basic image processing (using imagej) image processing2011.pdf · dr. arne seitz pt-biop course,...

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2011

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

arne.seitz@epfl.ch

MotivationWhat is the difference

Biological Question ExperimentImage AcquisitionImage AnalysisConclusion

Image Analysis

Can we trust our eyes....

Seeing is believing, measuring is science

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Pixels

Definition Digital imageA digital image is a representation of a two-dimensional image

using ones and zeros (binary). (Wikipedia)

Analog = continuous valuesDigital = discrete steps

Digital Images are raster images consisting of pixels (or picture elements). Pixels are the smallest addressable units.

1 10 01

1 110 0

11 01 11 1

Images are artefacts

This is the same object (sample) imaged with the same objective!

Object Imageof Object

Images are artefacts

- Images are a representation of an object.- Image of an point is not necessarily a point

(point-spread function).- The content/information of a pixel is affected by the

setting of the microscope.- Images without metadata are useless!!!

- Sampling of the image- Remember Nyquist- Sampling affects pixel information

Overview

• File formats (data storage)• Programs for image viewing / processing /

representation• Basic Image Processing (using ImageJ)

File Formats – data storage

• Lossless image formats• Lossy compresion 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)

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

Image Format: TIFF

Tag Image File Format– Image header with flexible set of ‘tags’ which can be used to

store e.g. microscopic settingsFlexible 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

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

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

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

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…

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

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

Image RepresentationImageJImaris (Bitplane):

– 4 floating licenses– installed on image processing workstations

Photoshop, Paintshop, Illustrator, Corel Draw (, Powerpoint)

Volocity (Improvision): Custom software of microscopes

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 automatc udate)– 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

Image Processing Basics

Visual Image Inspection Lookup tables (LUT) and LUT operationsHistogram, brightness, contrastFilterThresholdMeasurementsColor functions

Visual Image InspectionDisplaying images, histogram

Intensity value

Microscopy Primerhttp://micro.magnet.fsu.edu/primer

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

… …

181 10

… …

228 255

229 255

65535 255

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!

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

new LUT:1: (0,0,0)2: (0,255,0)3: (255,0,0)

“HiLo” LUT“Rainbow” LUT

Non-linear Histogram StretchEnhance contrast by (changing data):

Raw data“Equalization” non-linear stretch based on square root of the intensity

Linear stretch“Normalization”

Equalization

GammaGamma is a non-linear histogram adjustment8 bit images:

New intensity = 255 × [(old intensity/255) gamma]

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.

FilteringShifting and multiplying a filter kernel

Filtered image

Noise Reduction: Mean

Mean 1pt

Noise Reduction: Gaussian

Filtering with a gaussianbell-shaped kernel:

10 25 3

9 33 5

10 50 3

18 132 10

4 12 8

Noise Reduction: Median

median

Median 3x3

Median 5x5

Noise Reduction: Median, Mean

Median, 1pt

Mean, 1pt

Median-, Mean-, Max-, Min-Filter

Median, 5pt Mean, 5pt

Min, 2pt Max, 2pt

Mean-, Gauss-Filter

Mean, 2pt, 4 pt Gauss, 2pt, 4 pt

Mean-, Median-Filter

Mean, 2pt, 4 pt Median, 2pt, 4 pt

Min-, Max-Filter

Min, 2pt Max, 2pt

Sharpen / Blur-1 -1 -1-1 9 -1-1 -1 -1

sharpen

1 1 11 2 11 1 1

blurring

Example: Edge-Finding with derivatives

-1 -1 -10 0 01 1 1

-1 -1 -10 1 01 1 1

Background SubtractionEven background:

– subtract average background from image

Subtract “backgound 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

Line ProfileWithout background subtraction

After rolling ball (50) background subtraction

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)

Measuring Sizes

Set Scale with pixel (voxel) sizeInclude Scalebar

Measuring Length

Area Measurement

16bit image 32bit image 32bit image,background thresholdedto “Not a Number”

16bit image,same thresholdas in 32bit imagebut not applied

Analyze Particles

Segmented objects

Threshold and Opening/Closing

dilate erode

Closing: Dilate/ErodeOpening: Erode/Dilate

Color FunctionsRGB Merge /RGB Split

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

• 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

basic image processing (using imagej) image processing2011.pdf · dr. arne seitz pt-biop course,...

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