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Digital Imaging

Facilities at the IAAF

Prof Stuart Bunt (Based on PPT by Simon Watkins)

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Plan of the Talk• Digital imaging vs Film• Understand what constitutes a digital image• Understand what a pixel is• Understand greyscale and color resolution• Understand how digital cameras work• Understand principal of filter operation• Understand simple processes• Understand general demands of a digital analysis

network

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Detection systems• Film

– Slow, difficult to quantify and archive highest resolution

• Video cameras– Low resolution, may be intensified, used for

fast events• CCD camera

– High resolution, need to be cooled, slow• Photomultipliers

– Point recorders, used in confocal for example

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What are the advantages of digital images

• Speed• Quantitation• Manipulation (multicolor images)• Video recordings• etc etc

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Image collection and processingBasic Steps

ImageCollection•Online processing•Averaging•Background subtraction

Processing•Filters•Contrast•Brightness•Color correction

Storage

Feature Extraction•Thresholding

•Blob separation•Watershed split

Quantify•examples from the realworld

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Film 1:

Film is better than a pencilFilm will always have a higher resolution than digital methodsFilm stores the original data (assuming a perfect exposureFilm is difficult to work with for fluorescent imaging to do multicolor imaging with film demands massive amounts of experience

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Film 3:

• Modern microscopy is quantitative– Film is analog and is therefore not quantitative

• “Brightness” and resolution all depend on film type, speed and printing

• Slower films have longer exposure times and higher resolution, slowest film practical for fluorescence is 400 ASA.

• Digital imaging is fast, quantitative and easy!

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What is a digital imageDigital images are composed of pixels 1 2 3 4 5 6 7 8 9

Pixels contain information aboutintensity and color that can be stored on a computer. •Each pixel contains information about 1 location in the image•A sequence of pixels can be combined to make an image.

Digital images are at the heart of all modern microscopies

ProcessingCollection Storage Quantitation

The rest of this lecture discusseseach of these points in detail Presentation

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What is greyscale?

• 1bit=2 greyscales (binary)= black and white• 2bits=2x2=4 greyscales, • 3bits=2x2x2=8 greyscales etc.• most cameras 8bit=256 greyscales• Commonly cooled CCDs are 12

bits=4096greyscale

greyscale gives contrast and intensity for each pixel

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Pixels 3: color vs black and white• Color images are simply 3 black and white images combined, one for

each primary color• However they are 3 times larger than the black and white image• Color is very useful for image processing

RED GREEN BLUE

24 bits = 8bits + 8bits + 8bits

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Real World Resolution

400 DPI 200 DPI 72 DPI 36 DPI

16 DPI 8 DPI 4 DPI

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Summary

• Film will always be better than a camera• Film is ridiculously impractical for

fluorescent imaging• Digital images consist of pixels• Choose the right number of pixels and gray

scales for the question at hand• Color imaging is useful for image

processing.

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What is a CCD camera• An array of silicon targets

which collect light, transform it to electrons and send it in an organized fashion to a computer

• Two types– video – digital

• the principal difference is that video works at 30 frames/second, digital is adjustable up to minutes/frame

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Video rate vs Cooled CCD

Silicon Target

HEAT=thermal noiseRemoved by cooling CCD

X X

Noise increases withAmplifier BandwidthVideo rate CCD (30 frames/sec)must read at 10 million pixels/secSlow scan cameras work at only500,000 pixels/secTherefore slower and less noisy

PhotonsCosmic Rays

or X-Rays

Readoutamp

electrons

Speed relatednoise

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Intensified DevicesIncoming Photons

Dynodes

-

Incoming Photons

Fiber optic cables

Fiber optic cables

+ Avalanche PhotodiodePhotocathode

Anode Collector

CCDPhoto Multiplier Tube

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BinningBinning adds electron wells from numbers of pixels, Only one use of the read out amplifer, therefore less noise

This example is 2X2 binning

2 pixels

2 pi

xels

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Types of CCD

Image collected

Image transfered

Image readout asnew imagecollected

Most common, least expensive, subject to noise from readout amplifier

lens

CCD

Peltier cooler

Frame transfer, expensive, hard to make, fastframe is read off camera as a new frame is collected

Line transfer

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Regular CCDs vs backthinned CCDs

Backthinning increases QE

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Detector noise:

• Thermal, or gain

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Background SubtractionBrightfield

Original/backgroundcorrects non-white image

Divide color by itself=whiteRGB 0.6|0.9|0.3/ 0.6|0.9|0.3 =1|1|1

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Background subtraction

Subtract background inFluorescence, removes hotpixels and noise

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Well labeled and well sampled slides do not need correction

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Fast Fourier Transform and noise reduction

• Only useful on repetitive noise, which will generate a Fourier transform

Courtesy Mediacybernetics

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Image AveragingAveraging removes noiseKalman or proportionalaverage best improvement in image quality is proportionalto the Square root of the # of frames (4 frames twice as good as 1 frame etc.)

Primary problem is Progressive bleaching andPhototoxicity in Live cell work,

Single frame Two frames

Four frames Eight frames