1. What is a digital image ? 1

2. Illustrator/Inkscape
Bitmap/raster
Vector
Array of points (pixels)
Not scalable (aliasing)
Mathematical/geometric equations
Scalable
Microscopy Images
Photoshop/GIMP
ImageJ
Illustrator/Inkscape
Acrobat 2

3. 3

4. Y X RGB 24-bits Red 8-bits Green Blue 4 238 176 200 94 187 220 78 121
147 4

5. Microscope bitmap image
A matrix of numbers
Each number representing light intensity at a particular coordinate in the plane
Pixel depth : the size and structure of the pixel intensity range
Color is provided by the look-up table
Additional data : what, where, when, how ? = metadata 5

6. Bit depth
The sample signal intensity (infinite number of steps) is converted to pixel values (limited number of steps) by the sensor.
8-bit : from 0 to 255 (28=256 steps)
12-bit : from 0 to 4095 (212=4096 steps)
16-bit : from 0 to (216=65536 steps)
RGB : three 8-bit grayscale channels for red, green and blue
Hands-on : 6

7. 16 bit
8 bit

8. 16 bit
8 bit

9. Colour
Monochrome CCD cameras and confocal photomultipliers (PMT) do not “see” colour.
CCD sensor and PMT detect photons independently of their wavelengths (within their detection range).
The wavelengths detected depend on the filter set installed inside the microscope
Colour is false except for colour camera (RGB filter)

10. Look-up table (LUT)
255 10

11. Color is provided by the look-up table, color is always false
RGB
Red
Green
Blue
Color is provided by the look-up table, color is always false 11

12. Histogram : pixel intensity distribution12

13. Histogram : pixels distribution
distribution of pixels intensity (x axis) plotted against the number of pixel (Y axis) independently of their localisation within the picture
fill the dynamic range during image acquisition (black offset, saturation) 13

14. RGB
Red
Green
Blue
860 x 860 pixels
RGB color
8-bits per channel 14

15. Many images formats … Microscope specific formats
GIF, TIF, BMP, PNG, ICS, JPEG, MPEG, Quicktime, SWF, PICT, EPS, PDF, PSD, XCF, …
Microscope specific formats
LIF, LEI, LSM, ZVI, STK, OIF, dv, r3d, PIC, IPL, OIB, OIF, ND2 15

16. Which format to use ? TIF (TIFF and variants)
Loss-less compression (not always available)
Suitable bit-depth
Space for meta-data (scaling, exposure, …)
Open
Pretty much universal : should be readable in years from now
Most image acquisition softwares have proprietary formats exportable as TIFF 16

17. Compression Loss-less compression
Writes EXACTLY the same data in a smaller file
ZIP
Not that efficient (space/time) for microscopy data
Lossy compression
Change the dataset during compression
JPEG
NEVER USE for scientific data acquisition/analysis
Good for web publication and pictures distribution 17

18. Metadata Excitation wavelength Emission wavelength Dyes Exposure Gain
Offset
Time interval
Z-stack
Optical slice thickness
Objectives
Pixel size : scale bar
Personal notes …
Use the proprietary file format or tiff format to save the essential metadata (6 months later what is that picture ?!?!?) 18

19. Digital Image Data Metadata array of pixels
each pixel has an intensity value
Metadata
How, What, Where, When, Who… 19

20. Data Table of intensity value
Picture formed of pixels of different intensity
Histogram of pixel intensity distribution (loss of spatial information) 20