Deconvolution – Pros & Cons 3D microscopy...Low signal:noise yeast live 3D imaging Spinning Disk...
Transcript of Deconvolution – Pros & Cons 3D microscopy...Low signal:noise yeast live 3D imaging Spinning Disk...
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Deconvolution – Pros & Cons3D microscopy
"Systematic errors can be measured and corrected for"
Or
"Enhancing contrast and removing noise helps see the resolution that's really there"
Dan White – Oct 2010
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Optical sectioning – 3D imaging
www.biomedical-engineering-online.com
Digital sampling of
3 spatial dimensions
X and Y are easy, right...?
… but is Z special?
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Optical sectioning - methods:
• WM widefield microscopy• CLSM confocal laser single point scanning• SPD confocal multi point scanning • 2P two photon• TIRF total internal reflection fluorescence• SPIM single plane illumination
• structured illumination microscopy• widefield microscopy + deconvolution
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http://depts.washington.edu/keck/intro.htm
closed open
“confocal”pinhole rejects out of focus light from above and below the focal plane
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Michael Weber
50 µmMouse kidney section Alexa Fluor 488 WGA For a closed pinhole:Alexa Fluor 568 phalloidin PSF is much smaller in z, bit smaller in x,y
Make the PSF smaller? Confocal Sectioning
closed pinhole open pinhole
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Michael Weber
Slow for large FOV Flexible FOV PMT detector High Bleaching “In Focus” signal = good contrast Good for fast images, smaller area at high resolution.
laser point scanning confocal
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http://www.atto.com/technologies/imaging.shtml
spinning discpoint array scanning confocal
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spinning discconfocal scanner head
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Britta Schroth-Diez
• “field” illumination method
• Detector : CCD / EM-CCD
• High speed!!!
• Faster than point scan
• But still noisy
• Lower photobleaching
• Good for live samples
• Lower Z resolution
• compared to LSCM
• more out of focus signal
Spinning Disk :Multi point scanning confocal
YOKOGAWACSU X-1
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belfield.cos.ucf.edu
2 Photon Microscopy
Brad Amos MRC
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www.phys.ntu.edu.tw
Two Photon Microscopy• “point” illuminating technique• detector : PMT• Lower z resolution to LSCM (longer excitation � )• high penetration depth - up to 500 um • low temporal resolution (slow speed)• low photobleaching• low illumination light scattering• high price (expensive laser)• 1 or maybe 2 “colours” only• Good for
– Thick samples– Low light scattering– Living Samples
2 photon effect
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http://micro.magnet.fsu.edu/primer/java/tirf/reflect/index.html
Total Internal Reflection Fluorescence
• field illuminating technique
• detector : CCD• fast• very high Z resolution
~150 nm
• one optical section only at plane close to cover glass
• Living sample– Fast dynamics
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TIRFonly the objects on the coverslip
TIRF Wide field
Britta S-D, Silke G
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Sticking biology to flat glass is not very physiological...
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http://www.huisken.org/jan/spim.html
SPIMSingle Plane Illumination Microscopy
• field illumination technique detector: CCD high speed Isotropic Resolution xyz
Multi angle reconstruction very low photobleaching/toxicity lower Z resolution (single angle) 3D mounting in physiological environment not commercially available yet
White, Preibisch, Saalfeld, Ejsmont, Sarov, Tomancak
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Structured illuminationOptigrid,ApoTome
andOMX
• “field” illumination detector : CCD slow/medium speed high flexibility image processing required (fast) Good for fixed thinner samples, instead of confocal
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Widefield Microscopy + Deconvolution
• “field” illuminating technique• Detector : CCD• High temporal resolution (high speed)• High flexibility • Low bleaching / photo toxicity• Very high signal:noise (low speckle)• Image processing required (automated)• Good for fixed and living samples.
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Wide-field fluorescence + deconvolutionSingle plane – 2D blind deconv.
Johanna Spandl
raw image deconvolved image
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Wide-field fluorescence + deconvolutionz-stack mono layer cultured cells on glass
Alex Bird / Dan White
raw imagemax Z projection
deconvolved imagemax Z projection
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Low signal:noise yeast live 3D imagingSpinning Disk Confocal
Davide Accardi / Dan White
raw imagemax Z projection
deconvolved imagemax Z projection
Deconvolution is also good at noise removal, revealing details and improving contrast in all kinds of microscopy images.
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Image of a point is not a point!The image of a point is convolved by
the point spread function – PSF.
Widefield microscope Measured PSF1.4 NA Oil lensWatery sample
=Spherical Aberation!
yx
x
z
PSF size:WF>SD>SPIM>C
Jesse Lipp, Dan White
Real Widefield
PSF (linear scale)
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22over sampledNoisy, low contrast
correct samplingGood contrastSee 2 objects
2 points X and X – resolution limit apart, d Pixel size = d/3
under sampledCan't see 2 objects
Pixel Size / ResolutionRemember !!!
Spatial sampling:
~1/3 x smallest feature.
Remember !!!
A pixel is NOT a little square...
it is a spatial sample at some location x,y,z in space and time
Remember !!! Think like a spectroscopist, not a photographer!
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Image of a point isnot a point!
The image of a point is convolved by
the point spread function – PSF.
Widefield microscope PSF1.4 NA Oil lens 1.515Glassy sample 1.515
PSF is very much bigger in zthan in x and y
Why?
Higher NA = better resolution,
especially in Z!
z
x
PSF size:WF>SD>SPIM>C
Theoretical PSF
(log scale)
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Image of a point isnot a point!
The image of a point is convolved by
the point spread function – PSF.
Widefield microscope PSF1.4 NA Oil lens 1.515
Watery Sample1 Wave spherical aberration(eg approx. 10 um of water)
PSF is very asymmetric in Z and also shifted in Z
z
x
Theoretical PSF
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Image of a point isnot a point!
The image of a point is convolved by
the point spread function – PSF.
Widefield microscope PSF1.4 NA Oil lens 1.515
2 Waves spherical aberration
PSF is very very very asymmetric in Z and also
shifted very badly in Z
z
x
Theoretical PSF
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Confocal PSF is smaller than WF, but still significant in size.
PSFs with1.4 NA Oil lens 1.515Glassy sample 1.515
Theoreticalconfocal PSF
Theoreticalwidefield PSF
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Spherical Aberation Kills Confocal Imaging
PSF with1.4 NA Oil lens
1.515Watery sample 1.33
10 um away from 0.17 mm coverglass
Theoreticalconfocal PSF
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Quick and Dirtyvs. Slow and Careful
Default “High Noise” settings on DeltaVision don't handle very noisy
images or CCD Offset well
More careful deconvolution using
Huygens does a better job of dealing with noise and
CCD offset
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Tips to get it right:
1) Measure the PSF, orCorrect aberrations to get close to perfect PSF
Coverslip, immersion and mounting media.
2) Test different noise parameters until you get a reasonable result
Don't enhance noise or smooth detail.The is a happy optimum
3) Look for “left over” intensity – wrong PSF
4) Look for “new” false objects, not in raw image.
5) Correct spatial sampling – noisy data OK.