Digital Artwork & Photoshop in Life Sciences

1. Digital Artwork & Photoshop in Life Sciences Hans de Jong – Boudewijn van Veen 2013 Wageningen University

2. PART 1 About improvement and cheating About vectors and bitmaps Basics of Photoshop Global and local optimizations Colour management

3. General introduction • Image manipulation or cheating?about quality illustrations, photographs • Unaware or not familiar with criteria artwork submission journals • High complexity of producing artwork • Technical problems print jobs (manuals, books, PhD thesis)

4. Incomplete or poor artwork • What editors complain about: “Most of the papers that we received include photomicrographs or artwork…Of a sample of 32 papers currently in press, 19 were had incomplete or had incorrectly prepared artwork. Delays caused by the need to improve, change or reformat artwork accounted for an additional average time of 17 days...” Herbert MacGregor Editor-in-chief, Chromosome Research

6. Manipulation allowed or not?

7. Digital images and misconduct • No specific feature within an image may be enhanced, obscured, moved, removed, or introduced. • Adjustments of brightness, contrast, or colour balance are acceptable if they are applied to the whole image and as long as they do not obscure, eliminate, or misrepresent any information present in the original. • The grouping of images from different parts of the same gel, or from different gels, fields, or exposures must be made explicit by the arrangement of the figure (eg, dividing lines) and in the text of the figure legend. • If the original data cannot be produced by an author when asked to provide it, the acceptance of the manuscript may be revoked. • Rossner M, Yamada K. What's in a picture: the temptation of image manipulation. J Cell Biol. 2004;166:11-15. • Office of Public Health and Science. Managing allegations of scientific misconduct: a guidance document for editors. Available at: http://ori.dhhs.gov/documents/masm_2000.pdf.

8. Before you start • Archive your images • Keep original size and properties • Design a consistent organisation • Backup system (400 GB < € 80) • Choose for scientific artwork and collages • Which journal? • Which quality criteria? • Size of paper and columns • Resolution? • File type? • Colour mode? • Make first draft of drawing, composition or collage • How to exhibit the scientific message as good as possible?

9. Three levels of artwork • Learn about bitmaps (Photoshop) • Learn about vectors and font use (Illustrator; PowerPoint) • Prepare charts or specialized images, if needed (Excel, programs for statistics, Phylogeny, sequencing etc.) • Make composite artwork with images, vectors, text • Vector program • InDesign, FrameMaker, CorelDraw • Export PDF to • printing company • scientific journal • report • web

10. Basics of image types • Bitmaps • regular mesh of pixels, • each \with a gray value, XY position • TIFF, PSD, JPG, RAW, PIC, BMP • Conversions generally no problem • Note: TIFF for Mac different from PC. Important for PowerPoint • Vectors • mathematical representation of points shaping\\ a line, square, circle or other geometric entities • WMF, EMF, AI, CDR, WMF, font types, PDF (partly) • Conversions often problematic

11. Bitmap <> Vector • Illustrator, Corel Draw and other vector programs have conversions for bitmaps to vectors and v.v. • For converting vector to Bitmap • use export or save as function • Works usually easy and straightforward • Bitmaps to vectors (trace) • Results acceptable for simple images with little fine structure and patterns • Requires expertise and patience • Converting vector formats between programs often end up in unexpected results. Convert the image to PDF and open PDF in Illustrator.

12. Fonts and vectors Original letter (Font: Warnock) Convert into curves Drag the knot points or vectors Google: vector knot

13. Displaying special characters € 5000,- Kroměřížµ = ⅛ k*ξ*ψ, แฟื ันี หำ ธ้ฟร สำะะพห • Font technologies: • FOT = Bitmap font (Microsoft, only for software menus) • TTF = 8 bit truetype font (Microsoft + Mac) • PFM/PFB = 8 bit Type 1 font (Adobe) • OTF = 16 bit Opentype font (Microsoft + Adobe) • Use Opentype, where possibleEmbed fonts if needed(comes later when discussing PDFs) • In case of doubts or conflicts: convert fonts into curves (vector) or into bitmaps

14. Compression • Noticeable loss of details • Lossy compression: JPEG • (better is JPEG 2000) • Lossless compression: • LZW-TIFF, PSD (Photoshop) • Comparison of file size before and after compression Extreme JPG artefacts by repeated compression

15. RAW –TIFF – TIFF-lzw – JPEG RAW image = 12.9 MBoriginal image content of sensor TIFF image = 17.3 MBpixel-by-pixel storage, no data loss TIFF / LZW = 7.5 MB pixel-by-pixel storage,compression, no loss

16. Image depth • How many gray levels? • 1 bit: only black and white • 8 bits: 28 = 256 gray levelsis standard • 12 bits: 212 = 4096 gray levels • High quality CCD cameras • Better Noise reduction • Quantitative measurements • Photoshop can handle in gray and RGB • 8 bit (all comments) • 16 bit (part of the functions) • 32 bit (part of the functions) • New: High Dynamic Range imaging

17. 1-bit 4-bit 8-bit 24-bit

18. DPI vs LPI vs PPI... • DPI • Dots per inch • units used to measure the resolution of a printer • LPI • Lines per inch • The offset printing 'lines' or dots per inch in a halftone or line screen. • PPI • Pixels per inch • the number of pixels per inch in screen/scanner file terms • If you must go halftone, then PPI = LPI x 2.2 • Want more? Google DPI LPI

19. Black/white – Gray – Colour • Black/white • 1-bit format, line drawings, graphs • Mostly 800-1200 dpi (DPI = dots per inch) • Gray image • 8-bits • High-quality, RAW camera formats: 10-16 bits • Mostly 300-600 dpi • Colour • For PC, beamer and monitor display: 3-colour mode RGB(Red-Green-Blue), 3x8=24 bits • For printing: 4 colour mode CMYK(Cyan-Magenta-Yellow-Black), 4x8=32 bits • Mostly 300-600 dpi

20. Resolution • Line drawings • 800-1200 dpi • Gray images or colour • 300 (normal) – 600 (very high quality) • Posters • 300-1200 in printed on A4 (will later be enlarged to A0) • PowerPoint • Depends on beamer resolution (typically 1024 x 768) • Web pages 72-96 dpi

21. Quality criteria bitmaps • Input from cameras, scanners etc. • Store as uncompressed or lossless compressed bitmaps in original resolution • No lossy compression during processing • Final export compressed (JPEG most accepted compression) • Resolution light microscopic images less than few MB 2.5 x, NA 0.12: (TV adapter 1x) 2448 x 1843 100x, NA 1.4: (TV adapter 1x) 714 x 538 • Electron microscopic (very high) • Never more then needed for analysis or printing • Flatbed Scanners with optical resolution of at least 1200x1200 pixels • Scanning resolution dependent on final use (A4: 10-20 MB, web: < 100 kb) • Scanning of magazine / newspaper photo’s: descreening needed • Use film scanner, where possible. Rule of thumb: scan whole standard 24x36 mm negative produce at 1000 dpi (c. 4 MB)

22. Quality camera sensors • Video / TV: 250-600 linescolour is 8 bits • RGB colour2 x 8 bits = 16.7 Million • Most colour CCDimage reconstructedby interpolation • Original = RAW • Interpolated = TIFF, JPEG RAW image = sensor dump Interpolated TIFF

23. The problem of screens

24. Descreening removes moiré

25. Tips for descreening • Magazine / newspaper photos: regular arrays of dots (screen) • Converting into digital images: interference pattern (Moiré pattern) • Descreening with hardware scanner, Photoshop or dedicated software • Depends on screening frequency (lines per inch) and sample resolution • Blurring of pixels in Photoshop (Median filter) • Better: • Built-in descreening in scanner or • Specialized softwarehttp://www.scantips.com/basics6b.html Google: descreening images

26. Break

27. Practicals • PowerPoint presentation • Instruction movies • Example files • Series 1: basics • Series 2: advanced and specific tools • Basic information, overview • Background information on the mlab website • Instruction movies and example files:N.B. most material is copy protected, so don’t transfer to others!

28. Keyboard short keys for Photoshop • CTRL A Select All (Select menu) • CTRL B Adjust Colour Balance (Image menu) • CTRL C Copy (Edit menu) • CTRL D Deselect (Select menu) • CTRL F Latest Filter (Filter menu) • CTRL I Invert (Image menu) • CTRL M Adjust Curves (Image menu) • CTRL R Show Rulers (View menu) • CTRL U Adjust Hue/Saturation (Image menu) • CTRL Y Preview - CYMK (View menu) • CTRL Z Undo (Edit menu) • CTRL 0 (zero) Fit on Screen (View menu) • CTRL + Zoom In (View menu) • CTRL - Zoom Out (View menu) • CTRL/ALT I Image size • SPACE –TAB Toggle for menu’s

29. Photoshop 1 – global changes • File handling • Open (Ctrl O) / Save (Ctrl S) / save as (Sh + Ctrl S) • Change resolution and size (Ctrl + Alt + I) • Optimize contrast and brightness and colour • Levels tool (Ctrl L) • Curves tool (Ctrl M) • Colour balance (Ctrl B) • Colour hue and saturation (Ctrl U) • Sharpness (first menu setting, then Ctrl F) • Blur (first menu setting, then Ctrl F)

30. High Dynamic Range Imaging High-key midtone Low-key

31. Automate | Merge to HDR

32. History and adjustment layers • to correct or further adjust steps in the image processing • History: gives an overview of all steps in Photoshop • Adjustment layers: for all changes. Effect with and without can be easily compared.

33. original hue filtering blue convert to gray hue filtering red

34. Advanced colour - gray conversion • Open IMG_3054.tif(map 1-02) • Create two separate "Hue/Saturation Adjustment Layers“ (arrow) or use the menu…. • Top adjustment layer, • set blending mode to "Color“ • Saturation to minimum. • Bottom adjustment layer • change "Hue“ to appropriate contrast was obtained. • Also try saturation slider. • Finally merge/flatten the layers.  • Also try the black and white adjustment layer.

36. 2. Local changes bitmaps Sharpen filters In most cases the best filters are 1. Unsharp Mask… 2. Smart sharpen…

37. Different sharpening methods • simple sharpening • unsharp mask • too much sharpening • emphasize only the edges • advanced modes, specialized plug-ins, like remove the blur

38. Unsharp masking A mask obtained by subtracting the blurred copied layer from the original image enhances the borders of light and darker regions

39. better sharp mask too much unsharp mask original

40. Blur and median filters Different filters can be used for removing scratches, background noise, make the image “out-of-focus”, and to remove an unequal background.

41. Transformation tools Simple • Crop and perspective • Free transformation Advanced • Liquify tool • Open image in Ilustrator + mesh overlay

42. 3. Colour modes

43. Different colour modes • RGB (red-blue-green) • computer, beamer, slides, Web • CMYK (cyan magenta yellow black) • Printers, thesis, sci. journals • Lab (Lightness ab) • Device independent • L = lightness; a = green-red axis; b = blue yellow axis • colour manipulations • Indexed colours • Web

44. Colour model connections Subtractive colours: CMY + K Additive colours: RGB

45. Colour space Colour A outside the gamut were corrected to fit the CMYK frame (ICC dependent RGB > CMYK conversion)

46. Lab colour • No device can reproducing the full range of colors viewable to the human eye • The limited color space = gamut of colors • Some color models (such as CIE L*a*b) have a fixed color space because they relate directly to the way humans perceive color (device-independent) • Other color models (RGB, HSL, HSB, CMYK, and so forth) have different color spaces (device-dependent)

47. Avoid CMYK as much as possible! • Work always in RGB 24bits and Grayscale 8bits. • Only use JEPG (High) compression as final product • never as raw or original image file (TIFF, PSD, PNG, BMP or TIFF LZW) • TIFF is also different between Mac and PC • Never use Index color Mode • Open in Acrobat and convert to CMYK, and save as PDF.

48. All images in RGB as long as possible Work and keep all images in RGB, after assembly in “Illustrator, Indesign”,CorelDraw etc. Export to PDF as RGB Open in Acrobat and convert to CMYK, and save as PDF.

49. Tricks to improve colour balance • Variation • colour balance • hue / saturation • levels per channel • match colour • difficult jobsTry the images in map 2-11

50. Colour adjustments; try yourself • Open colour file • RGB > CMYK • adjust|variations • individual colour adjustment (Ctrl M, Ctrl L, Ctrl B) • colour saturation / desaturation (Ctrl U) • sharpness in LAB or CMYK model