This link might be helpfull:

http://imagej.net/Deconvolution

http://imagej.net/Parallel_Iterative_Deconvolution

Couldn't try on my own so far, but that might work.

First create the PSF-file using your exact settings (mounting, oil, objective, NA,...) and use this for the deconvolution.

Hi Asmita,

Here is a short citation issued from the following paper: http://imagejdocu.tudor.lu/lib/exe/fetch.php?media=tutorial:working:workshop3d2010.pdf

"Two plugins can be used in ImageJ to perform 3D deconvolution, DeconvolutionLab and Parallel Iterative Deconvolution.

They both need a stack containing the PSF, they are based on iterative algorithms, that is to say they first compute the estimated original signal. Then this estimated original signal is convolved with the PSF, so it should reproduce the deformation induced by the system and give the actual image. Different algorithms are then used to minimise this difference. A commonly used algorithm in fluorescence microscopy is Richardson-Lucy".

Check also for these following links:

http://pages.stolaf.edu/opn-lab/files/2016/12/Giannini_Giannini_Deconvolution_Manual_20161215.pdf

http://bigwww.epfl.ch/deconvolution/

Best regards

Just a quick note: make sure you are working with properly sampled z-stacks (that means proper pixel size and z-step. Deconvolution does not work with singe image. You need the whole z-stack (even a bit wider than the structure you're interested in is helpful).

https://svi.nl/NyquistCalculator

thanks for all your answers...i will try them myself and let u know if they work well 

I would not recommend doing deconvolution using ImageJ. This will be a waste of your time, ultimately. It can be done with ImageJ but not very smoothly or accurately. Better use a confocal microscope where deconvolution is done at the optical level. Good deconvolution software such as "Autoquant" (in my view the best so far) are quite expensive.

The point spread function (PSF) is the hardest to get. Without a good PSF deconvolution is meaningless.

Regards

To complement Nurul Kabir's recommendation, when deconvolution is not available at the optical level, depending on the confocal microscope, what is critical is to choose the deconvolution tool adequately.

The results depend greatly on the deconvolution algorithm implementation and its capability to estimate the PSF accurately. So, what is important is to compare state-of-art deconvolution algorithms.

To this end, here-are some useful links:

In addition, here is an excerpt from Pompey et al., 2013: https://www.google.fr/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwi7t-SMsfvRAhWEOxoKHae_Ab4QFggeMAA&url=http%3A%2F%2Fwww4.utsouthwestern.edu%2FLiveCellImagingFacility%2Fdocs%2FPhelps_Colocalization%2520Chapter_MethMolBiol.pdf&usg=AFQjCNFQ09uJ9fZtSJl-toStPjKZ48ODFA

" Four commercial packages with very sophisticated colocalization capability are Huygens (SVI, Inc), Imaris (Bitplane), Autoquant X (Media Cybernetics) and Volocity (Perkin Elmer). In addition, co-localization plugins are available for the open source java application ImageJ (http://rsbweb.nih.gov/ij/) and its various distributions, including Fiji (http://pacific.mpi-cbg.de/wiki/index.php/Fiji). Two plugins that implement all the co-localization methods described above are JaCoP (ImageJ) and Coloc_2 (Fiji)."

You can also follow this related question on researchgate: https://www.researchgate.net/post/Can_you_recommend_deconvolution_software_for_ImageJ1

For interested readers, another yet reference of interest about "Deconvolution Microscopy" algorithms by Sibarita, 2005:

http://www-sante.ujf-grenoble.fr/medecine/iab/clientzone/plforme9/fichiers/DeconvolutionMicroscopy_Sibarita_Springer.pdf

Thanks Stéphane Breton for your helpful comments.

I agree that Deconvolution, if done properly and with a good software is even better than a confocal microscope, at least in theory. However, the problems with algorithms and the PSF is just too much for a biologist like me. This is why many biologists choose the easier option- optical deconvolution by a confocal, that is, removing the out of focus information by decreasing the pinhole  and optical slicing . Also the time one have to spend for finding a good PSF and the 3-D stack calculations are huge.

Having said that it would be nice to know if there is some really reliable software for deconvolution. I consider many software related to this including imageJ as not more than the 'developing' stage.

Regards

thanks to everyone....all your suggestions were very helpful...i tried doing the deconvolution in ImageJ using an experimental PSF and it worked perfectly fine and my images look great now. Thanks a lot once again for all the valuable suggestions.

Hi Asmita,

could you eventually send a link for the plugin you used? Would like to test it for my images as well.

Thanks a lot!

Hi Lukas,

I am sending you the link below which contains a complete description of the procedure for performing deconvolution using ImageJ as well as has links for the plugins necessary for doing the same. I hope it helps you. In case you need any help feel free to ask.

https://imagej.net/Deconvolution

Hi Asmita,

I am learning to deconvolve my images using ImageJ and I had a few questions about it.

1. Does the type of image which can be deconvolved has to be Z - stack image?

2. What is the plug in you used in ImageJ for the deconvolution?

Thanks a lot

Sreyashree Bose

yes one generally deconvolves z-stack images. You can first acquire a z-stack of a stuck bead under the same magnification to generate the PSF. Open the image z-stack after this and then go to the 3D-Deconvolve function. Select the image stacks there: one sample stack and the other PSF genrated. Play with the parameters and you will get your deconvolved image from this.

I am skeptical of blind deconvolution in general (estimating the entire PSF image using an iterative process) however the method Ferreol suggests, is a parametric approach. It would be very interesting to see more results and understand the PSF and image co-convergence. The implication that "ImageJ is not appropriate for deconvolution" is not correct. ImageJ/Icy and other open source programs can produce results as good or better as commercial deconvolution if used properly. In fact in the most recent Deconvolution Grand Challenge, one of Ferreol's algorithms beat all the commercial entries (the best commercial entry was actually the Olympus implementation by David Biggs). In a recent paper, several algorithms from Deconvolutionlab2 were tested against Huygens and produced comparable results (Article DeconvolutionLab2: An Open-Source Software for Deconvolution...

I encourage anyone interested in ImageJ and/or Icy deconvolution implementations to post questions on the respective listservs and forums, and share images. If you interact with the developers and community you will be able to obtain very good results.

Hi, I'm new to all this and I have my dry x40 images from a confocal microscope (Zeiss Axio Observer). I want to deconvolve them on ImageJ but I don't know where to get all the required information from to generate a PSF. It asks for a wavelength, which wavelength is that? I've checked the microscope manual but I'm not sure if I've entered the correct details. I would prefer doing a blind deconvolution but I don't know of any plugins for doing this on ImageJ.

Omer Mohamed , use the position of emission maximum of your fluorophore. For GFP that would be roughly 510 nm.

Just to correct some things that I read around here by quickly scanning through: confocal microscopy in itself does NOT provide "optical deconvolution". The is no thing as an "optical deconvolution". You can only deconvolve by an algorithm. The reason is that any point in your image is a convolution (a specific type of relationship that reduces to multiplication once you apply Fourier transformation to the signal) of the actual signal and the PSF (point spread function - which describes exactly how a point is spread in your image).The PSF is inherent to the microscope/optical system that you are using and originates in physical limitations of light-matter interactions. Deconvolution uses the measured/theoretical PSF to compute the original image. It should be quite clear from this that you do NOT need a z-stack for a deconvolution algorithm to work. And you can also deconvolve images from a confocal microscope. Getting rid of the signal from other focal planes by using a confocal aperture and applying deconvolution are two completely different things.

That being said, if people do not understand the underlying physics and principles of deconvolution, they should not be using it. Otherwise they risk creating artifacts in their images leading to false evaluation of data.

Yes, you are right that the light from the other planes affects the light in your plane of interest and you should take it into account when performing a deconvolution on images from a non-confocal microscope to get the best results. On a confocal image, 2D will be sufficient.

Note: the term "epifluorescence" describes the light path in your system, not confocality. Confocal microscopes also use epifluorescence.

Patrice Mascalchi the point of confocal microscopy is that you only acquire the light originating in the focal plane. The pinhole filters out the light originating in all other planes. As such, your image is a convolution of the real signal from the focal plane and PSF, but only that part of it that is in the focal plane, i.e., it's in effect 2D.

For fluorescence microscopy in general (i.e., non-confocal set-up), however, it is indeed better to have a 3D PSF, as the light you acquire is combined from the light of not only your focal plane, but also of planes above and below.

That being said, I measured a z-stack of hyphal cells using a general fluorescence microscope, used 3D deconvolution within the original Zen software (Zeiss) and then made a max projection in ImageJ. I compared it with an image I obtained by first making the max projection of the original z-stack in ImageJ followed by 2D deconvolution in ImageJ. The first option provided a very slightly better result, but the difference wasn't evident at first sight.

Ferréol Soulez Hi I am trying to use Icy. It is great thank you for your work. However, it looks the 2D confocal image can not be used in this software. Other software such as Huygens or DeconvolutionLab plugin in ImageJ can do it.

It will be better if this function can be improved.

Anyway, this is a great software.

Best regards.