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Journal Article

Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes

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Heintzmann,  R.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Hanley,  Q. S.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Jovin,  T. M.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Citation

Heintzmann, R., Sarafis, V., Munroe, P., Nailon, J., Hanley, Q. S., & Jovin, T. M. (2003). Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes. Micron, 34, 293-300. Retrieved from http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T9N-48YVKVJ-5-1R&_cdi=5119&_user=38661&_pii=S0968432803000544&_orig=search&_coverDate=10%2F31%2F2003&_sk=999659993&view=c&wchp=dGLzVlb-zSkzV&md5=aee87ac9481ef91cd17e5ecbac8e91a2&ie=/sdarticle.pdf.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0012-F1C2-A
Abstract
Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably scaled widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a suppression of low spatial frequencies leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal to noise ratio (SNR). The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented.