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Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Deconvolution of Microscopy Images Issues to Consider for Widefield and Confocal Microscopy Jonathan.

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Presentation on theme: "Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Deconvolution of Microscopy Images Issues to Consider for Widefield and Confocal Microscopy Jonathan."— Presentation transcript:

1 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Deconvolution of Microscopy Images Issues to Consider for Widefield and Confocal Microscopy Jonathan Girrior 3D Applications Manager Media Cybernetics Presented by: If oversubscribed please email jgirroir@mediacy.com to reschedule. jgirroir@mediacy.com Webex support: 408-435-7088 866-229-3239

2 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Overview Image Formation Types of Image Restoration Blind vs. Non-blind Deconvolution Spherical Aberration Resources

3 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 The object The image What happened? Image Formation

4 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 One Dimension - Diffraction

5 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Two Dimensions - Airy Disk

6 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Three Dimensions – Point Spread Function (PSF) An impulse response. Characterizes your microscope.

7 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Effects of the PSF An object is a collection of point sources. Mathematical operation called a convolution. The Microscope is a convolution operator.  = Object PSFImage

8 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 No-Neighbors Single slice Nearest-Neighbors Several optical sections Inverse FilteringWhole volume, one pass Non-blind DeconvolutionWhole volume, multi-pass, known PSF Blind DeconvolutionWhole volume, multi-pass, adaptive PSF. Qualitative vs. Quantitative

9 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Widefield Deconvolution

10 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Widefield Deconvolution

11 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Confocal Deconvolution

12 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Widefield vs. Confocal PSF XY XZ WidefieldConfocal

13 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 No-Neighbors Debluring Uses only current slice. Blurs current slice with PSF to approximate blur contribution from adjacent slices. Subtract away the blur. Deblurs one slice at a time Very fast, 2D, only qualitative 0

14 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Nearest Neighbors Debluring Uses center slice and two adjacent slices Estimate amount of blur that adjacent slices contribute to center slice Subtract away the blur Deblurs one slice at a time Fast, 2D, only qualitative 0 +z -z

15 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Inverse Filter Restoration algorithm – looks at whole structure Works well on large objects Fast, 3D, qualitative Regularization/Stability DeblurredImage PSF 1  

16 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Requires PSF (theoretical or measured) Iteratively improve object estimate Impose constraints (non-negativity) Requires highly optimized imaging system Reblurred Object Estimate PSF Original Blurred Improved Estimate - UpdateError 3D Non-blind Deconvolution

17 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 3D Blind Deconvolution Estimate both PSF and Object! Need microscope parameters (NA, RI, xyz, λ) Theoretical PSF with spatial/spectral constraints. Object with non-negativity and noise constraints.

18 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Initial Object Estimate Initial Object Estimate 3d Blind Deconvolution PSF Original Blurry Data - Update Error PSF ? ? ? Iteration 1 - PSF

19 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Initial Object Estimate Initial Object Estimate 3D Blind Deconvolution PSF Original Blurry Data - Update Error Object Estimate Initial Object Estimate ? ? ? Iteration 2 - Object

20 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Object Estimate Object Estimate 3D Blind Deconvolution PSF Original Blurry Data - Update Error PSF ? ? ? Iteration 2 - PSF

21 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Blind vs. Non-Blind Deconvolution Non-blind –Must maintain library of PSFs. –PSFs need to be optimally measured and noise free. –Faster because only estimating image. Blind –Does not require imaging of beads. –Can respond to changes in optical model: Specimen, thermal expansion, aberrations and noise.

22 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 AutoQuant X Products AutoDeblur Widefield: $5,950 US AutoDeblur Confocal: $5,950 US AutoDeblur Widefield & Confocal: $9,400 US 10% Discount for all Webinar Attendees! Contact sales@mediacy.comsales@mediacy.com FREE 30-day demo: www.mediacy.com/leads/demos.asp www.mediacy.com/leads/demos.asp

23 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Spherical Aberration Changes in refractive index Incorrect cover slip thickness Thick specimens

24 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Effects of Spherical Aberration Results in an asymmetry of the PSF. Adds significantly more blur. Reduces signal.

25 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Correction of Spherical Aberration Match refractive indexes. Use a SA correction collar. Bias the PSF with similar aberration and deconvolve. Bias the PSF and allow it to adapt using blind deconvolution. Correct with optics in infinity space.

26 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Raw Image max = 2,614

27 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Deconvolved no SA max = 12,134

28 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Deconvolution with SA Correction max = 23,938

29 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Noise and Maximum Likelihood Comes from photon detection & instrument noise. Update step is important. Most companies smooth every few iterations. Maximum likelihood. –Statistical approach which tests what is the most likely to occur in the specimen using a known noise model. –Finds the “most likely” possibility for each portion of the image. –Models noise extremely well. –Can actually remove noise instead of suppress.

30 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Upcoming events Oct. 11 th - Imaging Series Webinar #3 - "3D Imaging: Issues to Consider for Biological Microscopy”. FREE Nov. 5 th AutoQuant training in San Diego during Neuroscience. Contact Kathy Hrach (kathy@mediacy.com). $295kathy@mediacy.com Dec. 5 th - AutoQuant training in Washington D.C. during Cell Biology. Contact Kathy Hrach (kathy@mediacy.com). $295kathy@mediacy.com

31 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Questions? Biggs D. (2004) Clearing Up Deconvolution. Biophotonics Feb 2004. Holmes TJ. Light Microscopic Images Reconstructed by Maximum Likelihood Deconvolution. Chapter 24 in The Handbook of Biological Confocal Microscopy, 2 nd Edition, James Pawley, Editor, Plenum Press, New York, 1995. Swedlow JR. (2001) A working person's guide to deconvolution in light microscopy. Biotechniques 31:1076-1097.

32 Dial in: (319) 741-8100 Access: 627827# Unmute/mute: #6 Thank you For more information contact: Jonathan Girrior Media Cybernetics 541-383-4133 jgirrior@mediacy.com FREE 30-day demo: www.mediacy.com/leads/demos.asp Slideshow at ftp.mediacy.com/uploaded/AQX/DeconWebinar.pdf ftp.mediacy.com/uploaded/AQX/DeconWebinar.pdf

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