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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Basic Image Processing(using ImageJ)
Dr. Arne SeitzSwiss Institute of Technology (EPFL)
Faculty of Life Sciences Head of BIOIMAGING AND OPTICS – BIOP
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Overview
• File formats (data storage)
• Programs for image viewing / processing / representation
• Basic Image Processing (using ImageJ)
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Definition Digital image
• A digital image is a representation of a two-dimensional image using ones and zeros (binary). (Wikipedia)
• Analog = continuous values• Digital = discrete steps
1
0
01
01
1 10 01
1 110 0
11 01 11 1
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Detection Devices
Array detector Point detector
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
File Formats – data storage
• Lossless image formats• Lossy compression formats• Custom formats (microscope companies)• Sequence vs. single image per file• 8bit, 12bit, 16bit, 32bit, RGB
Storage: – Always have at least 1 copy of the data– Very suitable fileservers (automatic backup)
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Lossless Image Formats
TIFF (with our without compression)
BMP (windows uncompressed)
GIF (graphics interchange format)
PNG (portable network graphics)
Raw data
‘text image’
Microscopy Primerhttp://micro.magnet.fsu.edu/primer
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Format: TIFF
Tag Image File Format– Image header with flexible set of ‘tags’ which can be used to
store e.g. microscopic settings
Flexible in color space and bit depth– Microscopy: grayscale 8bit, 16 bit (12bit data)– Color (e.g. Overlay): RGB (red green blue 8bit each)– Quantification: 32bit (floating point values)
Always lossless: Uncompressed or compressedMultiple images possible in one file
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Compression: TIFFRun Length Coding (RLE): first number discribes the color, the second the
number of following pixels having the same color.
LZW (Lempel-Ziv-Welch): Find repetative patterns of values and give them a number which is points to an entry of a „dictionary“ (LUT).
(0,0,0), 6 (0,255,0), 9 (0,0,0), 2 (255,0,0), 6
1 2 3
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Compression: TIFFPros:Extra infos can be written in the ‚tags‘
(e.g. microscope data like objective lens, voxel size)
Everybody can read it LosslessFlexible (8, 16, 32bit grayscale, 8:8:8bit RGB)
Cons:Big filesCompressed files can t be loaded by ImageJ
25665536 graylevels
Floating point values
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Lossy Image FormatsThe lossy compression algorithm takes advantage of the limitations
of the human visual senses and discards information that would not be sensed by the eye.(like mp3 in audio).
Compression level is usually flexible, but the more compressed the more information is lost and artifacts become visible by eye
From: www.wikipedia.org
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Compression: JPGSplit image into color and gray-scale information (color is less important than bounderies) reduce high frequency color information.
Group pixel into 8x8 blocks and transform through discrete cosine transform…
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Compression: JPG
Pros:Small FilesTrue ColorUsable for most photos (real life) and
presentations (powerpoint)
Cons:Do not use for quantification !„Unrelevant“ photoinfos get lostEvery file-saving reduces the quality
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Viewers
ImageJ (Java based, freeware, Win/MAC/Linux)Irfanview (www.irfanview.com/)
– Freeware– Convert (e.g. tif jpg)– Batch processing
ACDSee (ACD Systems)Microscope companies
– Zeiss Image Browser / Axiovision LE– Leica LCS Lite– Olympus Viewer
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image RepresentationImageJImaris (Bitplane):
– 4 floating licenses– installed on image processing workstations
Photoshop, Paintshop, Illustrator, Corel Draw (, Powerpoint)
Volocity (Improvision): Custom software of microscopes
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Processing
ImageJ(http://rsb.info.nih.gov/ij/index.html)– installed on all image processing workstations– Installation: http://pacific.mpi-cbg.de/wiki/index.php
(Fiji=ImageJ+plugins+regular update)
– Manual: www.uhnresearch.ca/facilities/wcif/imagej/(also available as pdf)
– Additional plugins: http://rsb.info.nih.gov/ij/plugins/index.html
Metamorph (Universal Imaging), – installed on 2 image processing workstations
Custom software of microscopes
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Image Processing Basics
Visual Image Inspection
Lookup tables (LUT) and LUT operations
Histogram, brightness, contrast
Filter
Threshold
Measurements
Color functions
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Visual Image InspectionDisplaying images, histogram
Microscopy Primerhttp://micro.magnet.fsu.edu/primer
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Visual Image InspectionDisplaying images, histogram
Intensity value
Pixe
l cou
nt
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
LUT operationsLookup table (LUT)
– Displays can only show 256 gray values (8bit) per color
– Data is unchanged, it s only “mapped” differently
Data Intensity
Displayed Intensity
0 0
… …
179 0
180 5
181 10
… …
226
227
228 255
229 255
65535 255
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Brightness, Contrast
Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background. Caution: Apply modifies the data!
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Color LUTThe pixel contains a „pointer“ to an array, where the actual pixel
values are storedold LUT:1: (0,102,255)2: (51,102,2553: (10,100,200)
1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 3 3 3 3 3 3
1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 3 3 3 3 3 3
new LUT:1: (0,0,0)2: (0,255,0)3: (255,0,0)
“HiLo” LUT
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Color LUTThe pixel contains a „pointer“ to an array, where the actual pixel
values are storedold LUT:1: (0,102,255)2: (51,102,2553: (10,100,200)
1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 3 3 3 3 3 3
1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 3 3 3 3 3 3
new LUT:1: (0,0,0)2: (0,255,0)3: (255,0,0)
“Rainbow” LUT
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Non-linear Histogram StretchEnhance contrast by (changing data):
Raw data“Equalization” non-linear stretch based on square root of the intensity
Linear stretch“Normalization”
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Equalization
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
GammaGamma is a non-linear histogram adjustment
8 bit images: New intensity = 255 [(old intensity/255) gamma]
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
FilteringImage processing filters are mainly used to:
– suppress the high frequencies in the image, i.e. smoothing the image, noise reduction
– or suppress the low frequencies, i.e. enhancing or detecting edges in the image
An image can be filtered either in the frequency or in the spatial domain. – Filtering in the frequency domain requires Fourier
transform first and re-transformation after application of the filter.
– Filtering in the spatial domain is done by convolving the image with the filterfunction.
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
FilteringShifting and multiplying a filter kernel
Filtered image
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Noise Reduction: Mean
mean
Mean 1pt
19
19
19
19
19
19
19
19
19
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Noise Reduction: Gaussian
Filtering with a gaussianbell-shaped kernel:
1 2 1
2 4 2
1 2 1
116
10 25 3
9 33 5
4 6 8
10 50 3
18 132 10
4 12 8
116
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Noise Reduction: Median
median
Median 3x3
Median 5x5
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Noise Reduction: Median, Mean
Median, 1pt
Mean, 1pt
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Median-, Mean-, Max-, Min-Filter
Median, 5pt Mean, 5pt
Min, 2pt Max, 2pt
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Mean-, Gauss-Filter
Mean, 2pt, 4 pt Gauss, 2pt, 4 pt
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Mean-, Median-Filter
Mean, 2pt, 4 pt Median, 2pt, 4 pt
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Min-, Max-Filter
Min, 2pt Max, 2pt
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Sharpen / Blur-1 -1 -1-1 9 -1-1 -1 -1
sharpen
1 1 11 2 11 1 1
blurring
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Example: Edge-Finding with derivatives
-1 -1 -10 0 01 1 1
-1 -1 -10 1 01 1 1
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Background SubtractionEven background:
– subtract average background from image
Subtract “background image” (same exposure time without illumination)
Uneven background: Rolling ball filter– Use kernel larger than diameter of largest object
Original Image
“Opening”
Original Image - Opening
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Line ProfileWithout background subtraction
After rolling ball (50) background subtraction
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
ThresholdingThresholding is used to change pixel values above or below a certain intensity value (threshold):
Threshholding is a simple method for Segmentation (separation and location of objects of interest)
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Measuring Sizes
Set Scale with pixel (voxel) size
Include Scalebar
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Measuring Length
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Area Measurement
16bit image 32bit image 32bit image,background thresholdedto “Not a Number”
16bit image,same thresholdas in 32bit imagebut not applied
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Analyze Particles
Segmented objects
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Threshold and Opening/Closing
dilate erode
Closing: Dilate/ErodeOpening: Erode/Dilate
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Color FunctionsRGB Merge /RGB Split
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Effects causing Image degradation:Noise
– Signal derived noise– Noise emerging from the digital imaging system
Scatter– Caused by heterogeneous refractive index (RI)
Glare– Random disturbance of light in the system
Blur
DeconvolutionFrom Object to Image
Object Image
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Point Spread Function (PSF)
A Point Spread Function is the 3D diffraction patternof a “point” source of light.
Widefield = hourglass shapeConfocal = American Football
shape
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Convolution of an ObjectObject can be referred as
accumulation of pointsEach point is visible as a PSF
Image process hast to be - Linear- Shift invariant
Convolution is in principle a reversible mathematical equation
Object PSF = Image = convolution
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Constrained Iterative Constrained:
“Nonnegativity” Smoothing or regularization to suppress noise amplification
Iterative:Best estimate is found in a successional serial of calculations.
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Different Algorithms……lead to different Results
Huygens: CMLE 30 It
AutoQuant: Blind 15 It
SoftWorx: 30 It
raw data AutoQuant: non blind 15 It
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
0
1000
2000
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7000
0
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600
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1400
AQ_blind_15It_thPSF not deconvolved
Signal improvement
Higher signal to background ratio
More distinct peaks
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
WF DeconvolutionComputational substraction of blur
or reassignment to the assumed source
Advantages:– Good light efficiency (esp. with reassignment)– CCD instead of PMT (high Quantum efficiency)– Fast stack recording possible low bleachingDisadvantages:– Need for high computational systems– Artefacts can not be excluded
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
WF Decon vs. ConfocalTo deconvolve or not to deconvolve
That is not the question:WF + Deconvolution is no
real alternative to Confocal pictures as they can also be deconvolved
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
Conclusions
• Keep environment constant and convenient
• Use powerful dyes
• Think about required resolution (x, y, z, t, brightness, channel number) to minimize photostress
• Use appropriate microscopy method
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
• Use lossless file formats for archiving important data
• Image processing is an important step in generating (optimal) results
• Only use documented image processing steps/routines
Summary
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Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform
More about image processing
1. Lecture M. Unser, EPFLsee also website: http://bigwww.epfl.ch/
2. Booksa) W. Burger, M. J. Burge
Digital Image Processing, Springer 2008b) J. C. Russ
The image processing Handbook, CRC Press 2007
3. PT-BIOPEPFL, SV-AI 0241, SV-AI 0140http://biop.epfl.ch/