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A Practical Guide To Deconvolution
by M. Messerli
This guide is based on the software product Huygens which is developed by Scientific Volume Imaging B.V. and distributed world-wide by Bitplane AG.
1. Acquiring Images For Deconvolution
SAMPLING:
Deconvolution can increase the resolution of your image, especially along the z-axis. Therefore the structure needs to be scanned at finer intervalls than you would normally use without deconvolution. The web calculater below gives you sampling guidelines:
-> Web-Calculator
If you need more information on sampline we invite you to read the following article.
INTENSITY:
As important than the spatial sampling is the correct measurement of the intensities. Huygens need to have accurate information about the relative intensities of the measured structures. Therefore compliance with the following rule is crucial:
-> no voxels should have the max intensity value and not voxel should have the min intensity value
For an image which has 8 bit dynamic range (256 grays) no voxel should be 255 (because it could be well over 255) and no voxel should be at 0 (because it could be well below). With an image which has voxel intensities between 1 and 254 be are sure to have adequately represented each of the voxel
in the image.
2. Parameters Needed For Deconvolution
Before you can use the Huygnes the following parameter must be known. Many of the parameters listed below may be initialized correctly when loading an image into the Huygens system. However, we recommend to verify all parameters prior to deconvolution using the Edit:Parameters function in Huygens.
A. Parameters needed for deconvolution using synthetic (calculated) point-spread function:
PARAMETERS FOR ENTIRE IMAGE
1. x-dimension
2. y-dimension
3. z-dimension
4. voxel x-dim
5. voxel y-dim
6. voxel z-dim
7. NA of objective lens
8. refractive index of immersion medium
9. refractive index of mounting medium
PARAMETERS NEEDED FOR EACH CHANNEL (COLOR)
10. excitation wavelength
11. emission wavelength
12. number of excitation photons (1 for single photon excitation, 2 for 2 photon excitation)
13. pinhole radius backprojected onto sample plane in nm
Parameter 13 often is not available as such but needs to be calculated from the physical size of the pinhole and the total magnification of the micrsocope between the sample plane and the pinhole. A table lists translation factor for some of the commercial instruments:
->pinhole conversion table
B. Parameters, needed for deconvolution using measured point-spread function:
14. 3D image of beads, 512 x 512 x 40 (or larger)
The beads must not be too close to one another and must not be too close to an edge of the data cube. It often happens that the beads are centered in one place but are too close either to the top or the botto of the volume image. A collection of 4 to 6 beads in an image of the size described above has been proven useful.
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