NIS-Elements AR User's Guide NIS-ElementsAR Published06.2010 Copyright©1991-2010LaboratoryImagings.r.o. LaboratoryImagings.r.o.,ZaDrahou171/17,CZ-10200Praha10 Nopartofthispublicationmaybereproducedortransmittedexceptwiththewrittenpermission ofLaboratoryImagings.r.o. Informationwithinthispublicationissubjecttochangewithoutnotice.Changes,technicalinac- curaciesandtypographicalerrorswillbecorrectedinsubsequenteditions. User'sGuide Table of Contents 1.WelcometoNIS-ElementsARdocumentation....................................................................... 1 2.InstallationandStartup................................................................................................. 3 2.1.NIS-ElementsARInstallation................................................................................. 3 2.2.CommandLineOptions....................................................................................... 9 2.2.1.SyntaxandExample:................................................................................ 9 2.2.2.Switches:.............................................................................................. 9 3.ApplicationAppearance................................................................................................ 11 3.1.NIS-ElementsARScreen.................................................................................... 11 3.1.1.MenuBar............................................................................................ 12 3.1.2.ApplicationToolBars.............................................................................. 14 3.1.3.StatusBar........................................................................................... 14 3.1.4.DocumentWindow................................................................................. 15 3.2.ModifyingLayout.............................................................................................. 18 3.2.1.Layouts............................................................................................... 18 3.2.2.LayoutManager.................................................................................... 19 3.2.3.DockingPanes...................................................................................... 24 3.2.4.AppearanceOptions............................................................................... 27 3.3.Organizer....................................................................................................... 28 3.3.1.ImageFilter.......................................................................................... 29 3.3.2.OperationswithImages........................................................................... 31 3.3.3.SortingofImages.................................................................................. 32 3.3.4.GroupingofImages................................................................................ 32 3.3.5.AdjustingtheOrganizerLayout.................................................................. 32 4.SettingtheHardwareUp............................................................................................... 33 4.1.WorkingwithCameras....................................................................................... 33 4.1.1.LiveSignalfromCamera.......................................................................... 35 4.1.2.CapturingImages.................................................................................. 35 4.2.OpticalConfigurations....................................................................................... 36 4.3.Objectives...................................................................................................... 39 4.3.1.ManagingObjectives.............................................................................. 39 4.3.2.ObjectiveCalibration.............................................................................. 41 4.3.3.Units.................................................................................................. 43 4.4.UserManagement............................................................................................ 45 5.Capturing................................................................................................................. 49 5.1.SingleImageCapture........................................................................................ 49 5.2.ND2Acquisition............................................................................................... 50 5.2.1.Time-lapseAcquisition............................................................................ 51 5.2.2.Multi-pointAcquisition............................................................................ 51 5.2.3.Z-seriesAcquisition................................................................................ 54 5.2.4.Multi-channelAcquisition......................................................................... 55 5.2.5.CombiningNDimensions......................................................................... 56 5.3.CapturingtoRingBuffer..................................................................................... 61 5.4.CapturingAVImovies........................................................................................ 63 6.ImageDocuments...................................................................................................... 65 6.1.NIS-ElementsARDocuments............................................................................... 65 6.1.1.ImageLayers........................................................................................ 65 6.1.2.DocumentTypesinNIS-ElementsAR .......................................................... 66 6.1.3.ND2Documents.................................................................................... 67 iii NIS-ElementsAR 6.2.WorkingwithDocuments.................................................................................... 78 6.2.1.OpeningSavedFiles............................................................................... 78 6.2.2.SwitchingBetweenLoadedDocuments....................................................... 79 6.2.3.ClosingDocuments................................................................................ 79 6.2.4.CopyingChannelsbyDragandDrop........................................................... 80 6.2.5.ImageFormats..................................................................................... 81 7.ImageAnalysis........................................................................................................... 83 7.1.HistogramandLookUpTables............................................................................. 83 7.1.1.Histogram............................................................................................ 83 7.1.2.LUTs................................................................................................... 85 7.2.Thresholding .................................................................................................. 95 7.2.1.RGBMode........................................................................................... 95 7.2.2.HSIMode............................................................................................ 97 7.2.3.IntensityMode...................................................................................... 97 7.2.4.MCHMode........................................................................................... 97 7.3.BinaryLayer ................................................................................................... 98 7.3.1.Controls(horizontaltoolbar)..................................................................... 98 7.3.2.Drawingtools(verticaltoolbar).................................................................. 98 7.3.3.BinaryLayerColorandTransparency......................................................... 101 7.3.4.ErasingSingleobjects........................................................................... 101 7.3.5.MultipleBinaryLayers........................................................................... 101 7.4.Measurement................................................................................................ 102 7.4.1. Calibration ......................................................................................... 102 7.4.2.RoughMeasurement............................................................................ 102 7.4.3.ManualMeasurement........................................................................... 103 7.4.4.AutomatedMeasurement ...................................................................... 104 7.5.RegionsofInterest-ROIs.................................................................................. 106 7.5.1.ROIinTime-MovingROIs....................................................................... 107 7.6.MeasurementFeatures ................................................................................... 110 7.7.MathematicalMorphologyBasics ....................................................................... 124 7.7.1.Connectivity........................................................................................ 126 7.7.2.StructuringElement=Kernel=Matrix........................................................ 126 7.8.ObjectCount ................................................................................................ 131 7.8.1.ThresholdingtheImage......................................................................... 131 7.8.2.ApplyingRestrictions............................................................................. 131 7.8.3.Results.............................................................................................. 133 7.9.TimeMeasurement ........................................................................................ 135 7.10.Tracking(-TR-option) ..................................................................................... 137 7.10.1.AutomaticTracking.............................................................................. 137 7.10.2.ManualTracking................................................................................. 139 7.10.3.ObjectRedefinition............................................................................. 140 7.10.4.Visualization...................................................................................... 140 7.10.5.Measurement.................................................................................... 141 7.10.6.GraphProperties................................................................................ 143 7.11.UserMacros................................................................................................ 144 7.11.1.RecordingaMacro.............................................................................. 144 7.11.2.Writing/EditingaMacro........................................................................ 144 7.11.3.CreatingaMacrofromHistory................................................................ 145 7.11.4.SavingaMacro.................................................................................. 146 7.11.5.RunningaMacro................................................................................ 146 7.11.6.BreakingaMacro............................................................................... 147 iv User'sGuide 7.12.CreatingReports........................................................................................... 148 7.12.1.WorkingwithReportObjects.................................................................. 150 7.12.2.WorkingwithReportTemplates.............................................................. 152 7.12.3.CreatingReportsfromDatabase............................................................. 153 7.13.CreatingMovies............................................................................................ 155 7.13.1.CapturingAVIMovie............................................................................ 155 7.13.2.ConvertingND2toAVI.......................................................................... 158 7.13.3.CreatingAVIfromaSequenceofImages................................................... 159 7.13.4.MovieMaker(-LO-option) .................................................................... 160 7.14.ExportingResults.......................................................................................... 164 8. Cameras ................................................................................................................ 169 8.1.CommonSettings........................................................................................... 169 8.2.AdvancedSettings.......................................................................................... 172 8.3.CameraROIonLiveImage................................................................................ 173 8.4.ControllingCamerasbyMacro........................................................................... 174 8.4.1.HowtoSetCameraProperties................................................................. 174 9.Devices.................................................................................................................. 175 9.1.LogicalDevices.............................................................................................. 175 9.2.NikonEclipse90i/80iMicroscope...................................................................... 180 9.3.NikonAZ100Microscope.................................................................................. 181 9.4.NikonEclipseLVSeriesMicroscope..................................................................... 182 9.5.NikonEclipseL200Microscope......................................................................... 183 9.6.NikonEclipseMA200Microscope....................................................................... 184 9.7.NikonMM400/800Microscopes....................................................................... 185 9.8.NikonTE2000Microscope................................................................................ 186 9.9.NikonTIMicroscope........................................................................................ 187 9.9.1.UsingTIRecipe................................................................................... 188 10.AdditionalModules.................................................................................................. 191 10.1. Database ................................................................................................... 191 10.1.1.NewDatabase................................................................................... 191 10.1.2.NewConnection................................................................................. 192 10.1.3.DatabaseTables................................................................................ 192 10.1.4.UserPermissions............................................................................... 192 10.1.5.DatabaseBackup............................................................................... 193 10.2.ExtendedDepthofFocus................................................................................ 194 10.2.1.AlignSequence.................................................................................. 194 10.2.2.Createthefocusedimage..................................................................... 194 10.2.3.Viewtheresult.................................................................................. 194 10.2.4.EditAreainFocusedImage................................................................... 197 10.2.5.RealTimeEDF................................................................................... 197 10.3.Deconvolution(-LO-option) ............................................................................. 199 10.3.1.Deconvolution(Standard)..................................................................... 199 10.3.2.BlindDeconvolution............................................................................ 200 10.3.3.Fast(Wiener)Deconvolution.................................................................. 201 10.4.HighDynamicRange(-LO-option) ..................................................................... 202 10.5.DeconvolutionModule(legacy)......................................................................... 204 10.6.AutoQuantBlindDeconvolutionModule.............................................................. 206 10.7.AutoQuantReal-Time2DDeconvolutionModule.................................................... 209 10.8.Metalo-GrainSizeAnalysisModule................................................................... 212 10.8.1.FeaturesoftheGrainSizeAnalysisModule:.............................................. 212 10.8.2.QuickGuide...................................................................................... 212 v NIS-ElementsAR 10.9.Metalo-CastIronAnalysisModule.................................................................... 213 10.9.1.FeaturesoftheCastIronAnalysisModule:................................................ 213 10.9.2.QuickGuide...................................................................................... 213 10.10.CalciumandFRETmodule............................................................................. 214 10.10.1.CapturingFRETimage,FRETcalibration.................................................. 214 10.10.2.FRETMethod................................................................................... 217 10.10.3.CreatingFRETimage......................................................................... 217 10.10.4.[Ca2+]IonConcentrationMeasurement................................................. 218 10.11.TheAdvancedInterpreter(API)Module............................................................. 221 vi User'sGuide 1. Welcome to NIS-Elements AR documentation. ThishelpfiledescribescapabilitiesoftheNIS-ElementsARlaboratoryimageanalysissystem.Chaptersand paragraphsmarkedbythefollowingsymbolsdescribefeaturesavailableonlyifcertainmoduleordevice wasinstalled. (-A1-option) Requiresthe[NikonA1ConfocalMicroscope]device. (-DB-option) Requiresthe[Database]module.See[Database]. (-DEC-option) Requirestheoneofthe[Deconvolution]modules. (-EDF-option) Requiresthe[EDF]module.See[ExtendedDepthofFocus]. (-FRET-option) Requiresthe[CalciumandFRET]module. (-LO-option) Requiresoneofthe[LocalOption]featurestobeinstalled.SeeStep2. (-MET-option) Requirestheoneofthe[Metallography]modules. (-NDA-option) Requiresthe[AdvancedNDAcquisition]module (-TR-option) Requiresthe[2DObjectTracking]module.See[Tracking(-TR-option)]. (-6D-option) Requiresthe[6DAcquisition]module 1 NIS-ElementsAR 2 User'sGuide 2. Installation and Startup 2.1. NIS-Elements AR Installation QuickStart • InserttheinstallationDVDintheDVD-ROMdrive.Awindowautomaticallyappears. • InstalltheselectedNIS-ElementsARsoftwareversion,additionalmodules,anddevicedrivers. • PlugtheprovidedHASPkeyintotheUSBportofyourPC. • RunNIS-ElementsAR TheInstallationDVD-ROMContent TheinstallationDVD-ROMcontains: • NIS-ElementsARsoftwaresetupfile • DriversandutilitiesfortheHASPKey • Driversforselectedcameras • User'sGuideinthePDFfileformat • Sampleimagedatabase • SampleND2imagesequences TheInstallationProcess Note YouhavetopossesstheadministratorrightstoyourcomputertobeabletoinstallNIS-Elements ARsuccessfully. 3 NIS-ElementsAR 1) WhentheinstallationDVDisinserted,aselectionwindowappearsautomatically. Selectthesoftwarepackagetobeinstalled.Selecttheoneyouhavegotthelicenseforandwhichis properlycodedinyourHASPkey.Theinstallationwizardwelcome-windowappears.Click[Next]to continue. 2) Selectwhethertoinstall[LocalOption]ornot.DefinethefolderwhereNIS-ElementsARshouldbe installed.Werecommendtousethepredefineddirectory.Ifyouwanttochangethedirectoryanyway, pressthe[Browse]buttonandselectanewone.Otherwiseclick[Next]. Note The[LocalOption]installationprovidessomeadvancedfeatureswhichdidnotpassthe qualityassuranceprocedureyet.Werecommendtowaituntiltheyarereleasedofficially. 4