Handling multi-dimensional images with Sync_Windows and Image5D

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1 Handling multi-dimensinal images with Sync_Windws and Image5D Jachim Walter a, Kathrin Teller b and Felix Habermann c* a TILL I.D. GmbH, c\ BiImaging Zentrum, Grßhaderner Str. 2, Martinsried, Germany; b Department f Bilgy II, University f Munich, Martinsried, Germany; c Institute f Veterinary Anatmy II, University f Munich, Munich, Germany ABSTRACT ImageJ by itself handles image data with up t three dimensins. That means that 3D image stacks r time series f 2D images can be viewed and prcessed. A number f plugins are available t handle image data with multiple clrchannels, slices and/r time-pints. Tw f these tls are presented here in detail: SyncWindws and Image5D. Sync_Windws synchrnizes cursr mvements, muse clicks and mvement f the slice selectr between selected images and image stacks. When images frm multiple clr-channels are synchrnized, the same regin f interest can be selected and measured in all channels. An extensin f SyncWindws, SyncMeasure3D, allws simultaneus measuring f vlumes and centers f hand-selected 3D-bjects. It is als useful fr measuring the shift between images f an bject in different clr channels ( chrmatic shift ). Image5D allws brwsing f time series f multi-channel image-stacks. The clr channels can be verlaid in selectable clrs and quickly switched n and ff. Existing plugins see and prcess the currently selected z-stack f an Image5D as a nrmal image-stack. Additinally, there are plugins t make prjectins and mntages frm all r selected channels. Keywrds: multi-channel, stack, sequence, time-series, display, analysis, chrmatic shift 1. INTRODUCTION Mdern micrscpic imaging systems enable their users t acquire three-dimensinal image data f their specimen. Data frm thse instruments can cmprise multiple clr channels (up t 32 in cnfcal micrscpes frm Zeiss and Nikn). Furthermre, time-series f such 3D multi-channel data can be btained. Visualizatin and, t a variable degree, prcessing f such data can be dne with the cntrl sftware supplied with the micrscpe systems. Fr ffline wrk with the data, ften additinal licenses f this sftware can be btained. Sftware fr ffline-analysis can als be purchased frm independent sftware cmpanies. Mrever, mst micrscpe manufacturers ffer free versins f their acquisitin sftware with a reduced functinality. ImageJ 1 has established itself as a free alternative t cmmercial scientific image prcessing sftware, and in many respects surpasses the functinality f available cmmercial sftware when the apprpriate plugins are installed. ImageJ s capability t handle multi-dimensinal data is limited. Tw-dimensinal images and stacks f tw-dimensinal images are supprted, where the stack can either be a z-stack, a time series r a clr (RGB) stack. Up t three channels f 8-bit data can als be addressed by the RGB image type. Thus, ImageJ can at mst address z-stacks r time-series f up t three 8-bit clr channels. T vercme this limitatin, several plugins have been develped. The UCSD cnfcal micrscpy plugins 2 pen and view multi-channel 12-bit images acquired with Olympus Fluview cnfcal micrscpes and quickly select and deselect certain channels fr display. They include the SyncWindws_ plugin, which synchrnizes cursr mvements and muse clicks in selected windws. The HyperVlumeBrwser plugin 3 interprets the additinal dimensin f an image stack as cmpsed frm tw extra dimensins instead f ne, and prvides tw scrllbars t brwse these dimensins. The WiscScan Data Brwser 4 is adapted frm the HyperVlumeBrwser fr pening and brwsing f data * Further authr infrmatin: (Send crrespndence t J.W.) J.W.: crrespndence@walter-witzenhausen.de, Telephne: K.T.: Kathrin.Teller@lrz.uni-muenchen.de F.H.: f.habermann@anat.vetmed.uni-muenchen.de

2 acquired with WiscScan laser-scanning micrscpes. The Image Layering Tlbx 5 verlays multiple 2D-images with selectable clrs fr each image. Here, we present tw plugin-cllectins maintained by ne f us (J.W.) fr displaying and analyzing multidimensinal image data. Sync_Windws/Sync_Measure_3D can be used t perfrm measurements synchrnusly in several grayscale z-stacks interpreted as clr channels f a multi-channel data-set. Image5D is a new image type fr ImageJ that hlds time series f z-stacks with an arbitrary number f channels f 8-bit, 16-bit r flating pint image data. A cllectin f plugins (the Image5D-plugins ) pen, save and d prcessing f Image5Ds Sync_Windws / Sync_Measure_3D 2. METHOS AND RESULTS Sync_Windws is an enhanced versin f the SyncWindws_ plugin by Partrick Kelly, which is part f the UCSD cnfcal micrscpy plugins package. With Sync_Windws ne can synchrnize cursr mvements and muse clicks in multiple images. This can be used t cnveniently select the same regin f interest (ROI) in different clr channels f the same data set. Sync_Windws als synchrnizes the displayed z-slice in image-stacks. Sync_Measure_3D extends SyncWindws and adds the ptin t measure 3D-gravity centers, vlumes and intensities f signals in synchrnized stacks Installing Sync_Windws and Sync_Measure_3D T install Sync_Windws/Sync_Measure_3D, dwnlad the file Sync_Win.jar frm the ImageJ plugins page ( place it int the plugins directry f yur ImageJ installatin and restart ImageJ. The plugins are accessible in the Analyze/Tls submenu Brwsing multi-channel data with Sync_Windws - Open the images t be synchrnized. - Run Sync_Windws frm the Analyze/Tls submenu f the ImageJ menubar. - The Sync_Windws cntrl windw pens (Fig. 1). It cntains the fllwing elements A list with names f all pen image windws, in which windws can be selected by a single click n the respective names. A checkbx Synchrnize Cursr t select, whether cursr mvements and muse-clicks are synchrnized in the selected windws. A checkbx Synchrnize z-slices t select, whether the current slice shuld be the same in all stacks. A checkbx Image Crdinates t select the type f synchrnizatin f the cursr. If the checkbx is selected, cursrs in synchrnized windws are at the same psitin in the image relative t the crdinate system f the image, even if the synchrnized windws have different zm factrs r different regins f the images are zmed ut. If the checkbx is deselected, cursrs in synchrnized images are at the same psitin relative t the crdinate system f the screen. Tw buttns Synchrnize All and Unsynchrnize All fr selecting/deselecting all windws at nce fr synchrnizatin. Figure 1. The Sync_Windws cntrl windw. Left: n windw synchrnized, right: all windws synchrnized.

3 2.1.3 Ding Measurements with Sync_Measure_3D The use f Sync_Measure_3D will be explained using an example. Measuring f the chrmatic shift f a micrscpe requires psitin measurements (intensity gravity centers) f flurescent beads imaged in the different clr channels f the instrument. In this example, the same beads appear in three clr channels. Their psitins in the different channels are slightly different due t chrmatic aberratins f the micrscpe bjective. - Open the images f all three clr channels and specify their pixel height/width and vxel depth in the Image/Prperties dialg. - Run Sync_Measure_3D frm the Analyze/Tls submenu. - Select all windws fr synchrnizatin. - Press the Start Measurements buttn. A panel with a slider fr every synchrnized windw appears. (Fig. 2) - Select a suitable threshld fr each f the synchrnized windws using the sliders. (Fig. 2) - T measure the psitins f a bead image in all image stacks simultaneusly, draw a ROI arund the bead f interest and duble-click int the ROI. A text windw that cntains the results f the measurement in ne line appears. - Measure the psitins f the ther beads. A line fr every measurement is added t the results windw. Fr every image stack the line cntains the intensity weighted center f the measured bead, the vlume and ttal intensity f the bead, the selected threshld, and the distance f the bead t all preceding beads. - Press Stp Measurements. The threshld sliders disappear, and tw mre lines appear in the results windw (Fig. 3). Fr every stack the first line, labeled Shift, cntains the average shift relative t the first stack (in this case the Cy3 stack) in x-, y- and z-directin. Psitive values mean that the image in the respective stack is mved t the right, dwn and t higher slice numbers relative t the reference stack (i.e. the first stack). Figure 2. The Sync_Measure_3D cntrl windw and the three image stacks crrespnding t three clr channels. All windws are synchrnized, measurement is started and the threshld fr the first image (Cy3-Stack) is selected.

4 Figure 3. The Sync_Measure_3D results windw fr the measured three image stacks. Sync_Measure_3D can als be used t measure 3D-distances between nn-identical bjects in different clrchannels. Fig. 4 shws, hw the psitins f tw centrmeres in nuclei f human lymphcytes are measured. The Synchrnize Cursr and the Synchrnize z-slides checkbxes in the cntrl windw are deselected. Therefre, ROIs and the slices fr drawing them can be independently selected in the three stacks. Measurement are perfrmed as in the synchrnized case by duble-clicking int ne f the ROIs. Fr further analysis, the data can be cpied t the system clipbard by selecting Edit/Cpy All in the menubar f the results windw. Frm the clipbard the data can be inserted int a spreadsheet windw. Figure 4. tp: The Sync_Measure_3D cntrl windw with the Synchrnize Cursr checkbx deselected. bttm: Image stacks f the nuclear cunterstain ( tpr ) and the tw centrmere signals ( cen1, cen2 ). Fr explanatins see text.

5 2.2. Image5D Viewing Data with Image5D Image5Ds are displayed in a windw that displays x/y sectins f the Image5D at the currently selected slice, frame and channels (see Fig. 5). Belw the image, there are sliders fr selecting the displayed z-slice, and time-frame. T the right f the image there is a cntrl fr selecting the displayed channel r channels ( channel-cntrl ). The channel-cntrl cntains a drpdwn bx fr changing between three mdes t display channels: grayscale, clr, and verlay mde. In grayscale mde, a single channel is displayed at a time in gray. In clr mde, each channel is displayed with its clrmap applied. It is pssible t assign a different clrmap t each channel. A vertical scrllbar is used t select the currently displayed channel in bth mdes. In verlay mde, all channels are displayed in the windw, each channel shwn with its clrmap applied. There is a radibuttn (rund) and a checkbx (square) fr each channel. The backgrund f these cntrls is filled with the clr crrespnding t the currently selected clrmap, and the label f the channel is displayed t the right f the checkbx. The checkbxes are used t tggle the display f the respective channel. Deselecting a checkbx switches its assciated channel ff. Pressing the buttn All abve the cntrls fr the first channel switches all channels n, and the buttn Nne switches all channels ff. T plugins, which are nt designed fr using Image5D, an Image5D appears as a grayscale image-stack, which cntains all the z-slices f the currently selected clr-channel at the current time-frame. E.g. the Image/Stacks/Z_Prject cmmand f ImageJ des a z-prjectin f the current clr-channel. In grayscale and clr mdes, the current channel is selected by the channel-scrllbar. In verlay mde, the radibuttns are used t select the currently active channel, althugh a current channel is nt necessary fr managing the display in verlay mde. In clr and verlay mde, clicking the Clr buttn n the very tp f the channel-cntrl pens a panel, which allws a cnvenient selectin f the clrmap fr the current channel ( clr-chser ). The biggest part f the clrchser is ccupied by a field, which cntains little squares in different clrs. Clicking n ne f the squares assigns the clr f this square t the current channel. T the left f this field, the current clrmap f the channel is displayed. Abve the field, there is a checkbx Grayscale. Selecting this checkbx temprarily switches the display f the current clr-channel t grayscale. When the checkbx is deselected, the previus clrmap is restred. The buttns Edit Clr and Edit LUT pen dialgs fr selecting the clr with RGB-sliders r editing the clrmap in detail t btain e.g. a rainbw-clrmap. Figure 5. Image5D f a FISH-stained chicken lymphcyte cell. Left: displayed in clr mde with the clr-chser panel pened; right: in verlay mde ( vl ), clr-chser panel clsed.

6 Installing Image5D Dwnlad the file Image5D.jar frm the ImageJ plugins page ( place it int the plugins directry f yur ImageJ installatin, and restart ImageJ. The Image5D plugins are accessible in the Plugins/Image5D submenu Creating, Opening, and Saving Image5Ds T create an Image5D, use the plugins "Open Series as Image5D", "Stack t Image5D" r "RGB t Image5D". Open Series as Image5D pens a series f images r stacks frm hard-disk and cnverts it t an Image5D. Stack t Image5D cnverts an already pened stack t an Image5D, and RGB t Image5D cnverts an RGB-image r stack t an Image5D. Image5Ds can be saved t hard-disk with the plugin Save Image5D and reladed with Open Image5D. Image5Ds are stred as ImageJ TIFF-stacks with sme extra metadata. Clrmaps fr each channel are retained during saving and lading. Ordered series f (2D-) images can be pened as Virtual Image5D. In this mde, nly the currently displayed images are laded int memry. All the ther images are nly present n the hard-disk. In this way, large stacks r time series can be pened and brwsed, that wuld exceed the size f available RAM Prcessing Image5Ds As explained abve, plugins which are nt designed t use Image5Ds treat them as image-stacks. Fr example, the Image/Stacks/Z_Prject functin and the Image/Stacks/Make_Mntage functin f ImageJ make z-prjectins and mntages f nly ne channel. In principle, ne can make prjectins and mntages f all channels separately, and then cmbine them t a single RGB-image. But as this is tedius, plugins are prvided in the Image5D cllectin, which make prjectins and mntages f Image5Ds. REFERENCES 1. W. Rasband, ImageJ, P. Kelly, and H. J. Karten, UCSD Cnfcal Micrscpy Plugins, P. Pirrtte, J. Mutterer, and J. Brandenburg, HyperVlume Brwser, Francis Wng, and Curtis Rueden, WiscScan Data Brwser Plugin fr ImageJ, Karl Schmidt, Image Layering Tlbx,