Deconvolution | Deconvolution

Key Words: confocal, fluorescence, Resolution, signal-to-noise ratio

Definition:Deconvolution is a technique that either subtracts or reassigns out-of-focus blur in fluorescence microscope images to improve image clarity

TECHNOLOGY:

All fluorescently-labeled structures in a specimen will emit light on excitation with light of appropriate wavelengths - whether they are in the plane of focus or not. Out-of-focus contribution to an image can complicate an image and result in loss of contrast - especially when using objectives with a high resolving power, for example, high N.A. oil immersion objectives.

Deconvolution processing seeks to remove or reassign out-of-focus blur by mathematically processing planes (optical sections) through a digital image. Deconvolution of widefield fluorescence images can provide results comparable to confocal imaging techniques. Digital convolution can, in addition, be used to further enhance optical sections acquired by confocal microscopy and is useful in improving clarity by reassigning out of focus blur in 3-D images constructed from stacks of optical sections.

A variety of algorithms are commercially available for 2D or 3D deconvolution, which can be classed as non-restorative or restorative. The former improve contrast by removing blur, while restorative methods re-assign light to its point of origin.

APPLICATIONS:

Deconvolution can be applied retrospectively to any digital fluorescence image to improve interpretation. It can be applied during image acquisition. One of the advantages of deconvolution is that it can be carried out with very low illumination and, therefore, enables imaging of light sensitive living cells for longer periods compared with laser scanning confocal imaging.

MICROSCOPE CONFIGURATION:

Deconvolution software can be applied to the processing of any digitally acquired image.

Nikon uses Autoquant software modules in its NIS elements software. The NIS elements file format can be read by Autoquant independent software as well as Scientific Volume Imaging's Huygens software package. Both software packages are regarded as advanced packages for deconvolution.

RECOMMENDED SYSTEM:

The mutiport design of the Eclipse Ti series of microscopes enables the use of standard epi-fluorescence techniques, high-resolution deconvolution imaging or confocal imaging. Deconvolution software is an optional module available with NIS Elements software.

LINKS:

Confocal tutorial with advice on when to use confocal, deconvolution or multiphoton techniques