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BioImaging &Optics Platform Basic Image Processing (using ImageJ) Dr. Arne Seitz Swiss Institute of Technology
BioImaging &Optics Platform Basic Image Processing (using ImageJ) Dr. Arne Seitz Swiss Institute of Technology
BioImaging &Optics Platform Basic Image Processing (using ImageJ) Dr. Arne Seitz Swiss Institute of Technology

BioImaging &Optics Platform

Basic Image Processing

(using ImageJ)

Dr. Arne Seitz Swiss Institute of Technology (EPFL) Faculty of Life Sciences Head of BIOIMAGING AND OPTICS – BIOP arne.seitz@epfl.ch

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Overview • File formats (data storage) • Programs for image viewing /
BioImaging &Optics Platform Overview • File formats (data storage) • Programs for image viewing /
BioImaging &Optics Platform Overview • File formats (data storage) • Programs for image viewing /

BioImaging &Optics Platform

Overview

File formats (data storage)

Programs for image viewing / processing / representation

Basic Image Processing (using ImageJ)

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Definition Digital image • A digital image is a representation of a
BioImaging &Optics Platform Definition Digital image • A digital image is a representation of a
BioImaging &Optics Platform Definition Digital image • A digital image is a representation of a

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

• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image
• Analog = continuous values • Digital = discrete steps Dr. Arne Seitz PT-BIOP course, Image

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 1 1 1 1 1 0 0
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BioImaging &Optics Platform Detection Devices Array detector Point detector Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Detection Devices Array detector Point detector Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Detection Devices Array detector Point detector Dr. Arne Seitz PT-BIOP course, Image

BioImaging &Optics Platform

Detection Devices

Array detector

Point detector

Platform Detection Devices Array detector Point detector Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform File Formats – data storage • Lossless image formats • Lossy compression
BioImaging &Optics Platform File Formats – data storage • Lossless image formats • Lossy compression
BioImaging &Optics Platform File Formats – data storage • Lossless image formats • Lossy compression

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)

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
BioImaging &Optics Platform Lossless Image Formats TIFF (with our without compression) BMP (windows uncompressed) GIF
BioImaging &Optics Platform Lossless Image Formats TIFF (with our without compression) BMP (windows uncompressed) GIF

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’

PNG (portable network graphics) Raw data ‘text image’ Microscopy Primer http://micro.magnet.fsu.edu/primer Dr.

Microscopy Primer http://micro.magnet.fsu.edu/primer

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Image Format: TIFF Tag Image File Format – Image header with flexible
BioImaging &Optics Platform Image Format: TIFF Tag Image File Format – Image header with flexible
BioImaging &Optics Platform Image Format: TIFF Tag Image File Format – Image header with flexible

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 compressed Multiple images possible in one file

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Image Compression: TIFF Run Length Coding (RLE): first number discribes the color,
BioImaging &Optics Platform Image Compression: TIFF Run Length Coding (RLE): first number discribes the color,
BioImaging &Optics Platform Image Compression: TIFF Run Length Coding (RLE): first number discribes the color,

BioImaging &Optics Platform

Image Compression: TIFF

Run Length Coding (RLE): first number discribes the color, the second the number of following pixels having the same color.

second the number of following pixels having the same color. (0,0,0), 6 (0,255,0), 9 (0,0,0), 2

(0,0,0), 6

number of following pixels having the same color. (0,0,0), 6 (0,255,0), 9 (0,0,0), 2 (255,0,0), 6
number of following pixels having the same color. (0,0,0), 6 (0,255,0), 9 (0,0,0), 2 (255,0,0), 6
number of following pixels having the same color. (0,0,0), 6 (0,255,0), 9 (0,0,0), 2 (255,0,0), 6

(0,255,0), 9

(0,0,0), 2 (255,0,0), 6

LZW (Lempel-Ziv-Welch): Find repetative patterns of values and give them a number which is points to an entry of a „dictionary“ (LUT).

which is points to an entry of a „dictionary“ (LUT). 1 Dr. Arne Seitz PT-BIOP course,

1

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

which is points to an entry of a „dictionary“ (LUT). 1 Dr. Arne Seitz PT-BIOP course,

2

which is points to an entry of a „dictionary“ (LUT). 1 Dr. Arne Seitz PT-BIOP course,

3

BioImaging &Optics Platform Image Compression: TIFF Pros: Extra infos can be written in the ‚tags‘
BioImaging &Optics Platform Image Compression: TIFF Pros: Extra infos can be written in the ‚tags‘
BioImaging &Optics Platform Image Compression: TIFF Pros: Extra infos can be written in the ‚tags‘

BioImaging &Optics Platform

Image Compression: TIFF

Pros:

Extra infos can be written in the ‚tags‘

(e.g. microscope data like objective lens, voxel size)

Everybody can read it Lossless Flexible (8, 16, 32bit grayscale, 8:8:8bit RGB)

it Lossless Flexible (8, 16, 32bit grayscale, 8:8:8bit RGB) 256 Floating point values 65536 graylevels Cons:

256

Lossless Flexible (8, 16, 32bit grayscale, 8:8:8bit RGB) 256 Floating point values 65536 graylevels Cons: Big

Floating point values

65536 graylevels

Cons:

Big files Compressed files can t be loaded by ImageJ

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Lossy Image Formats The lossy compression algorithm takes advantage of the limitations
BioImaging &Optics Platform Lossy Image Formats The lossy compression algorithm takes advantage of the limitations
BioImaging &Optics Platform Lossy Image Formats The lossy compression algorithm takes advantage of the limitations

BioImaging &Optics Platform

Lossy Image Formats

The 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

more information is lost and artifacts become visible by eye From: www.wikipedia.org Dr. Arne Seitz PT-BIOP

From: www.wikipedia.org

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Image Compression: JPG Split image into color and gray-scale information (color is
BioImaging &Optics Platform Image Compression: JPG Split image into color and gray-scale information (color is
BioImaging &Optics Platform Image Compression: JPG Split image into color and gray-scale information (color is

BioImaging &Optics Platform

Image Compression: JPG

Split 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…

8x8 blocks and transform through discrete cosine transform… Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
8x8 blocks and transform through discrete cosine transform… Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

8x8 blocks and transform through discrete cosine transform… Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
8x8 blocks and transform through discrete cosine transform… Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Image Compression: JPG Pros: Small Files True Color Usable for most photos
BioImaging &Optics Platform Image Compression: JPG Pros: Small Files True Color Usable for most photos
BioImaging &Optics Platform Image Compression: JPG Pros: Small Files True Color Usable for most photos

BioImaging &Optics Platform

Image Compression: JPG

Pros:

Small Files True Color Usable for most photos (real life) and presentations (powerpoint)

Cons:

Do not use for quantification ! „Unrelevant“ photoinfos get lost Every file-saving reduces the quality

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

photoinfos get lost Every file-saving reduces the quality Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
photoinfos get lost Every file-saving reduces the quality Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Image Viewers ImageJ (Java based, freeware, Win/MAC/Linux) Irfanview (
BioImaging &Optics Platform Image Viewers ImageJ (Java based, freeware, Win/MAC/Linux) Irfanview (
BioImaging &Optics Platform Image Viewers ImageJ (Java based, freeware, Win/MAC/Linux) Irfanview (

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

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Image Representation ImageJ Imaris (Bitplane): – 4 floating licenses – installed on
BioImaging &Optics Platform Image Representation ImageJ Imaris (Bitplane): – 4 floating licenses – installed on
BioImaging &Optics Platform Image Representation ImageJ Imaris (Bitplane): – 4 floating licenses – installed on

BioImaging &Optics Platform

Image Representation

ImageJ Imaris (Bitplane):

4 floating licenses

installed on image processing workstations

Photoshop, Paintshop, Illustrator, Corel Draw (, Powerpoint) Volocity (Improvision):

Custom software of microscopes

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
BioImaging &Optics Platform Image Processing ImageJ (http://rsb.info.nih.gov/ij/index.html) – installed on all
BioImaging &Optics Platform Image Processing ImageJ (http://rsb.info.nih.gov/ij/index.html) – installed on all

BioImaging &Optics Platform

Image Processing

ImageJ

BioImaging &Optics Platform Image Processing ImageJ (http://rsb.info.nih.gov/ij/index.html) – installed on all

(http://rsb.info.nih.gov/ij/index.html)

installed on all image processing workstations

(Fiji=ImageJ+plugins+regular update)

Manual: www.uhnresearch.ca/facilities/wcif/imagej/ (also available as pdf)

Metamorph (Universal Imaging),

installed on 2 image processing workstations

Custom software of microscopes

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
BioImaging &Optics Platform Image Processing Basics Visual Image Inspection Lookup tables (LUT) and LUT operations
BioImaging &Optics Platform Image Processing Basics Visual Image Inspection Lookup tables (LUT) and LUT operations

BioImaging &Optics Platform

Image Processing Basics

Visual Image Inspection Lookup tables (LUT) and LUT operations Histogram, brightness, contrast Filter Threshold Measurements Color functions

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Visual Image Inspection Displaying images, histogram Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Visual Image Inspection Displaying images, histogram Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Visual Image Inspection Displaying images, histogram Dr. Arne Seitz PT-BIOP course, Image

BioImaging &Optics Platform

Visual Image Inspection

Displaying images, histogram

Visual Image Inspection Displaying images, histogram Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

histogram Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Microscopy Primer http://micro.magnet.fsu.edu/primer

Microscopy Primer http://micro.magnet.fsu.edu/primer

BioImaging &Optics Platform Visual Image Inspection Displaying images, histogram Intensity value Pixel count Dr. Arne
BioImaging &Optics Platform Visual Image Inspection Displaying images, histogram Intensity value Pixel count Dr. Arne
BioImaging &Optics Platform Visual Image Inspection Displaying images, histogram Intensity value Pixel count Dr. Arne

BioImaging &Optics Platform

Visual Image Inspection

Displaying images, histogram

Intensity value Pixel count
Intensity value
Pixel count
Displaying images, histogram Intensity value Pixel count Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform LUT operations Lookup table (LUT) – Displays can only show 256 gray
BioImaging &Optics Platform LUT operations Lookup table (LUT) – Displays can only show 256 gray
BioImaging &Optics Platform LUT operations Lookup table (LUT) – Displays can only show 256 gray

BioImaging &Optics Platform

LUT operations

Lookup table (LUT)

Displays can only show 256 gray values (8bit) per color

Data is unchanged, it s only “mapped” differently

– Data is unchanged, it s only “mapped” differently Dr. Arne Seitz PT-BIOP course, Image Processing,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Data Displayed Intensity Intensity 0 0 … … 179 0 180 5 181 10 …
Data
Displayed
Intensity
Intensity
0
0
179
0
180
5
181
10
226
227
228
255
229
255
65535
255
BioImaging &Optics Platform Brightness, Contrast Caution: Apply modifies the data! Contrast is the difference in
BioImaging &Optics Platform Brightness, Contrast Caution: Apply modifies the data! Contrast is the difference in
BioImaging &Optics Platform Brightness, Contrast Caution: Apply modifies the data! Contrast is the difference in

BioImaging &Optics Platform

Brightness, Contrast

BioImaging &Optics Platform Brightness, Contrast Caution: Apply modifies the data! Contrast is the difference in

Caution: Apply modifies the data!

Brightness, Contrast Caution: Apply modifies the data! Contrast is the difference in visual properties that makes

Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background.

object distinguishable from other objects and the background. Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Color LUT The pixel contains a „pointer“ to an array, where the
BioImaging &Optics Platform Color LUT The pixel contains a „pointer“ to an array, where the
BioImaging &Optics Platform Color LUT The pixel contains a „pointer“ to an array, where the

BioImaging &Optics Platform

Color LUT

The pixel contains a „pointer“ to an array, where the actual pixel values are stored

old LUT:

1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:

1: (0,102,255)new LUT:

new LUT:

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3: (10,100,200)

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1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)

1: (0,0,0)3 3 3 3 2: (51,102,255   3: (10,100,200) 1 1 1 1 1 1 2

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2 1 1 3 3 3 3 3 3 2: (0,255,0) Dr. Arne Seitz PT-BIOP course,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

3 3 3 3 2: (0,255,0) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 3:

3: (255,0,0)

3 3 3 3 2: (0,255,0) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 3:

“HiLo” LUT

BioImaging &Optics Platform Color LUT The pixel contains a „pointer“ to an array, where the
BioImaging &Optics Platform Color LUT The pixel contains a „pointer“ to an array, where the
BioImaging &Optics Platform Color LUT The pixel contains a „pointer“ to an array, where the

BioImaging &Optics Platform

Color LUT

The pixel contains a „pointer“ to an array, where the actual pixel values are stored

old LUT:

1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:
1: (0,102,255) new LUT:

1: (0,102,255)new LUT:

new LUT:

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2: (51,102,255

 

3: (10,100,200)

1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)
1: (0,0,0)

1: (0,0,0)3 3 3 3 2: (51,102,255   3: (10,100,200) 1 1 1 1 1 1 2

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2 1 1 3 3 3 3 3 3 2: (0,255,0) Dr. Arne Seitz PT-BIOP course,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

3 3 3 3 2: (0,255,0) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 3:

3: (255,0,0)

3 3 3 3 2: (0,255,0) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 3:

“Rainbow” LUT

BioImaging &Optics Platform Non-linear Histogram Stretch Enhance contrast by (changing data): “ Equalization ”
BioImaging &Optics Platform Non-linear Histogram Stretch Enhance contrast by (changing data): “ Equalization ”
BioImaging &Optics Platform Non-linear Histogram Stretch Enhance contrast by (changing data): “ Equalization ”

BioImaging &Optics Platform

Non-linear Histogram Stretch

Enhance contrast by (changing data):

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

Raw data

stretch based on square root of the intensity Raw data Dr. Arne Seitz PT-BIOP course, Image
stretch based on square root of the intensity Raw data Dr. Arne Seitz PT-BIOP course, Image
stretch based on square root of the intensity Raw data Dr. Arne Seitz PT-BIOP course, Image
stretch based on square root of the intensity Raw data Dr. Arne Seitz PT-BIOP course, Image

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Linear stretch “Normalization

of the intensity Raw data Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Linear stretch
of the intensity Raw data Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Linear stretch
BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization
BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization
BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization

BioImaging &Optics Platform

BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization
BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Equalization

BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization
BioImaging &Optics Platform Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Equalization
BioImaging &Optics Platform Gamma Gamma is a non-linear histogram adjustment 8 bit images: New intensity
BioImaging &Optics Platform Gamma Gamma is a non-linear histogram adjustment 8 bit images: New intensity
BioImaging &Optics Platform Gamma Gamma is a non-linear histogram adjustment 8 bit images: New intensity

BioImaging &Optics Platform

Gamma

Gamma is a non-linear histogram adjustment 8 bit images:

New intensity = 255

[(old intensity/255) gamma]

8 bit images: New intensity = 255 [(old intensity/255) gamma ] Dr. Arne Seitz PT-BIOP course,
8 bit images: New intensity = 255 [(old intensity/255) gamma ] Dr. Arne Seitz PT-BIOP course,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Filtering Image processing filters are mainly used to: – suppress the high
BioImaging &Optics Platform Filtering Image processing filters are mainly used to: – suppress the high
BioImaging &Optics Platform Filtering Image processing filters are mainly used to: – suppress the high

BioImaging &Optics Platform

Filtering

Image 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.

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz
BioImaging &Optics Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz
BioImaging &Optics Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz

BioImaging &Optics Platform

Filtering

Shifting and multiplying a filter kernel

Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz PT-BIOP course, Image
Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz PT-BIOP course, Image
Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz PT-BIOP course, Image
Platform Filtering Shifting and multiplying a filter kernel Filtered image Dr. Arne Seitz PT-BIOP course, Image

Filtered image

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Noise Reduction: Mean 1 1 1 9 9 9 1 1 1
BioImaging &Optics Platform Noise Reduction: Mean 1 1 1 9 9 9 1 1 1
BioImaging &Optics Platform Noise Reduction: Mean 1 1 1 9 9 9 1 1 1

BioImaging &Optics Platform

Noise Reduction: Mean

BioImaging &Optics Platform Noise Reduction: Mean 1 1 1 9 9 9 1 1 1 9

1

1

1

9

9

9

1

1

1

9

9

9

1

1

1

9

9

9

1 1 1 9 9 9 1 1 1 9 9 9 mean Mean 1pt Dr.
1 1 1 9 9 9 1 1 1 9 9 9 mean Mean 1pt Dr.

mean

1 1 9 9 9 1 1 1 9 9 9 mean Mean 1pt Dr. Arne
Mean 1pt
Mean 1pt

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Noise Reduction: Gaussian Filtering with a gaussian bell-shaped kernel: 1 2 1
BioImaging &Optics Platform Noise Reduction: Gaussian Filtering with a gaussian bell-shaped kernel: 1 2 1
BioImaging &Optics Platform Noise Reduction: Gaussian Filtering with a gaussian bell-shaped kernel: 1 2 1

BioImaging &Optics Platform

Noise Reduction: Gaussian

BioImaging &Optics Platform Noise Reduction: Gaussian Filtering with a gaussian bell-shaped kernel: 1 2 1

Filtering with a gaussian bell-shaped kernel:

1
1

2

1

 

2 4

2

 

1 2

1

1

16

10
10

25

3

9

33

5

4

6

8

10
10

50

3

18

132

10

4

12

8

1

16

8 10 50 3 18 132 10 4 12 8 1 16 Dr. Arne Seitz PT-BIOP
8 10 50 3 18 132 10 4 12 8 1 16 Dr. Arne Seitz PT-BIOP

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Noise Reduction: Median median Median 3x3 Median 5x5 Dr. Arne Seitz PT-BIOP
BioImaging &Optics Platform Noise Reduction: Median median Median 3x3 Median 5x5 Dr. Arne Seitz PT-BIOP
BioImaging &Optics Platform Noise Reduction: Median median Median 3x3 Median 5x5 Dr. Arne Seitz PT-BIOP

BioImaging &Optics Platform

Noise Reduction: Median

BioImaging &Optics Platform Noise Reduction: Median median Median 3x3 Median 5x5 Dr. Arne Seitz PT-BIOP course,
BioImaging &Optics Platform Noise Reduction: Median median Median 3x3 Median 5x5 Dr. Arne Seitz PT-BIOP course,
BioImaging &Optics Platform Noise Reduction: Median median Median 3x3 Median 5x5 Dr. Arne Seitz PT-BIOP course,

median

Median 3x3
Median 3x3
Median 5x5
Median 5x5

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Noise Reduction: Median, Mean Median, 1pt Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Noise Reduction: Median, Mean Median, 1pt Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Noise Reduction: Median, Mean Median, 1pt Dr. Arne Seitz PT-BIOP course, Image

BioImaging &Optics Platform

Noise Reduction: Median, Mean

Median, 1pt

Platform Noise Reduction: Median, Mean Median, 1pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Mean, 1pt

Platform Noise Reduction: Median, Mean Median, 1pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
Platform Noise Reduction: Median, Mean Median, 1pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Median-, Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP
BioImaging &Optics Platform Median-, Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP
BioImaging &Optics Platform Median-, Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP

BioImaging &Optics Platform

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

&Optics Platform Median-, Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP course, Image

Median, 5pt

Min, 2pt

Median-, Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Mean, 5pt

Max, 2pt

Mean-, Max-, Min-Filter Median, 5pt Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Mean-, Gauss-Filter Mean, 2pt, 4 pt Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Mean-, Gauss-Filter Mean, 2pt, 4 pt Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Mean-, Gauss-Filter Mean, 2pt, 4 pt Dr. Arne Seitz PT-BIOP course, Image

BioImaging &Optics Platform

Mean-, Gauss-Filter

BioImaging &Optics Platform Mean-, Gauss-Filter Mean, 2pt, 4 pt Dr. Arne Seitz PT-BIOP course, Image Processing,

Mean, 2pt, 4 pt

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Platform Mean-, Gauss-Filter Mean, 2pt, 4 pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Gauss, 2pt, 4 pt

BioImaging &Optics Platform Mean-, Median-Filter M e a n , 2 p t , 4
BioImaging &Optics Platform Mean-, Median-Filter M e a n , 2 p t , 4
BioImaging &Optics Platform Mean-, Median-Filter M e a n , 2 p t , 4

BioImaging &Optics Platform

Mean-, Median-Filter

BioImaging &Optics Platform Mean-, Median-Filter M e a n , 2 p t , 4 p

Mean, 2pt, 4 pt

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

M e a n , 2 p t , 4 p t Dr. Arne Seitz PT-BIOP

Median, 2pt, 4 pt

BioImaging &Optics Platform Min-, Max-Filter Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Min-, Max-Filter Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Min-, Max-Filter Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

BioImaging &Optics Platform

Min-, Max-Filter

BioImaging &Optics Platform Min-, Max-Filter Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Min-, Max-Filter Min, 2pt Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Min, 2pt

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Max, 2pt

BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1
BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1
BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1

BioImaging &Optics Platform

Sharpen / Blur

-1 -1 -1

1 1 1

BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1 1
BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1 1
BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1 1
BioImaging &Optics Platform Sharpen / Blur -1 -1 -1 1 1 1 -1 9 -1 1
-1 9 -1 1 2 1 -1 -1 -1 1 1 1 sharpen blurring
-1
9 -1
1 2 1
-1 -1 -1
1 1 1
sharpen
blurring
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1
-1 -1 1 1 1 -1 9 -1 1 2 1 -1 -1 -1 1 1

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Example: Edge-Finding with derivatives Dr. Arne Seitz PT-BIOP course, Image Processing,
BioImaging &Optics Platform Example: Edge-Finding with derivatives Dr. Arne Seitz PT-BIOP course, Image Processing,
BioImaging &Optics Platform Example: Edge-Finding with derivatives Dr. Arne Seitz PT-BIOP course, Image Processing,

BioImaging &Optics Platform

Example:

Edge-Finding with derivatives

&Optics Platform Example: Edge-Finding with derivatives Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

-1 -1 -1

0 0

0

1 1

1

-1 -1 -1

0 1

0

1 1

1

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 -1 -1 -1 0 0 0 1
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 -1 -1 -1 0 0 0 1
BioImaging &Optics Platform Background Subtraction Even background: – subtract average background from image
BioImaging &Optics Platform Background Subtraction Even background: – subtract average background from image
BioImaging &Optics Platform Background Subtraction Even background: – subtract average background from image

BioImaging &Optics Platform

Background Subtraction

Even 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

– Use kernel larger than diameter of largest object Dr. Arne Seitz PT-BIOP course, Image Processing,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Original Image “Opening”
Original Image
“Opening”

Original Image - Opening

object Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 Original Image “Opening” Original Image -
BioImaging &Optics Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image

BioImaging &Optics Platform

BioImaging &Optics Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image

Line Profile

Without background subtraction

Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
Platform Line Profile Without background subtraction Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

After rolling ball (50) background subtraction

subtraction Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 After rolling ball (50) background subtraction
subtraction Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 After rolling ball (50) background subtraction
BioImaging &Optics Platform Thresholding Thresholding is used to change pixel values above or below a
BioImaging &Optics Platform Thresholding Thresholding is used to change pixel values above or below a
BioImaging &Optics Platform Thresholding Thresholding is used to change pixel values above or below a

BioImaging &Optics Platform

Thresholding

Thresholding is used to change pixel values above or below a certain intensity value (threshold):

values above or below a certain intensity value (threshold): Threshholding is a simple method for Segmentation

Threshholding is a simple method for Segmentation (separation and location of objects of interest)

Segmentation (separation and location of objects of interest) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
Segmentation (separation and location of objects of interest) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
Segmentation (separation and location of objects of interest) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
Segmentation (separation and location of objects of interest) Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Measuring Sizes Set Scale with pixel (voxel) size Include Scalebar Dr. Arne
BioImaging &Optics Platform Measuring Sizes Set Scale with pixel (voxel) size Include Scalebar Dr. Arne
BioImaging &Optics Platform Measuring Sizes Set Scale with pixel (voxel) size Include Scalebar Dr. Arne

BioImaging &Optics Platform

Measuring Sizes

BioImaging &Optics Platform Measuring Sizes Set Scale with pixel (voxel) size Include Scalebar Dr. Arne Seitz

Set Scale with pixel (voxel) size Include Scalebar

Sizes Set Scale with pixel (voxel) size Include Scalebar Dr. Arne Seitz PT-BIOP course, Image Processing,
Sizes Set Scale with pixel (voxel) size Include Scalebar Dr. Arne Seitz PT-BIOP course, Image Processing,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Measuring Length Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Measuring Length Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Measuring Length Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform

Measuring Length

BioImaging &Optics Platform Measuring Length Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Measuring Length Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Area Measurement 16bit image 3 2 b i t i m a
BioImaging &Optics Platform Area Measurement 16bit image 3 2 b i t i m a
BioImaging &Optics Platform Area Measurement 16bit image 3 2 b i t i m a

BioImaging &Optics Platform

Area Measurement

BioImaging &Optics Platform Area Measurement 16bit image 3 2 b i t i m a g

16bit image

BioImaging &Optics Platform Area Measurement 16bit image 3 2 b i t i m a g

32bit image

Area Measurement 16bit image 3 2 b i t i m a g e Dr. Arne

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

e Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 32bit image, background thresholded to “Not

32bit image,

background thresholded to “Not a Number”

32bit image, background thresholded to “Not a Number” 16bit image, same threshold as in 32bit image
32bit image, background thresholded to “Not a Number” 16bit image, same threshold as in 32bit image

16bit image, same threshold as in 32bit image but not applied

BioImaging &Optics Platform Analyze Particles Segmented objects Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Analyze Particles Segmented objects Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Analyze Particles Segmented objects Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

BioImaging &Optics Platform

Analyze Particles

BioImaging &Optics Platform Analyze Particles Segmented objects Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Analyze Particles Segmented objects Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

Segmented objects

&Optics Platform Analyze Particles Segmented objects Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Threshold and Opening/Closing Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Threshold and Opening/Closing Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Threshold and Opening/Closing Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform

Threshold and Opening/Closing

BioImaging &Optics Platform Threshold and Opening/Closing Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010
BioImaging &Optics Platform Threshold and Opening/Closing Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 dilate erode Closing: Dilate/Erode Opening: Erode/Dilate

dilate

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 dilate erode Closing: Dilate/Erode Opening: Erode/Dilate
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 dilate erode Closing: Dilate/Erode Opening: Erode/Dilate

erode

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 dilate erode Closing: Dilate/Erode Opening: Erode/Dilate

Closing: Dilate/Erode Opening: Erode/Dilate

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 dilate erode Closing: Dilate/Erode Opening: Erode/Dilate
Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010 dilate erode Closing: Dilate/Erode Opening: Erode/Dilate
BioImaging &Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image
BioImaging &Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image

BioImaging &Optics Platform

Color Functions

RGB Merge /RGB Split

&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
&Optics Platform Color Functions RGB Merge /RGB Split Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Deconvolution From Object to Image Effects causing Object Image degradation: Noise Image
BioImaging &Optics Platform Deconvolution From Object to Image Effects causing Object Image degradation: Noise Image
BioImaging &Optics Platform Deconvolution From Object to Image Effects causing Object Image degradation: Noise Image

BioImaging &Optics Platform

Deconvolution

From Object to Image

Effects causing Object Image degradation:

Noise

Image

– Signal derived noise

Image degradation: Noise Image – Signal derived noise – Noise emerging from the digital imaging system
Image degradation: Noise Image – Signal derived noise – Noise emerging from the digital imaging system

– Noise emerging from the digital imaging system

noise – Noise emerging from the digital imaging system Scatter – Glare Caused by heterogeneous refractive

Scatter

Glare

Caused by heterogeneous refractive index (RI)

– Random disturbance of light in the system

Blur

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
BioImaging &Optics Platform Point Spread Function (PSF) A Point Spread Function is the 3D diffraction
BioImaging &Optics Platform Point Spread Function (PSF) A Point Spread Function is the 3D diffraction

BioImaging &Optics Platform

Point Spread Function (PSF)

A Point Spread Function is the 3D diffraction pattern of a “point” source of light. Widefield = hourglass shape Confocal = American Football shape

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Widefield = hourglass shape Confocal = American Football shape Dr. Arne Seitz PT-BIOP course, Image Processing,
BioImaging &Optics Platform Convolution of an Object Object can be referred as accumulation of points
BioImaging &Optics Platform Convolution of an Object Object can be referred as accumulation of points
BioImaging &Optics Platform Convolution of an Object Object can be referred as accumulation of points

BioImaging &Optics Platform

Convolution of an Object

Object can be referred as accumulation of points Each point is visible as a PSF

Object PSF = Image

= convolution

Image process hast to be

- Linear

- Shift invariant

Convolution is in principle a reversible mathematical equation

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

Convolution is in principle a reversible mathematical equation Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL
BioImaging &Optics Platform Constrained Iterative Constrained:  “Nonnegativity”  Smoothing or
BioImaging &Optics Platform Constrained Iterative Constrained:  “Nonnegativity”  Smoothing or
BioImaging &Optics Platform Constrained Iterative Constrained:  “Nonnegativity”  Smoothing or

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.

estimate is found in a successional serial of calculations. Dr. Arne Seitz PT-BIOP course, Image Processing,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Different Algorithms… …lead to different Results r a w d a t
BioImaging &Optics Platform Different Algorithms… …lead to different Results r a w d a t
BioImaging &Optics Platform Different Algorithms… …lead to different Results r a w d a t

BioImaging &Optics Platform

Different Algorithms…

…lead to different Results

raw data

…lead to different Results r a w d a t a Huygens: CMLE 30 It AutoQuant:

Huygens: CMLE 30 It

different Results r a w d a t a Huygens: CMLE 30 It AutoQuant: non blind

AutoQuant: non blind 15 It

d a t a Huygens: CMLE 30 It AutoQuant: non blind 15 It SoftWorx: 30 It

SoftWorx: 30 It

CMLE 30 It AutoQuant: non blind 15 It SoftWorx: 30 It AutoQuant: Blind 15 It Dr.

AutoQuant: Blind 15 It

non blind 15 It SoftWorx: 30 It AutoQuant: Blind 15 It Dr. Arne Seitz PT-BIOP course,

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Signal improvement 7000 1400 6000 1200 5000 1000 4000 800 3000 600
BioImaging &Optics Platform Signal improvement 7000 1400 6000 1200 5000 1000 4000 800 3000 600
BioImaging &Optics Platform Signal improvement 7000 1400 6000 1200 5000 1000 4000 800 3000 600

BioImaging &Optics Platform

Signal improvement

7000 1400 6000 1200 5000 1000 4000 800 3000 600 2000 400 1000 200 0
7000
1400
6000
1200
5000
1000
4000
800
3000
600
2000
400
1000
200
0
0
AQ_blind_15It_thPSF
not deconvolved
Dr. Arne Seitz
PT-BIOP course, Image Processing, EPFL 2010

Higher signal to

background ratio

More distinct peaks

Computational BioImaging &Optics Platform WF Deconvolution substraction of blur or reassignment to the assumed source

Computational

Computational BioImaging &Optics Platform WF Deconvolution substraction of blur or reassignment to the assumed source
Computational BioImaging &Optics Platform WF Deconvolution substraction of blur or reassignment to the assumed source

BioImaging &Optics Platform

WF Deconvolution

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 bleaching Disadvantages:

Need for high computational systems

Artefacts can not be excluded

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform WF Decon vs. Confocal To deconvolve or not to deconvolve That is
BioImaging &Optics Platform WF Decon vs. Confocal To deconvolve or not to deconvolve That is
BioImaging &Optics Platform WF Decon vs. Confocal To deconvolve or not to deconvolve That is

BioImaging &Optics Platform

WF Decon vs. Confocal

To 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

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

to Confocal pictures as they can also be deconvolved Dr. Arne Seitz PT-BIOP course, Image Processing,
BioImaging &Optics Platform Conclusions • Keep environment constant and convenient • Use powerful dyes •
BioImaging &Optics Platform Conclusions • Keep environment constant and convenient • Use powerful dyes •
BioImaging &Optics Platform Conclusions • Keep environment constant and convenient • Use powerful dyes •

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

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform Summary • Use lossless file formats for archiving important data • Image
BioImaging &Optics Platform Summary • Use lossless file formats for archiving important data • Image
BioImaging &Optics Platform Summary • Use lossless file formats for archiving important data • Image

BioImaging &Optics Platform

Summary

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

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010

BioImaging &Optics Platform More about image processing 1. Lecture M. Unser, EPFL see also website:
BioImaging &Optics Platform More about image processing 1. Lecture M. Unser, EPFL see also website:
BioImaging &Optics Platform More about image processing 1. Lecture M. Unser, EPFL see also website:

BioImaging &Optics Platform

More about image processing

1.

Lecture M. Unser, EPFL

see also website: http://bigwww.epfl.ch/

2.

Books

a) W. Burger, M. J. Burge Digital Image Processing, Springer 2008

b) J. C. Russ The image processing Handbook, CRC Press 2007

3.

PT-BIOP EPFL, SV-AI 0241, SV-AI 0140 http://biop.epfl.ch/

Dr. Arne Seitz PT-BIOP course, Image Processing, EPFL 2010