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What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Basics Image Processing,Data Handling, and Storage
David Miguel Susano Pinto
Micron Advanced Microscopy Course, 2020
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
What is an image?
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
What is an image?Pixel data Metadata
• emission wavelength 535nm• excitation wavelength 500nm• exposure time 2s• objective Olympus 100X/1.40• pixel size 64.4×64.4 nm
• deconvolved• 8bit conversion after contrast adjustment• full range displayed
• horse fibroblast cell line• transient transfection of H2B-GFP• treated with sugar lumps
File ⇒ my-lovely-horse-H2B-GFP 01 R3D D3D.dv
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Pixel data
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Camera
Photons ⇒ Charge ⇒ Digital number
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Camera
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Camera
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Histogram of pixel values
0 10000 20000 30000 40000 50000 600000
2000
4000
6000
8000
10000
Adjust contrast/levels, see effect on display and pixel values.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 2
2 2× 2 43 2× 2× 2 84 24 165 25 326 26 647 27 1288 28 2569 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 22 2× 2 4
3 2× 2× 2 84 24 165 25 326 26 647 27 1288 28 2569 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 22 2× 2 43 2× 2× 2 8
4 24 165 25 326 26 647 27 1288 28 2569 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 22 2× 2 43 2× 2× 2 84 24 16
5 25 326 26 647 27 1288 28 2569 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 22 2× 2 43 2× 2× 2 84 24 165 25 32
6 26 647 27 1288 28 2569 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 22 2× 2 43 2× 2× 2 84 24 165 25 326 26 647 27 1288 28 256
9 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bit depth / dynamic rangenbits1 2 22 2× 2 43 2× 2× 2 84 24 165 25 326 26 647 27 1288 28 2569 29 51210 210 102411 211 2048
. . .15 215 3276816 216 65536
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Pixel data
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
Km
Vmax
½Vmax
0 1000 2000 3000 40000.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Substrate concentration
Rea
ctio
n ra
te
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
Serra da Freita
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
0 100 200 300 400 5002000
4000
6000
8000
10000
12000
14000
0 100 200 300 400 5002000
4000
6000
8000
10000
12000
14000
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Signals
0 100 200 300 400 5002000
4000
6000
8000
10000
12000
14000
0 100 200 300 400 5002000
4000
6000
8000
10000
12000
14000
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as Surfaces
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Images as ND Arrays
• x and y• time• z (volume)• wavelength• phase• stage angle
Think ”data”, not ”picture”
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
RGB
If you ever get an RGB image, you did something wrong.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
RGB
If you ever get an RGB image, you did something wrong.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
multi channel
Multichannel image is a series of grayscale images.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Lookup tables or colormaps
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
See ImageJ “Display LUTs”
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Lookup tables or colormaps
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
0 50 100 150 200 2500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
color index
0 50 100 150 200 250color index
See ImageJ “Display LUTs”
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Lightness perception
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Qualitative or Categorical
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Qualitative or Categorical
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Qualitative or Categorical
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Direct record
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Image Reconstruction
• Localisation Microscopy (STORM/PALM)• Structured Illumination
Two step process
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
raster vs vector
raster individual pixel values. Use ImageJ.vector polygons with attributes. Use inkscape.
ImageJ Inkscape GIMP
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Figure preparation guidelines
• Carry out all processing and analysis of images beforemaking figures by using pixel based (raster) programs.• Handling of images for figures should use pixel based
(raster) programs.• Vector graphics for lettering, arrows, diagrams, arranging
panels.• Both can rotate, resize, and crop.• Do not use office applications like powerpoint, keynote,
writer, word, or impress.• Do not use screenshoot.• Be consistent with processing steps, especially contrasting.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Compression
Save TIFF, not JPEG.
A useful generalisation.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Compression
Save TIFF, not JPEG.A useful generalisation.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Image data is large
1024 × 1024, 20 Z slices = 20MB
Most cameras are 16bit, that’s 40MBDo it in two channels, that’s 80MB
Every minute for 1 hour, you now have a 5GB fileIf you visit 10 points, that’s 50GB worth of data.
You have just reached Zenodo’s limit.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Image data is large
1024 × 1024, 20 Z slices = 20MBMost cameras are 16bit, that’s 40MB
Do it in two channels, that’s 80MBEvery minute for 1 hour, you now have a 5GB fileIf you visit 10 points, that’s 50GB worth of data.
You have just reached Zenodo’s limit.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Image data is large
1024 × 1024, 20 Z slices = 20MBMost cameras are 16bit, that’s 40MBDo it in two channels, that’s 80MB
Every minute for 1 hour, you now have a 5GB fileIf you visit 10 points, that’s 50GB worth of data.
You have just reached Zenodo’s limit.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Image data is large
1024 × 1024, 20 Z slices = 20MBMost cameras are 16bit, that’s 40MBDo it in two channels, that’s 80MB
Every minute for 1 hour, you now have a 5GB file
If you visit 10 points, that’s 50GB worth of data.You have just reached Zenodo’s limit.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Image data is large
1024 × 1024, 20 Z slices = 20MBMost cameras are 16bit, that’s 40MBDo it in two channels, that’s 80MB
Every minute for 1 hour, you now have a 5GB fileIf you visit 10 points, that’s 50GB worth of data.
You have just reached Zenodo’s limit.
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
where
1 you do it2 someone else does it
1 one disk per person2 shared filesystem3 OMERO
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
where
1 you do it2 someone else does it
1 one disk per person2 shared filesystem3 OMERO
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Fileset
• one file one image
• one file many images• many files one image
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Fileset
• one file one image• one file many images
• many files one image
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Fileset
• one file one image• one file many images• many files one image
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Files and metadata
• why some many formats?• metadata saved may be format dependent• use libraries and applications that respect you• open formats
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Bioformats and OME-TIFF
What?Image?
Camera
Histograms
Bitdepth
Images as ND Arrays
Colour
LUT
LUT
ImageReconstruction
Figuresraster vs vector
guidelines
Compression
StorageOMERO
Image Files
OME
Open Microscopy Environment
• bioformats• OME-XML• OME-TIFF• OMERO• IDR
