Huygens Professional User Guide

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Deconvolving my first imageHistogram • The image histogramSelect the image in the Main Window and selectImage histogram from the Edit menu. An image histogramis created as a thumbnail image namedfaba32:histo. You may use the Expand viewer for abetter view. The histogram enables you to inspectthe quality of the image visually. The one inFigure 4 is of reasonable quality: No sharp peaks atthe extreme sites of the histogram image indicatingclipping. An example of a clipped image is shownbelow.Not also that the black space at the left hand of thehistogram. It signifies an electronic mismatch in therecording of the image. See “Blacklevel” onpage 20.A clipped image example, see Figure 5.If you see a narrow peak at the left hand side of thehistogram this signifies ‘clipping’. It occurs whennegative input signals are mapped to zero’s by theCCD camera. See also “Clipping” on page 22.If a peak is visible at the extreme right hand side ofthe histogram it indicates saturation. Saturation is atype of clipping caused by raising the laser intensityabove the maximum pixel value available on yourmicroscope. Usually, all values above the maximumvalue are replaced by the maximum value. On rareoccasions they are replaced by zeroes. Clipping willhave a negative effect on the results of deconvolution,especially with WF imagesFigure 4. The image histogramfrom the exampleFigure 5. The image histogramfrom a clipped image. A spike isseen at the left hand site.Operations windowIn the former steps all operations were performed without any operation parameters (functionsapplicable on a particular image). For example if one wants to know the image statistics noextra information is needed but the name of the image. All these types of operations are accessiblefrom the main Window. Most operations, however, need extra input data. For exampleadding some constant value to an image. This can be done in the Operations WindowSince in the next steps all image processing operations need one or more parameters you haveto work with the Operations window. You open an Operations window on an image by clickingon the small button labelled O in the images’ Thumbnail component.10 The Huygens Professional User Guide
Deconvolving my first imageSubsequently you select an operation from the Menu bar or from the Quick access buttons. TheMenu barQuick access buttonsFigure 6. The Operation window with the PSF buttonpressed.left side of the operation window is similar to the Expand viewer. At the right side you will findthe parameter entry area. By editing the parameters you control the way the function operates.To execute the operation, press the RUN button.bleaching correctionIntermezzo: check for bleaching correctionRemember, we were inspecting our image.This extra check is given here for the sake of completenessfor those using this schematic approachfor widefield images or confocal time series. Select‘Plot Flux’ from the ‘Analysis’ menu in the Operationwindow. A typical example is shown inFigure 7. The Demo image is a regular confocalimage: bleaching correction test makes no sensehere!• Extra check to see if bleaching correction isneeded in widefield images or confocal timeSeries. While recording widefield images orconfocal time series strong bleaching mayoccur.Figure 7. A typical example of ableached widefield image. Use the‘Plot Flux’ tool from the Analysismenu available in the Operationwindow.The Huygens Professional User Guide 11
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