect sampling

10. Fourier transform PSF MTF

11. http://www.microscopyu.com/ Modulation transfer function (MTF)

12. Widefield: Δx = λ em / (4 n sin α) Δz = λ em / (2 n (1- cos α)) Confocal: Δx = λ ex / (8 n sin α) Δz = λ ex / (4 n (1- cos α)) PSF MTF Perfect sampling

13. Aliasing

14. E.H.K. Stelzer et al. (1998) J. Microscopy 189 p 15- 24 PSF/MTF and noise

15. JC Russ (1998) Image processing handbook http://micro.magnet.fsu.edu/primer/ Camera: an array detector

16. Charge Coupled Device JC Russ (1998) Image processing handbook, http://micro.magnet.fsu.edu/primer/ CCD readout

17. http://www.astro.uu.se/~johwar/AMAST/sxvh9.html NOISE ! readout dark quantum blooming SNR = PQ e t / [PQ e t + Dt + Nr 2 ] 1/2 linearity Problems of (digital) cameramen

18. http://www.diracdelta.co.uk/ Quantization accuracy gain & ofset binning (pixel size) acquisition time Qantization, detector settings

19. low offset high offsethigh gain low gain Gain and offset

20. Image is a two-dimensional function of coordinates: I=f(x,y) Minimum: 0 (or 1) Maximum: dynamic range Computer image = table of numbers

21. Dynamic range

22. 1, 2, 3, 4, 5, 6, 7, 8, 9...er aha! 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21..... 98, 99 100, 101..... and so on 1234 1  1000 1  10 3 2  100 2  10 2 3  10 1  10 1 4  1 4  10 0 00 11 210 311 4100 5101 6110 7111 81000 91001 101010 111011 121100 131101 141110 151111 10101 0802308023 1412214122 0202102021 1112011120 1  16 1  2 4 16 + 0 + 4 + 0 +1 = 21 Binary notation

23. product (AND) pq r 11 1 10 0 01 0 10 0 sum (OR) pq r 11 1 10 1 01 1 10 0 negation (NOT) p r 0 1 1 0 AND 1001 1100 1000 OR 1001 1100 1101 NOT 1001 0110 ABA AND B A OR BA exOR BNOT A Logic

24. Segmentation with binary mask I > t => 1 I 1 binarization log. AND

25. Mapping function (LUT)

26. image dark <> bright contrast low <> high Whole image: histogram

27. "What is truth?" Pontius Pilate (John 18:38) Conversion

28. Image improvement – convolution (averaging) 111 111 111 3x3 (1) 3x3 (3) 7x7 (1) 1111111 1111111 1111111 1111111 1111111 1111111 1111111 3x3 pix. 7x7 pix.

29. Better than average - median 111 111 111 3x3 median 3x3 gauss.

30. Enhancement of details - convolution 000 10 000 8 3x3 laplacian 3x3 derivative 9 3x3 hi-pass

31. Additive space: Red Green Blue Subtractive space : Cyan Magenta Yellow Hue Saturation Brigtthess Lightness a b Colour spaces

32. Warm and cold colours

33. Colour components I

34. Colour components II

35. (Pseudo)colours

36. Where to store it File typeBit depthColor coding Transp. (bits) compressionPortabilityRemarks losslesslossy BMP1/4/8grayscale ---- RLE ---- Poor (Microsoft format) Inefficient compression 1 24RGB- GIF8RGB color table1LZW-ExcellentNo grayscale support. JPEG24YUV--JPEGGood Very efficient compression 1 (with distorion). No RGB and grayscale support. JPEG20001/4/8color table38 2 JPEG200 fair Very efficient compression 1. Relatively new format intended to replace jpeg. 8/16grayscale 24/48RGB 36/64CMYK PNG1/2/4/8 grayscale or RGB color table 8deflation-good Efficient compression The format developed to replace GIF in www. 24/48RGB TIFF1/4/8RGB color table8 Deflation (ZIP), RLE, Huffman, LZW JPEGgood Format offers large array of features, which may be extended further using specific tags. Only limited number of these is supported universally, though. Efficient compression 1. 1/4/8/16grayscale 24/48RGB, YUV 32/64CMYK