Approximate Time: 1 hour
Deconvolution is an image processing technique that is used to remove out-of-focus blur and haze from 3D fluorescence images, thus improving contrast and resolution. Blurring is caused by the spreading of light as it passes through microscope optics. Since blurring is a non random effect, it can be predicted with various optics models and if you can predict the effect, you may be able to reverse it to a certain extent. This is the basis of deconvolution. There are many different algorithms that are used to do this and they are discussed in the review paper by Wallace et al. cited below. With our computing power these days, running a deconvolution operation is easy and it's a reasonable way to sharpen up your images. James Pawley, author of the "Handbook of Biological Confocal Microscopy", recommends that you should always deconvolve your images whether their acquired in widefield or confocal mode.
Check out these references for more details:
- Wes Wallace, Lutz H. Schaefer, and Jason R. Swedlow."A Workingperson's Guide to Deconvolution in Light Microscopy." BioTechniques Vol.31: 1076-1097 (November 2001).
- Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006. (Chapters 23, 24, and 25 pp. 453-500).
Here at the AOMF, we provide training with Autoquant DeBlur deconvolution software. The course covers:
- Preparing images for opening in Autoquant
- Choosing deconvolution parameters: pixel size, z-spacing, excitation and emission wavelengths, etc.
- Choosing a deconvolution method: nearest neighbours, 3D blind deconvolution, etc.