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

Improving signal to noise

What is it? (1/6) Widefield – Single Channel What is deconvolution?

Multiple wavelengths - Widefield

Confocal Microscopy

2D Blind deconvolution

2D Blind deconvolution – TIRF

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

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

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

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

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)

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

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

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

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

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

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

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

Blind deconvolution

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

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

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

Blind deconvolution

AutodeblurImaging