Huygens Professional User Guide

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Deconvolving my first imageFigure 2. The Main Window with empty thumbnail images loaded by default.Thumbnail imagesStep 2: Load an imageThe Main Window is initially loaded with 4 black thumbnail images, images that are alreadyprepared for you, but where all image data are zero’s. One of the images is called ‘psf’ whichwill be used in the deconvolution steps as you will learn soon.Loading your imageFirst you have to load an image. Select ‘Open’ from the File menu in the Main Window andfind the file to be processed.In the distribution you will find the demo image faba32 (the file pair faba32.ids and faba32.ics,where faba32.ids is the file containing the raw data and faba32.ics is a header file, containingall microscope parameters). Click on one of these, no matter which one.You may combine steps 1 and 2: starting Huygens and loading images using a single commandline:huygens2 faba32.ics dama.ids&This command appends the images faba32 and dama to the Main window.If you have Tiff slices to be processed please read “Tiff file series naming convention” onpage 27 for the naming convention in order to be able to read a multi-dimensional image as awhole.Step 3: Inspect yourimage.Inspect your image.• Use the slicerExpand viewer Open the Expand viewer by pressing the Arrow-up button from the thumbnail offaba32. The Expand viewer is intended as a general purpose image inspection tool. It iscomposed of a menu bar and a tabbed deck consisting of a Projection page, a Slicer Page8 The Huygens Professional User Guide
Deconvolving my first imageand a Parameters page. You can open as many Expand viewers as you like, on the sameimage or on different images.image is not a timeseries.‘t’-slicing is notavailable.Figure 3. Two Expand Viewers opened from the same image. Left theSlicer Tab is selected, right the Projection Tab.With the Viewer’s Slicer you can scan through the slices (in different directions). Since thisexample image is not a time series, the time (t) select box is greyed.Microscopic parameters • Verify the microscopic parameters (needed for the generationof a PSF) like Numerical Aperture, Microscope type, samplesize, etc. by selecting the Parameters Tab from the Expandviewer (see Figure 3).Did you use the correct Sampling size? To find out, selectfaba32 in the Main window, by clicking on the image (thename is displayed in blue, when selected!). Now select‘Nyquist rate’ from the Edit menu and you will be informed.We see that the image is fairly good sampled in the x and y directions since the nyquist rateis near to the value of one. However, the z (the x-y slices) is undersampled (the samplingwas too course). Advised is to sample in the z-direction with a distance of 166 nm or less.Conclusion: if the Nyquist tool leaves you with values smaller then one you have undersampledyour image. Tough luck for you now, but hopefully future recordings may benefitfrom this information.(Super sampling of an image is not a problem, although your dataset becomes unnecessarilylarge without providing extra information).For now we continue with the z-undersampled demo image.The Huygens Professional User Guide 9
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Page 8 and 9: Installation and system requirement
Page 10 and 11: Installation and system requirement
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Page 18 and 19: The easy way• Press the Run butto
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Page 24 and 25: original imageFigure 13. Example of
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Page 28 and 29: d2rd 2=πr 2 dand thus r = ------π
Page 30 and 31: Biorad confocal microscopesTABLE 6.
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