1. FROM IMAGES TO ANSWERS Deconvolution of Widefield and Confocal images The growing role of deconvolution NE 2007

2. Improving signal to noise

3. http://www.molecularexpressions.com/primer/anatomy/numaperture.html What is it? (1/6) Widefield – Single Channel What is deconvolution?

4. Multiple wavelengths - Widefield

5. Confocal Microscopy

6. 2D Blind deconvolution

7. 2D Blind deconvolution – TIRF

8. When to use deconvolution In widefield fluorescent Microscopy –Improve contrast of 2D and 3D images –Improve resolution in three dimensions –Improve 3D rendering and visualization

9. When to use deconvolution In confocal microscopy –Improve signal to noise ratios –increase dynamic range –Improve image grayscale resolution –Increase axial resolution

10. When to use deconvolution Documentation –Images and projections will show more detail and greater contrast –Improvements can be striking

11. When to use deconvolution Analysis –Deconvolution retains quantitative accuracy –Improves contrast facilitating analysis Counting Tracking Area measurements Intensity measurements

12. Why does this work? The microscope is a well understood optical system The behavior of light is well modeled Persistence Why this works (1/5)

13. The Point Spread of a Microscope Every microscope- sample combination has a different point spread function. Every element in the optical path affects the point spread function: –Scope –Objective –Cover slip –Sample

14. The Point Spread of a Microscope Compare a widefield PSF to a confocal PSF

15. Looking forward to Deconvolution YFP Labeled Motor Neurons (Mouse) Laura Baylor, Harvard University Laboratory of Dr. Jeff Lichtman Emissive Wavelength Emissive Wavelength Numerical Aperture Numerical Aperture Spherical Aberration Spherical Aberration

16. Non-Blind deconvolution estimates the original object from the measured PSF and the collected data (2D or 3D image). Non-Blind Deconvolution Non Blind Deconvolution

17. Blind deconvolution separates the original object and the point spread from the collected data (2D or 3D image). Blind deconvolution Blind Deconvolution

18. What happens during deconvolution? How it’s done (8/8)

19. Non-Blind Deconvolution Reblurred Initial Object Estimate PSF Original Blurred - UpdateError Improved Object Estimate

20. Blind deconvolution

21. Initial Object Estimate Initial Object Estimate Blind Deconvolution PSF Original Blurry Data - Update Error PSF ? ? ? Iteration: 1 - PSF

22. Initial Object Estimate Initial Object Estimate Blind Deconvolution PSF Original Blurry Data - Update Error Object Estimate Initial Object Estimate ? ? ? Iteration: 1 - Object

23. Object Estimate Object Estimate Blind Deconvolution PSF Original Blurry Data - Update Error PSF ? ? ? Iteration: 2 - PSF

24. Blind deconvolution

25. AutodeblurImaging www.aqi.com