A general view of the basic Fiji/ImageJ tools 13-11-2014 io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C INSTALLATION ca l O C M (S )( C SO BM ) BM SO ) O C )( C Go to the SMOC Web page: Software/ImageJ y Fiji (S M lmageJ y Fiji >>Software on fo VERSIÓN Y PÁGINAS OFICIALES ca l OS RECOMENDAMOS LA INSTALACIÓN DE " FUI" COMO ALTERNATIVA A IMAGEJ ImageJ ImageJ E-maillist ImageJ: user and developer conference C a Equipos y a Personal a a Sugerencia& OTRAS VERSIONES ADAPTADAS ic a Búsquedas a Software MANUALES a Protocolos a Otros enlaces Web de 19-9-2012 io a Mapa M a English version o Portal de manuales y ayuda de este programa • Manual "on-line" ImageJ ic r a Información de contado Documentos y tutoriales oficiales os c o a Información y novedades a UPDATED op ía Ó pt Fiji : una versión adaptada de ImageJ Una versión de ImageJ adaptada para Biología (Wright Cell Imaging Facility) Una versión de ImageJ adaptada a Microscopía (MacBiophotonic s ) IQM (procesamiento de stacks - Basado en Java e ImageJ) o Manuales Fiji o Tutoriales " online" ImageJ o Manual en formato PDF de ImageJ o Analyzing gels and western blots with ImageJ Se rv ic a Red Española de Microscopía Óptica Avanzada (REMOA) a Documentos ARTICU LOS ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic Click on the links ImageJ or Fiji to access their download web pages and select the software version you need It isn’t neccesary to install the program, just uncompress the downloaded file ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M The uncompressed folder can be placed everywhere, even in one external USB drive io de The executable file is “fiji-*.exe” (depending on the downloaded version) Se rv ic Plugins folder contains most of the tools and applications of the software. If additional plugins were needed, many more are available in the link “Utilidades y opciones de ImageJ (Plugins)” of this SMOC Web page (see also next section) More information about plugins is also available in the link “Plugins y utilidades destacados” of this SMOC Web page io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C PLUGINS INSTALLATION ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic 1) Look for the application 2) Download the file “*.class” or “*.jar” into the plugins folder 3) Open Fiji and the plugin will appear in the list of Plugins ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r de M You can create your own folders to group some commands in the Plugins menu Se rv ic io 1) Create the new folder in the plugins folder 2) Put the plugins you want to group in this new folder 3) Press de “Help/Refresh Menus” button or restart Fiji and the new plugin group will appear in your list of Plugins. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C MEMORY ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic In the Edit/Options/Memory & Threads menu, it is possible to increase the RAM used by Fiji. It will depend on the computer and OS configuration The Parallel Threads for Stacks field determines the number of parallel threads used by commands in the Process>Filters menu when processing stacks io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C OTHER OPTIONS ca l O C M (S )( C SO BM ) ) SO To set Foreground, Background and Selection color. The selection color is highlighted in the Point Tool and Wand Tool icons. Drawing colors are displayed in the Color Picker Tool (foreground and background colors) and drawing tools such as the Arrow, Brush, Flood Filler and Pencil (foreground color only) (See also next page) Se rv ic io de M ic r os c op ía Ó pt ic a y C on fo ca l (S M O C )( C BM Allows to change the line width (in pixels) of line selections and the lines generated by the Edit/Draw It is possible to change some interesting options in this dialog, like JPEG compression quality ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de Foreground and background colors can also be modified with the Image/Color/Color Picker menu. Clicking on the Foreground/ Background switcher button sets the current foreground to the background and vice versa. The Black/White reset button sets the foreground to black and the background to white. Double-clicking on a color square brings up the Color Selector. Use the sliders to specify the RGB values of the foreground or background color. ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Se rv ic io 16-bit range and Auto contrast stacks in the Edit/Options/Appearance menu should be set properly to open images without automatic contrast enhancement ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Se rv ic io X/Y scale in profile plots can be adjusted in Edit/Options/Profile Plot Options ) SO BM O C )( C SYNCHRONIZE WINDOWS ca l (S M You can access to this plugin in the Analyze/Tools menu. Se rv ic io de M ic r os c op ía Ó pt ic a y C on fo It is useful if you want to compare in parallel several images by changing some display settings in every window simultaneously. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C TOOLBAR ca l O C M (S )( C SO BM ) ) SO BM )( C If the mouse is passed over icons a message explaining the tool is shown 2) Some icons give access to several related tools Se rv ic io de M ic r os c op ía Ó pt ic a y C on fo ca l (S M O C 1) Pressing the double arrowhead at the right corner give access to other toolbars ) SO BM )( C O C M (S ca l on fo Tool options can be shown by clicking the right mouse button over icons 2) Other additional options can also be accessed by double clicking over icons 3) The Action Bar plugin can give you the possibility to add personalized toolbars. This plugin has to be installed Se rv ic io de M ic r os c op ía Ó pt ic a y C 1) io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C PLUGIN UTILITIES ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de In the Plugins/Utilities menu, Control Panel opens a window containing Fiji commands in a hierarchical tree structure Click and double click can be used to launch commands and options ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r de M In the Plugins/Utilities menu, Find Commands opens a window where Fiji commands can be searched by keywords, showing their location route (active Show full information) Se rv ic io Commands can also be launched directly from here by clicking on the text ) SO BM creare .snorttut.. ~ comp e a1 C ~un Remo\•e... ~noncut 1 O C lu:il.r! l i'luíJin )( C ----- ln;:.t,; ll Plugin 30 M SO C hjf!(.f C:ou "lt~! r (S 30 VIewer on fo ca l ArJ,JI'!'ll! rr-ager o.:., lu l~ rod lm;.;!J¡.; F iii ~H~ C LOCI ic 11 ~ir.tr.; t ."111 Ó pt 3PIM Regrstratoo op ía ~mentat1on T~.<tc:kiii!J 1ransronn Command : Shortcut: Argument: [F4l r-------1 lmport Save As Filters Tools Utilities de M ic r UlilitM.:·; Menu: os c 3thc1rng :) Plugin: a y o;mc r row lnstall Plugin Se rv ic io In the Plugins/Shortcuts menu, several tools for shortcuts can be found: it is possible to create a shortcut for any plugin or to change its location in the menu ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de To get help about plugins, if there were any, there are two ways: Se rv ic io 1) Click on the Help/Help on Menu Item command. Then go to the menu where the plugin is installed and click on it 2) Go to the Help /About Plugins menu and look whether the plugin is in the list io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C ADDITIONAL PLUGINS ca l O C M (S )( C SO BM ) ) SO BM )( C O C SMOC Web page • • Fiji ImageJ • G. Landini – Software • Parker plugins • BioVoxxel & BioVoxxel Toolbox op ía Ó pt ic a y C on fo ca l (S M • 3D ROI Manager 3D Tools 3D processing Se rv ic io de M ic r os c • • • io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C OPEN AND SAVE IMAGES ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S on fo ca l Open images directly from the menu or just drag them into the Fiji Toolbar Ó pt ic a y C It is possible to save images in many different formats but additional plugins can be downloaded from the Plugin ImageJ web page Se rv ic io de M ic r os c op ía To save movies use the plugin Save As/Movie, which can be downloaded from here. Quicktime movies may require additional plugins which can be downloaded at http://rsb.info.nih.gov/ij/plugins/movie-writer.html ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía STACKS: multiple spatially or temporally related images are opened in a single window ic r os c In stacks, a pixel (which represents 2D image data in a bitmap image) becomes a voxel (volumetric pixel) io de M All the slices in a stack must be the same size and bit depth. A scrollbar provides the ability to move through the slices and the slider is preceded by a play/pause icon that can be used to start/stop stack animation. Right-clicking on this icon runs the Animation Options Se rv ic A folder of images can be opened as a stack either by dragging and dropping the folder onto the ImageJ/Fiji window or by choosing File/Import/Image Sequence ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía VIRTUAL STACKS are disk resident (as opposed to RAM resident) and are the only way to load image sequences os c that do not fit in RAM Se rv ic io de M ic r - Virtual stacks are read-only, so changes made to the pixel data are not saved when you switch to a different slice - You can easily run out of memory using some commands because any stack generated from them that do not generate virtual stacks will be RAM resident - TIFF virtual stacks can usually be accessed faster than JPEG virtual stacks - Stacks can be opened as “virtual” by File/Import/TIFF Virtual Stack and Plugins/Image5D commands. The signal Virtual (V) will appear close to the name ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M HYPERSTACKS are multidimensional images, extending image stacks to four (4D) or five (5D) dimensions: x io de (width), y (height), z (slices), c (channels or wavelengths) and t (time frames) Se rv ic Hyperstacks are displayed in a window with three labelled scrollbars See also how to transform one Stack to one Hyperstack ) SO BM utilüies Compile and Run... ,, lnstall Plugln.. )( C New ' ~ .1(~ .. . : O C 3D ( 30 Object Counler 3D Viewer Open Series As lmage5D Landmarks Virtual lmage5D Opener Opl lt Flow Hypervolume Opener Process C LOCI y Slack lo lmage5D Regislralion Duplieale Skelelon 5el POSÜIOn pt Segmenlalion ic lmage5D lo Slack SCripling a RGB lO lmage5D SPIM Regislral ion on fo ca l (S M AnalyZe se1 Channel Display Stilching set Channel LabeiS 1 ~ op ía Ó Stacks z Tracking Make Montage Transform z Projett lmage5D Stack to RGB Transfer Channel Settlngs lmage5D Extenslons de M ic r os c utililies Se rv ic io To manage stacks of images, with several channels, sections, a timelapse, etc., you can open them with Plugins/Image5D tools. The image visualization and navigation become more user-friendly ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c de M ic r Stacks of images, with several channels, sections, a timelapse, etc. can be transformed to Hyperstack for a better image management Se rv ic io The Channels tool window will appear (see next page) ) SO BM )( C C on fo ca l (S M O C 1 2 os c op ía Ó pt ic a y 3 ic r The Channels tool command can be also found in the Image/Color menu de M 1) Channels selector, Se rv ic io 2) Three ways to visualize colors, 3) Addtional options and colors available in the More menu io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C IMAGE INFORMATION AND PROPERTIES ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M io de Image information as pixel/voxel size, resolution, image size, slices, etc. can be get with the Image/Show Info and Image/Porperties commands. Se rv ic Be sure to have ImageJ/Fiji updated. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C WORKING WITH CHANNELS ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic Channel colors can be changed with different LUTs in the Image/Lookup Tables menu Some of the most useful ones are shown here. See also Lookup Tables (LUTs) section. ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c de M ic r With RGB images, Merge and Split Channels tools become very useful to combine or split colors Se rv ic io With the Merge Channels command it is possible to Create composite instead of RGB images. Its advantages are: 1) It is possible to merge more than three channels, 2) Until 16 bit per channel supported, 3) Each channel can be adjusted independently of the others io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C BRIGHTNESS AND CONTRAST ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c Adjustments of the channels brightness and contrast can be located in the Image/Adjust menu M ic r Be carefull: once the Apply button has been clicked the original image information will be lost or changed Se rv ic io de Brightness/Contrast (B&C) and Window/Level (W&L) are redundant, but the second one behaves in a manner closer to that implemented on medical image terminals by interactively adjusting the Window – range of minimum and maximum (Contrast) – and Level – position of that range in the grayscale intensity space (Brightness) Color Balance makes adjustments to the brightness and contrast of a single color of a standard RGB image (8–bit per color channel) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó os c op ía For image stacks, Process/Enhance Contrast can be used to adjust the brightness and contrast of each slice in a stack, according to either the optimal for each individual slice (Use Stack Histogram is unchecked) or the overall stack (Use Stack Histogram checked) de M ic r Saturated Pixels determines the number of pixels in the image that are allowed to become saturated. Increasing this value will increase contrast Se rv ic io Normalize will recalculate the pixel values of the image so the range is equal to the maximum range for the data type. The contrast stretch is similar to the ‘Auto’ option in Brightness/Contrast but each slice in the stack is adjusted independently, according to the optimal for that slice alone. Equalize Histogram will enhance the image using histogram equalization. Create a selection and the equalization will be based on the histogram of that selection. ) SO BM O C )( C Process/Enhance Local Contrast (CLAHE) is a very interesting command for contrast enhancement of single images and stacks ca l (S M Block size: the size of the local region around a pixel for which the histogram is equalized. This size should be larger than the size of features to be preserved Ó pt ic a y C on fo Histogram bins: the number of histogram bins used for histogram equalization. The implementation internally works with byte resolution, so values larger than 256 are not meaningful. This value also limits the quantification of the output when processing 8bit gray or 24bit RGB images. The number of histogram bins should be smaller than the number of pixels in a block Se rv ic io de M ic r os c op ía Max slope: limits the contrast stretch in the intensity transfer function. Very large values will let the histogram equalization do whatever it wants to do, that is result in maximal local contrast. The value 1 will result in the original image Mask: choose, from the currently opened images, one that should be used as a mask for the filter application. Selections and masks can be used exclusively or in combination io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C HISTOGRAM ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c de M ic r The easiest way to study the pixel intensity distribution of one image is taking a look to its Histogram, which shows how many pixels in the image have which intensity. Select Analyze/Histogram for grayscale images and Analyze/Color Histogram for RGB ones Se rv ic io The Log button shows the histogram in logarithmic scale, which is handy for fluorescence images that are often mostly black It is very useful for quantitative analysis to detect saturation, wrong offset, noise/background levels, etc. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C THRESHOLDING ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c The Image/Adjust/Threshold menu give access to Threshold commands M ic r Thresholding is used to select image data by its grey level. It can be done manually or using automatic thresholds Se rv ic io de In the example above, pixels with a grey value between 156 and 255 will be selected/extracted For color images it is better to use Image/Adjust/Color Threshold io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C REGION OF INTEREST (ROI) ca l O C M (S )( C SO BM ) ) SO BM )( C M (S ca l Right- and double-clicking over icons give access to addtional options and tools O C The ROI tools are used to draw regions Se rv ic io de M ic r os c op ía Ó pt ic a y C on fo Remember to check “Line Width” and “Colors” in the Edit/Options menu After drawing ROIs, they can be permanently painted or filled by Edit/Fill and Draw tools In the Image/Overlay menu more options are available ) SO BM )( C O C M ca l on fo Select objects by Threshold Go to Edit/Selection and apply Create Selection ic a y C 1) 2) (S To create ROIs around objects: Ó io de M ic r os c op ía Select objects by Threshold Go to Analyze/Analyze particles and be sure to have “Add to Manager” checked Se rv ic 1) 2) pt To add them to ROI Manager: io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C CROP ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Image/Crop and Image/Duplicate can be used to extract some area from an image or stack Se rv ic io First of all, a region must be drawn with the ROI tools. Then, select crop or duplicate to get the selected area The Duplicate command have some more options than Crop and don’t close the original image ) SO BM Utlltties )( C New Compile anc Run__ O C lnslal Plugln___ 30 M 30 Objecl Counter (S 30Vewer Anal}ze __ -- ~ ~_x ca l Examples ,, - Fealure Extraction (511) (511 ) on fo lmage5D lmageFiow Integral lmage Filters C LOCI (45) Land'rlarks y Optlc Flow a Process ic Registration Crop 1 Cancel 1 (Move mouse W1th shifl to update panes_) pt SPIM Regist' alion o crop these lmages: Ó Scrip:ing os c op ía Scgmcntuticn Transfonn de M Volune Víewer ic r Utiltties Se rv ic io Plugins/Stacks/Crop (3D) can be used to crop stacks The area to be cropped can be selected in X, Y and Z with the point selectors of images or with the Crop options tab io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C ZOOM ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l pt ic a y C on fo Use the Magnifying glass tool to change zoom clicking on the image to zoom in and right-click (or Alt-click) to zoom out. The current magnification is shown in the image’s title bar. Double click on the magnifying glass icon to revert to the image’s original magnification os c op ía Ó Clicking and dragging while holding down the Shift key runs Image/Zoom/To Selection Se rv ic io de M ic r The Scrolling/Hand tool helps to navigate through the image with high zoom In the Image/Zoom menu, additional zoom options can be found io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C SET SCALE ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a Ó pt ic To make some measurements, it is first necessary to set the distance per pixel of the image in the Analyze/Set Scale menu Se rv ic io de M ic r os c op ía There are two ways to do it: 1) Distance in pixels = 1 and Known distance = distance known for each pixel 2) Draw a region over one area of the image whose distance is known. The number of pixels of this region will be displayed in Distance in pixels. Insert the value of the distance known for that region in Known distance When Global is checked, the scale defined in this dialog is used for all opened images during the current session instead of just the active image io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C SCALE BAR ca l O C M (S )( C SO BM ) ) SO BM )( C Summarize Olstnbulion... Font siz.e: l w hite Background: I Nane on fo I Lower Right~ Location: P'" Bold Text r Serif Font Curve Fitting. .. Color Inspector 30 . .- r Scale Bar . Oirectionality Synchronize Windows Sync Windows 30 Surface Plot os c ClassifiCation Cancel l op ía PointPicKer Helmholtz Analysis ~ Ó Shape lndex Map O ptic Flow Label all slices ic Color Histogram " a ~. pt Colocalization r Hide Text r Overlay C Fractal Box Count... Anatyze Une Graph y 30 OC Options ...:..1 ...:..1 ca l Color: M 14 114 Height in pixels: 30 Objects Counter O C l!m Width in ¡Jm: Clear Results (S Label Local ThícKness OuicKPALM de M ic r TopoJ Se rv ic io In the Analyze/Tools/Scale Bar menu it is possible to modify every setting to add a Scale bar to the image Remember to visit the Analyze/Set scale menu before adding the scale bar io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C BACKGROUND SUBTRACTION ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c M ic r Process/Subtract Background uses a ‘sliding paraboloid’ or a legacy ‘rolling ball’ algorithm that can be used to correct uneven illuminated background Se rv ic io de Rolling ball radius should be set to at least the size of the largest object that is not part of the background. The sliding paraboloid typically produces more reliable corrections since the rolling ball is prone to edge artifacts We recommend using the HiLo LUT to apply these kind of corrections (see also “Working with channels” section) and keep the Preview tab active ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de Another option is to acquire a Background image (with the same acquisition settings of the original image but avoiding light reaching the detector) and Subtract this image from the original one using the Image Calculator. We recommend selecting the 32-bit (float) result option. More information io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C SHADING/FLATFIELD CORRECTION ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c Shading/Flatfield correction can be used for correcting non-uniform field illumination, spots and dirt. de M ic r It is necessary to acquire both, a Background image (see Background subtraction) and a Shading image (defocusing the sample until just an illumination field is observed). Se rv ic io Go to the Image Calculator and apply these image calculations: Corrected Image = (Original - Background) / (Shading - Background) Fit_Polynomial plugin can also be used for these purposes. More information ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Se rv ic io If it was impossible to get a Shading image it could be get from the original one by the application of a Gaussian Blur filter. It isn’t the best solution but it might work sometimes. Increase the Sigma (Radius) until no image details are observed. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C ROI MEASUREMENT ca l O C M (S )( C SO BM ) ) SO Calbrete.. . Ct ~ •H Hl5togro!lm Plut FIJO~ ~ I Jri~ ·:P. Ct1i•K P ln J Shape descriptors lntegrated density r Area fraction TooiS nr. n~un n ~ )( C Median Kurtosis Limit to threshold r lnve rt y coordinates r St:>•:, positi on Display label Scientifi c notation a r Add to over1ay ic Co or Hl>1()9raT pt Cj or lncpedor 30 Redirect to: jNone Ó O ro:tlonolit( Shepe lndex ve~ Dedmal places (0-9): op ía Oplt Ftow HdnhuLt Aualys s ~n ~~~rr~cP. r r C ~n 1.:0 C(allzatJon Ferers diameter r y O U]tll;t~ Cuunt~ 1 Fitellipse r r Skewness Gel S 30 Penmeter r r on fo r r r Sct S·: ole... Centroid r Center of mass r Bounding rectangle O C r CeuRe$uh~ BM r M Label r l.lodal gray value P" 1.1in & max grayvalue P" Mean gray value (S O:llrfbution .•. p ¡;¡r·eaJ r Standard deV! ation ca l SUft'1113rize Anr ~ Cancel 1 Help 1 os c l: 2SSI!Cal101l p Local Thl:kness OUcki'J\LM de M ic r TopoJ Se rv ic io Before doing measurements, options must be configured in the Analyze/Set Measurements menu If Limit to threshold is active the thresholded areas inside the ROI will be only measured ) SO BM )( C Oistribution__ O C Label Clear Resuns M Set Measurements __ (S SetScale __ Histogram Ctri+H Plot Profile Ctri+K Gels C Tools 30 Objects Counter y 30 OC Options a Colocalization ic Color Histogram pt Color Inspector 30 Ó Oirectionalily Helmholtz Anatysis Local Thickness ic r de M TopoJ os c 30 Surface Plot op ía snape lndex Map Optie Ftow OuickPALM - on fo Surtace PloL Classification '·'' ca l Calibrate __ Se rv ic io To measure one single ROI just go to Analyze/Measure Several options are available to save and display the data ~~~ ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r To measure several ROIs go to Analyze/Tools/ROI Manager de M After drawing each ROI press Add to include its coordinates in the list. They can be saved as “.roi” files in a Zip archive Se rv ic io Press Measure to get measuremets for each ROI. Use Shift and Ctrl keys to select several ROIs simultaneously ROIs can be permanently painted on the image with the Flatten command Additional options can be found in the More menu of the ROI Manager BM SO ) 3D IMAGEJ SUITE O C )( C To measure several ROIs in 3D stacks go to Plugins/3D/3D Manager Se rv ic io de M ic r os c op ía Ó pt ic a y C on fo ca l (S M 3D ImageJ Suite has many options for 3D measurement. You can find additional information here ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r de M Analyze/3D Objects Counter command can be useful to make measurements in a 3D stack io Before doing the analysis, go to 3D OC Options to select the Parameters needed Se rv ic Additional information can be found at the 3D Object Counter and 3D Manager basic tutorial 3D ImageJ Suite can complement the 3D Object Counter tools io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C INTENSITY PLOT PROFILES ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Se rv ic io With the Image/Stacks/Plot Z-axis Profile menu it is possible to get a graph and a table of intensities along a Z stack or a timelapse within a ROI Data can be saved as an excel or text file ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Se rv ic io With the Analyze/Plot Profile menu it is possible to get a graph of intensities along a line A List of the data can be obtained and saved With the option Live activated, the profile will be updated as the line is changed ) SO BM Utilities )( C New Compile and Run... O C lnstall Plugln .. 3D M 3D Object Counter (S 3DViewer ca l Examples Feature Extraction on fo lmage5D lmageFiow lntegral lmage Filters C LOCI ... - .- -------- ~~ ·' ~...--,, . piXels, - . l::::!....~_x _~ - 8 , .K Ó pt ic a y LandmarKs Segmentation SKeleton Stitcning TracKing t 200 1 "'" ~ ~ 00 100 Distance M Utilities ic r Transfonn os c StacKs op ía Scriptlng de Volume Viewer Se rv ic io To get plot profiles of RGB images install the RGB Profiler plugin. After installation it will appear in the Plugins menu It can be useful for a quick evaluation of colocalization or pixel shifting ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic To obtain profiles of several selections in a single plot use Analyze/Tools/ROI Manager/Multi Plot io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C GEL TOOLS ca l O C M (S )( C SO BM ) Use Analyze/Gels tools to analyze one-dimensional electrophoretic gels For detailed information check the ImageJ manual You can also use the ROI measurement tools or this video tutorial “gel quantification analysis” io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C LOOKUP TABLES (LUTs) ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de Se rv ic io LUTs can be displayed with the option Image/Color/Display LUTs. On the other hand, it is possible to get the values and graph of the LUT which has been applied to one image with the Image/Color/Show LUT menu. ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de Open Image/Color/Edit LUT menu to edit color tables and create customized LUTs. New LUTs can be edited and saved easily. Click on one of the squares of the LUT editor to edit its color. Or drag across several entries and you will be asked to define the RGB starting and ending values for the selected range. The Editor will fill the remaining entries using the selected interpolation method in Set: Replication just repeats the entries while Interpolation and Spline Fitting produce gradations between the colors. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C LUT CALIBRATION AND INTENSITY BARS ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de Before applying a calibration bar, it is first neccesary to calibrate image gray values. Open your control image and open Analyze/Calibrate menu. Associate your experiment values, pH for example, with the gray intensities in the image. In the left column indicate mean gray values which might have been previously measured with the ROI manager. In the right column enter the known standard values corresponding with the mean gray ones. Calibration Function and Unit can be changed, and settings can be saved to be reused. Remember to select None in the Function menu if no calibration is wanted to be applied. ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic Once the calibration has been done, Analyze/Tools/Calibration Bar menu can be opened to configure and insert the calibrated Lut on the image. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C STACK ALIGNMENT/REGISTRATION ca l O C M (S )( C SO BM ) ) SO BM )( C O C M Se rv ic io de M ic r os c op ía Ó pt ic a y C on fo ca l (S When pixel shifting is observed in a Z Stack, it can be corrected by the Plugins/Registration commands Linear Stack Alignment with SIFT and StackReg give good results but there are many others which could suit better to your images This correction may be specially important for colocalization or FRET experiments io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C SLICE SELECTION ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M io de In the Image/Stacks/Tools menu you will find many commands to select a subset of slices in a stack Se rv ic With Slice Keeper it is possible to extract some slices by an increment number. With Combiner, two stacks can be visualized simultaneously in the same window See also Crop 3D (Crop section) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de For slice selection the Image/Stacks/Make Montage command can be useful to have a picture of all slices in a window io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C 3D STACKS ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c M ic r Use the Image/Stacks/Orthogonal Views command to get Orthogonal sections of the stack Se rv ic io de The intersection point of the three views follows the location of the mouse click and can be controlled by clicking and dragging in either the XY, XZ or YZ view. The mouse wheel changes the screen plane in all three views Voxel dimensions can be changed in Image/Properties/Voxel Depth or in Image/Stacks/Reslice Z ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c The Image/Stacks/Reslice command lets getting Orthogonal sections with more flexibility ic r Output spacing refers to the distance between one orthogonal section and the next one de M Start at determines the image edge from which reconstruction starts Se rv ic io Start at is replaced by Slice count if there is a line ROI. With lines selections, a stack is created by shifting (by Output spacing) the line down and to the left to generate additional slices for the output stack With the Image/Stacks/Dynamic Reslice command the Orthogonal section shown is updated while changing the line ROI ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c The Image/Stacks/Z Project command projects an image stack along the axis perpendicular to image plane Se rv ic io de M ic r Average Intensity projection outputs an image wherein each pixel stores average intensity over all images in stack at corresponding pixel location Maximum Intensity projection (MIP) creates an output image each of whose pixels contains the maximum value over all images in the stack at the particular pixel location Sum Slices projection creates a real image that is the sum of the slices in the stack Standard Deviation projection creates a real image containing the standard deviation of the slices Median projection outputs an image wherein each pixel stores median intensity over all images in stack at corresponding pixel location ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r io de M The Image/Stacks/3D Project command creates a sequence of projections of a stack onto a plane using Nearest Point (surface), Brightest Point or Mean Value projection Se rv ic The user may choose to rotate the volume about any of the three orthogonal axes (x, y, or z), make portions of the volume transparent (using thresholding) or add a greater degree of visual realism by employing depth cues ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r Se rv ic io de M The Analyze/Surface Plot and 3D Surface Plot command display a three-dimensional graph of the intensities of pixels in a grayscale or pseudo color image (non-RGB images) It can be done of the whole image or of ROI selections ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r de M With Plugins/3D Viewer and Volume Viewer tools, stacks can be displayed as texture-based volume renderings, surfaces or orthoslices Se rv ic io Additional information about 3D Viewer and Volume Viewer Take also a look to BioImageXD io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C EXTENDED DEPTH OF FIELD ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de Due to the limited depth of field of brightfield images, it is usually impossible to image large 3-D organisms and objects entirely in focus. By optically sectioning the specimen the in-focus information at the specimen's surface can be acquired over a range of images, which can then be processed to generate a single in-focus image with Plugins/Extended Depth of Field (Easy or Expert mode) commands Additional information about these plugins can be found at http://bigwww.epfl.ch/demo/edf/ io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C TIMELAPSE AND VIDEOS ca l O C M (S )( C SO BM ) ) SO BM )( C Ctr1+Mayúsculas+P Delete SIice Ctr1+Mayúsculas+D Next Shce [>] Previous SIice [ <) O C Ctr1+Mayúsculas+X SetSiice... Transfonn lmages to Stack M Ctr1+E Stack to lmages Speed {0. 1-10 00 fps): (S Make Montage... Reslice [/]... Ctr1+Mayúsculas+H First Frame: .-11_ __ ca l Orthogonal Views Lookup Tables Z Project... 30 Project on fo Plot Z-axis Profile La bel... C Concatenate ... Reduce ... y Reverse Last Frame: r 145 Loop Back and Forth 17 Start An imation ~ Cancel a lnsert li ic Montage to Stack Make Substack.. pt Grouped z Project... Ó Remove Slice Labels Stack Splitter lnteneave de M ic r os c op ía Start Animation [\] Se rv ic io For video editing Image/Stacks/Tools/Animation Options and Image/Video Editing tools are recommended See also the “Open and Save images” section io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C VIDEOS AND STACKS LABELING ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r Se rv ic io de M It is possible to label stacks and movies with three different commands: 1) Image/Stacks/Label, to label stack labeling 2) Image/Stacks/Time Stamper, to label timelapse movies 3) Image/Stacks/Series labeler. It is the most flexible one and can be used for both io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C IMAGE FIGURES FOR PUBLICATION ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic Plugins/FigureJ is a plugin to create image figures for publication, to mount and organize several images in panels. You can find information about Figure J in the ImageJ Wiki. ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M de io Se rv ic Plugins/ScientiFig is another plugin to create image figures for publication. You can find information about this plugin in ScientiFig. io Se rv ic de M op ía os c ic r a ic pt Ó y on fo C MACROS ca l O C M (S )( C SO BM ) ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c ic r M Se rv ic io de To record several actions as a Macro go to the Plugins/Macros/Record. The recorder window will be opened and actions will be saved while processing After finishing press Create and the Macro editing window will appear, where you can edit and save the macro ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c With the Process/Multiple Image Processor menu it is possible to apply a macro to several files inside one folder M ic r Additional information about this plugin and macro recording de Tutorial on ImageJ Batch Processing Se rv ic io There are other Batch tools in the Process menu to process several images at once Another way to apply some macro or function to several images is to create one stack with Import/Image sequence. Then apply it to the stack. ) SO BM )( C O C M (S ca l on fo C y a ic pt Ó op ía os c de M ic r ImageFlow is a plugin still under development which offers a node-based graphical user interface for Macro-creation and image editing. It allows to create macros by connecting nodes to form a workflow Se rv ic io You will find information about this plugin at: http://imageflow.danielsenff.de/ http://imageflow.danielsenff.de/imagej/Imageflow.html https://www.dokuwiki.org/plugin:imageflow#imageflow_plugin ) SO BM M (S ImageJ tutorial Wiki O C )( C ADDITIONAL INFORMATION on fo ca l ImageJ documentation y C ImageJ user guide in pdf Ó pt ic a Fiji documentation op ía Focus on Bioimage Informatics. Nature Methods. 2012. 9, 627-763 ic r os c SMOC Web page about ImageJ/Fiji Se rv ic io de M Free open source software and other image processing programs ImageJ2: the future
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