Adapted from : Joel B. Sheffield 2

Transcription

1 Image Analysis 1 1- Introduction to image analysis software ImageJ -development history -ImageJ vs. Fiji -installation -ImageJ website -sample images -tour of software environment 2- Basic image analysis using ImageJ: -opening/saving a file -file size vs. bit-depth and image-resolution -calibration -histograms, live updates -contrast, brightness, saturation -measurement: linear profiles, statistical information -Region of Interest (ROI) and local measurements/manipulations -image calculations 1

2 Adapted from : Joel B. Sheffield 2

3 ImageJ An adaptation of NIH image for the Java platform. Can run on any computer systems that can run Java (Sun Microsystems) Open source Two powerful scripting languages Java Plugins Macro Language Continual Upgrades Active community of several thousand users 3

4 ImageJ Donc, absence de licence coûteuse. Pas Besoin d être à l université pour faire ses travaux. Un portable est amplement suffisant pour 95% de vos besoins. Si on besoin de plus de mémoire pour des analyses c est possible de utiliser l'un des différents serveurs à Laval. Supporte plusieurs types de langage de programmation Plugins (Interface permettant d'avoir accès à toute la puissance de java. Idéal pour ajouter de nouvelles fonctions à ImageJ) 4

5 Resources 1. Ferreia, T. et Rasband, W. Ieds) «ImageJ User Guide : IJ1.46r», National Institues of Health, (lien) 2. Baecker, V. (ed) «Workshop: Image Processing and Analysis with ImageJ», Montpellier RIO Imaging, (lien) 3. Miure, K (ed) «Basics of image processing and analysis», Centre for Molecular and Cellular Imaging EMBL Heidelberg, (lien) 4. ImageJ Website: 5. Fiji Website: 5

6 1.ImageJ Website: 1. Features 2. Documentation ImageJ User Guide 3. Documentation Keyboard Shortcuts 4. Mailing list 5. Downloads example images (get these) 6

7 Menus

8 Introduction to the Main Menu Of these, we ll concentrate on: Image Process Analyze Plugins Help 8

9 L'interface principale Ouvrir, sauvegarde, création d'image Modification et conversion, opération géométrique Statistiques, Mesures, graphique Gestion des fenêtres Montage, outils de dessin Filtres et opérations mathématiques Accès aux plugins Raccourcis au site web, information et mise à jour 9

10 Opening and saving files Drag and Drop files onto IJ control strip, or Open samples 10

11 Les Menus (Edit) Mémoire maximum Windows 32-bit: le nombre le plus petit, 75% du total ou 1.4 Gbit Windows 64-bit: 75% du total Mac OSX 32-bit: le nombre le plus petit, 75% du total ou 1.8 Gbit Linux 32-bit: 3 Gbit Linux 64-bit: illimité 11

12 Image Menu 12

13 Image Menu Demo: 1) Dot blots 13

14 Image Menu Demo: 1) M51.tif Adjust brightness contrast Saturation? No! Pixel values do not change. 14

15 Process Menu 15

16 Process Menu Math: Vs. 16

17 Process Menu Image Calc. + Vs. 17

18 Analyze Menu 18

19 Analyze Menu Demo: 1) Dot blots -Measure -Set measurements -Changing measurement types and loss of information 2) Embryos -RGB 19

20 Analyze Menu Demo: 1) Results -Set measurements -Distribution -Options (file type for saving) 2) Edit -Copy data 20

21 Analyze Menu Demo: 1) Dot blots -Log -Live -List -Copy -Statistics 2) Embryos -RGB 21

22 Plugins ImageJ peut être considéré comme un ensemble de petits programmes ou «plugins» écrit en java. Cette structure modulaire à l'avantage de permettre d'ajouter facilement de nouvelles fonctions à ImageJ. Different versions d ImageJ ont different plugins bundled ensemble 22

23 Plugins Menu 23

24 Enregistrement des commandes de macro et registre des opérations Plugins > Macros > Record... Enregistre les actions effectuées et les transpose en langage de macros. Peut aussi être utilisé pour laisser une trace des opérations effectuées Demo 24

25 Plugins : information, download 25

26 Help Menu 26

27 Opening and saving files Dot_Blot.tif (open sample images) File size = 141kB Image Adjust Type Change to 16-bit Histogram comparison 27

28 Opening and saving files Dot_Blot.tif (open sample images) File size = 141kB Image Adjust Type Apply LUT Histogram comparison Ice fire 28

29 Opening and saving files Dot_Blot.tif (open sample images) File size = 141kB Adjust size reduce save Zoom comparison. File size comparison 29

30 Opening and saving files Dot_Blot.tif (open sample images) File size = 141kB Adjust size reduce save Zoom comparison. File size comparison Exercise: Save as jpg File size = 7kB Save back to tiff File size = 141 kb Zooming Pixels, position indicator, intensity value 30

31 Opening and saving files Dot_Blot.tif (open sample images) File size = 141kB Adjust size reduce save Zoom comparison. File size comparison Exercise: Save as jpg File size = 7kB Save back to tiff File size = 141 kb Zooming Pixels, position indicator, intensity value 31

32 Opening and saving files Dot_Blot.tif (open sample images) Exercise: 1. Save as jpg File size = 7kB Do an image subtraction of new file from original file. 2. Save back to tiff File size = 141 kb Do an image subtraction of new file from original file. 32

33 Opening and saving files M51c.tif (open sample images) Demo: 1. Save as jpg File size = 4kB Do an image subtraction of new file from original file. 2. Save back to tiff File size = 160 kb Do an image subtraction of new file from original file. 33

34 Pixel locator/grey value Dot_Blot.tif (open sample images) Zooming Pixels, position indicator, intensity value 34

35 The Image Histogram Log Scale The histogram shows the number of pixels of each value, regardless of location. The log display allows for the visualization of minor components. Note that there are unused pixel values 35

36 Log Scale In this case, the log display indicates that virtually all pixel values are used, even though they are a small percentage of the total. 36

37 Brightness Adjustment The brightness adjustment essentially adds or subtracts a constant to every pixel, causing a shift in the histogram along the x axis, but no change in the distribution 37

38 Contrast Enhancement For contrast enhancement, a lower value, in this case, 88, is set at zero, and a higher value, 166, is set at 255. The values of each of the pixels are adjusted proportionately. Note that because of the integer values, not all of the pixel values are used. 38

39 Addition et soustraction Process > Math > Add Origina l Addition et soustraction = modifie la brillance de l'image 39

40 Multiplication et division Process > Math > Multiply... X 0.5 Origina X 2 l Multiplication et division = modifie le contraste de l'image 40

41 Amélioration automatique du contraste Process > Enhance Contrast 41