HOW IMAGE ANALYSIS WORKS

AND HOW IT CAN BE AUTOMATED

By Eden Zik July 2012

IMAGE ANALYSIS = EXTRACTION OF USEFUL DATA FROM AN IMAGE

SAMPLING PROCESS
Plating in a dish
Dyeing

Analyzing

Capturing

ANALYSIS PROCESS

Measurement

Imaging

DATA

WHAT IS THE HARDEST PART OF THE ANALYSIS PROCESS?

MEASUREMENT

but why?

HUMAN FLAW
Images are difficult to quantify with the naked eye, and things are never perfectly repeatable therefore (Less reliable) Intensity hard to compare, even within image Objects can seem relatively large or small when comparing different images Characteristics such as form factor, relative area, or hole area are impossible to instinctively measure Subtle trends in morphology can be left undetected Slow processing time, up to an hour per image

COMPUTER INABILITY
Desired information not initially distinguished from all of the information embedded in the image. (Less accurate) Edges hard to detect Dirt often mistakenly processed Pale objects ignored Objects are often joined together in the same region

MAN/MACHINE ADVANTAGE:
THE EFFICIENCY OF A COMPUTER WITH THE GUIDANCE OF THE HUMAN EYE
Reliable measurement, with minimal human interference (i.e all images are created equal, and deserve fair treatment under the journal) Precise and repeatable measurement of any characteristic Fast processing time
BOTTOM LINE:

THE BEST DATA IS PRODUCED BY A COMPUTER WITH HUMAN GUIDANCE

FOR ULTIMATE MEASUREMENT

ALL HAIL THE JOURNAL!

A journal is a list of commands to be executed by Metamorph. A journal is designed to be ran with no human intervention, and produce data directly.

However, guidance is required to

Open up the desired file, for example

Find a cell

And save the data to a desired directory.

MINIMAL INTERVENTION IS KEY!

NOTHING BUT:

1. Finding the file to be processed 2. Identifying the region to measure

Usually a cell Done in images with multiple cells Isolates data of one cell from another 3. Saving the excel file with the data

MINIMAL INTERVENTION = RELIABILITY, REPEATABILITY

So how to design a largely automated process without losing accuracy?

STEPS OF MEASUREMENT
Formatting Exclusion Isolation

Marking

Binarization

Filtration

Transferring

Logging

FORMATTING (MANUAL)
Making the image suitable for processing File Open image File Find Images Open as stack Stack Display Color Combine
Color combine defines each layer as a color Color can be false or real Useful for processing if each fluorescent layer needs to be analyzed individually Collapses each layer into its own color

Display Color separate

EXCLUSION (MANUAL)
Choosing the region in the image to be processed Defining a region Moving a region to an area to be measured Region Create region
For a similar characteristic measured in a region across many images, define a constant region for all images For cells to be measured individually in images with multiple cells, create a custom region that covers one cell and nothing else

ISOLATION
Isolating the region selected Isolation is key!
For most analysis tools are relative- they compare the intensity of one point to the rest (more later)

Edit Duplicate Open as Image

Works once region is selected

FILTRATION I
The most important step! Wake up Orian!

Filtration is setting a threshold to an image

A threshold is any chosen numerical value.

All pixels that are under (or over) that value are excluded All regions that do not meet the morphological value are excluded Most of these features are found under Process The rest are found under Measure Filtration is done before binarization, or both before AND after binarization Most morphological values are useful precursors to performing an intensity threshold, the most common filteration

FILTRATION II
Intensity (What most people think of as a threshold) Marks brightest (or darkest) objects in the image Auto threshold for bright objects is extremely useful! Location More similar to isolation filtration by where the object is in the image Integrated Intensity Threshold Sets a threshold of a pixel by its close neighbors Great for maximizing contrast prior to a normal threshold Morphological Gradient Outlines the edges of objects Does it by measuring a rapid change in intensity

FILTRATION III
Detect edges

Uses a complex mathematical model to identify where groups of pixels are located (in other words, Magic!) Makes identifying objects much easier, prior to an intensity threshold Some functions under Detect edges work better than others. Sebel usually works best! Holes
Fills in dark or bright holes in an image Useful order to bring out objects Open/Close Enhances the contrast of an image Open makes bright pixels brighter and dark pixels darker Close makes bright pixels darker and dark pixels brighter Erode, Watershed, Top hat many less useful ones.

FILTRATION IV
Integrated Metamorphic Analysis! The single thing that makes metamorph pretty incredible. Image must be threshold, or binarized Under Measure Integrated Metamorphic Analysis Specific filtration of threshold/ binerized images by:
Form factor (how close the shape is to a circle 1= perfect circle) Area Optical density Elongation Angle of chord from center to border Many other featured.

BINARIZATION
Every pixel exceeding the thresholds defined in filtration gains an intensity value of 1. The rest of the pixels lose their intensity value, and become 0. The image becomes very clearly defined Objects become clear Morphology filtration are best done when image is binerized Measuring a binary image is useful for morphology (i.e shapes of particles) Drawing regions around objects

MARKING
Once an image is binerized, however, all of its intensity information is lost. Therefore, the most common nonmorphological use of a binary image is for identifying objects and drawing regions around them. Regions Draw Regions around Objects

TRANSFERRING
In order to measure intensity of the objects, one must transfer the regions clearly defined in the binary image to the original Regions Transfer regions Threshold
A threshold must be set to the original image in order for intensity to be measured Intensity is a relative measurement Taken with respect to the darkest pixels An Auto threshold works just fine

LOGGING (MANUAL)
In order to log and export the data gathered from the image, the regions must first be measured. Measurement Region Measurement Measure
User has ability to choose which parameters to measure Intensity Area Location
Excel prompt will open Excel will open

Measurement Region Measurement Open Data Log

AND THEN DATA PROCESSING

A WHOLE OTHER STORY

THAT WE WILL DISCUSS ANOTHER TIME.

NOW, PLEASE TAKE A MOMENT TO THANK ALMIGHTY THE MAN UPSTAIRS, , WHOSE ETERNAL GRACE HAS ENABLED US TO HAVE COMPUTERS DOWNSTAIRS. COMPUTERS WHICH MAKE OUR ANALYSIS SO MUCH FASTER.

And now for marc