digital imaging - university of western australia · film 3: • modern microscopy is quantitative...
TRANSCRIPT
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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
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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