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. Start configuring Photoshop • Open Camtasia movie Photoshop preference (Adv. Photoshop) • Open the preferences control box to configure Photoshop, settings plus scratch file. • Edit | preferences | General... (Ctrl+K) • Open Colour management via Edit | color settings (Shift+Ctrl+K), configure for SWOP • Open Control Panel, run AdobeGamma, and calibrate your Screen.

29. A quick view of Photoshop Click on the small arrow head in the right bottom for more options

30. 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

31. 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)

32. Open and save an image • Start Camtasia movie open-save as and follow instructions • Start Photoshop and open file in map 1-01 • Show bitmap resolution: Image | Image size (CTRL/ALT I) • Resample or Resize “difference” in process • Go through history steps • Save bitmap file in compressed or uncompressed way

33. Advanced file type handling • Open RAW file, adjust settings and store in high-quality (lossless) file formats • Open Instruction movie Opening RAW Camera Datain the advanced Photoshop movies map and open one of the raw image (map 2-01) • Set space=adobe RGB (1998); Depth=16 bits/channel • Use advance mode: White balance=shadow; try other setting; then press ok • Set image | mode to 8 bits /channel • Also press Alt+Ctrl+I for File information • Save file in both TIFF, TIFF-LZW, JPEG and JPEG2000. Compare quality and file size.

34. Optimise image with little contrast • Instruction movie 16 to 8 bits AutoContrast and Level • Open a file from map 2-02 • Optimise by auto contrast or auto levels and use levels (Ctrl for fine-tuning) • Save file for later comparison • Open original file again and convert to 8 bits. Optimise again and compare gray value range of 16 bits and 8 bits images. • Later on we can pseudo-colour the images to better demonstrate the gray value differences (exercise 14).

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

36. Automate | Merge to HDR

37. Background / image improvements Background noise and white balance • Instruction movie Level Black-White setting • Open all file from map 2-03 • Optimise black and white levels with Ctrl+L • Set white balance with white brush in the Levels control window Advanced global image corrections • Instruction movie global-levels • Open file map 2-04 • Manual adjustments of level (ctrl+L) and curves (ctrl+M) ; try to improve contrast with best details in shadows and highlights; correct colour hue (ctrl+U) • Do the same now with Image | adjustment | Shadow / Highlight... • Adjust all that is needed to make the image attractive.

38. How to convert colour to gray 4 methods Simple • Change color model to gray (Image | Mode | Grayscale) • Desaturate (Image | Adjustments | Hue Saturation ^ U) Advanced • 2 Hue/saturation adjustment layers • Adjustment layer BW

39. Adapt colour file to gray scale • Start Colour to black-white and levels and follow instructions • Open file(s) in map 1-02 • Convert first directly to gray scale • Then desaturate, followed by converting to gray • Optimize contrast with levels procedure • Curves comment to alleviate mid-tones • Save file (save as)

40. 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.

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

42. 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.

43. Rotation and contrast improvement • Start Rotate and improve contrast and follow instructions • Open file in map map 1-03 • Improve contrast • Rotate image; use ruler if angles of straight lines should be precise • Save as in TIFF /LZW format

45. Various basic tools • Scanning an image with a flatbed scanner • Start Scanning colour image to see how in practice images from textbooks are scanned • Descreening (to remove moiré effect) is demonstrated • Cropping an image • Start Cropping-2 and follow instructions • Open file in map 1-07 • Apply the two modes of cropping • Adapting image resolution • Start Resolution and follow instructions • Open file in map 1-08 • Adapt the image to 300 dpi and an image size of 6 cm width

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

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

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

49. better sharp mask too much unsharp mask original

50. Sharpening an image • Start sharpening and follow instructions • Open file in map 1-4 • Convert to gray • Optimize contract • Enhance sharpness with the unsharp mask tool, trying different parameters • For advanced users: use the smart sharpen tool