Huygens Professional User Guide

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2 The Huygens Professional User GuideDo not undersample to limit photodamage 22Bleaching 22Illumination instability 22Mechanical instability 22Thermal effects 23Internal reflection 23Computing the backprojected pinhole radius 23Airy disk as unit for the backprojected pinhole 23Converting from integer parameter 23Airy disk as unit for the backprojected radius of a square pinhole 24Computing the backprojected pinhole distance in Nipkow spinning disks 24Pinhole radius tables 24Leica confocal microscopes 24Zeiss confocal microscopes 25Olympus confocal microscopes 25Biorad confocal microscopes 26A supplied calibration curve 26Questions 26What does the quality factor mean while running Huygens? 26Can I deconvolve a Tiff series? 27Tiff file series naming convention 27Can I deconvolve a single plane widefield image? 28Can I deconvolve a single Tiff image? 28
CHAPTER 1IntroductionThis guide is useful to the beginner in deconvolution and to the expert that startsusing the Huygens Professional toolkit.For the expert we recommend also to study the Huygens Reference Manual.contentsIn chapter 1 we explain the installationIn chapter 2 you deconvolve your first imageIn chapter 3 we help you determining some important parameters and give you hints for basicimage data-acquisitionWhat is Huygens Professional?Huygens professional is an image processing software package tailored for deconvolution ofmicroscopic images. It enables you to deconvolve a wide variety of images ranging from 2Dwidefield (WF) images to 4D multi-channel two-photon confocal images or from Scanningdisk confocal microscopes. Also people who use experimental set-ups like 4p-microscopesmay benefit from the software. The Huygens Professional toolbox contains the following features:Image deconvolutionfunctionsBasic image processingcapabilities• Accelerated Maximum Likelihood Estimation restoration algorithm optimized for low light levelimages• Iterative Constrained Tikhonov-Miller restoration algorithm• Quick Tikhonov-Miller and Quick MLE restoration algorithms• Point Spread Function (PSF) measurement tool box to derive a microscopic PSF from finite sizedmicro bead images, containing:• Automatic alignment and averaging procedure to combine the signal from different micro beads in oneor more images• PSF reconstruction tool to correct for the finite size of micro beads• Generates a theoretical Point Spread Function for widefield, confocal and two-photon microscopesbased on electromagnetic diffraction theory• Automatic bleaching correction of 3D and 4D widefield images and 4D confocal and multi-photonimages• Z-drift corrector tool for timeseries that enables you to correct for movement in the Z (axial) directionthat could have been occurred for instance by thermal drift of the microscope table.• Capability to handle multiple images• Time series support• Multi parameter (multi channel) image elements (stacked or packed)• Basic data types: unsigned byte, 16 bit signed integer, 32 bit float, 2 x 32 bit complex• Per image undo/redo capabilities• Scripting and batch processing environment based on Tcl (Tool Command Language)The Huygens Professional User Guide 3
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Page 12 and 13: Deconvolving my first imageFigure 2
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