Introduction to ImageJ 8 Sept 2009

Transcription

1 Biomedical Imaging Research Unit School of Medical Sciences Faculty of Medical and Health Sciences The University of Auckland Private Bag Auckland, NZ Ph: ext Introduction to ImageJ 8 Sept 2009 Jacqui Ross General Comments: ImageJ is a public domain image processing program. It was written by Wayne Rasband at the Research Services Branch (RSB) of the National Institute of Mental Health (NIMH) which is part of the National Institutes of Health in Maryland, USA. The predecessor to ImageJ, NIH Image was originally only available for Macs but later the PC version Scion Image was developed. ImageJ is based on Java/javascript (Sun Microsystems) and will run on any platform. ImageJ has become a standard tool in many laboratories around the world because it is free, open source, and very well supported. The website address is: You can download the program from This seminar will introduce you to some of ImageJ s features and some basic concepts. Portions of the handout have been lifted from the manual and other materials which can be found on the ImageJ homepage. Associate Professor Alistair Young, the Director of the BIRU, has also kindly provided material, some of which is currently used in our Advanced Biomedical Imaging course (MEDSCI 703) There is a mailing list that you can subscribe to at: You can arrange to get a digest sent to you each day if you don t want each message coming through independently. I recommend this if you are using the program a lot but otherwise it s not necessary since you can still search the archives without being a subscriber. There is some documentation available here: and there is also a Documentation WIKI being developed at: Plugins Plugins are additional software modules or code which provide the ability to perform specific tasks. There is a plugins collection available at: Many more are available on the website and from other users. There is a list of available plugins here: Some people have developed their own collections of plugins and bundled them together. For example, Tony Collins from the McMaster Biophotonics Institute, (previously at WCIF), has a great collection for microscopy: He also provides excellent information about the plugins on his 1

2 website: As new updates are released, some of the most useful plugins are incorporated into the core program. Macros In addition to plugins, it is also possible to write macros using the ImageJ macro language. This allows you to string a series of commands together to perform operations that you may want to do repeatedly. These can also be converted to plugins. Memory You can change the memory allocation (RAM) if necessary by going to Edit-Options-Memory. Make sure that you don t exceed the capability of your computer. You shouldn t go beyond 75% of your RAM capacity. Opening Files File-Open: opens TIFF (uncompressed), GIF, JPEG, DICOM, BMP, STK, video and FITS images. It also opens lookup tables (LUT), text files, regions of interest (ROIs), etc. File/Import: provides access to plugins for reading RAW files, images in ASCII format, and for loading images over the network using a URL. To import a raw (one byte or 8 bits per pixel) file, you must know the image size (eg. 480x640 pixels) and the offset to the image data. Files can be opened in groups by selecting them and dragging and dropping them on the ImageJ icon. You can also open files from a series (x-y, lambda, z) by using File-Import-Image Sequence. The files should be named in numerical order. ImageJ will then create a stack for you of the data. There are also lots of individual plugins available for opening files from particular imaging systems (e.g. Zeiss.zvi). The most useful plugin for importing/opening files is the Bioformats plugin, which is available here: It will allow you to open a large variety of different file formats. File/Revert: allows you to revert to the last saved version of the image. File Save: files can be saved in TIFF, GIF, JPEG, AVI, etc., tab-delimited text, and raw formats. Status Bar Gives information such as memory usage: 2

3 Grayscale and RGB pixel values and X, Y pixel coordinates Tools There are a number of drawing tools on the LHS of the toolbar. Red triangles (as indicated by the arrows below) indicate more tools/options which are available either by right or left hand mouse click or double-clicking. 3

4 On the RHS, where the >> is located, there are a number of other tool sets available which can be selected. These install macros which change the tools visible on the RHS of the toolbar as shown below: Selections or Regions of Interest (ROI): Drawing tools Most commands in ImageJ will work on a region of the image which you need to select or segment in some way. You can select the whole image by going to Edit-Selection-Select All. You can define a specific region of interest (ROI) within the image, using any one of the region selection tools in the Menu toolbar (rectangular, oval, polygonal and freehand). To remove a selection, just click the mouse outside the ROI. Rectangle: when using the rectangle tool, you can resize by dragging the corners. Hold down the Shift key to constrain the selection to be square. Polygon: when using the polygon tool, click once for each vertex, then double click to close the selection. Edit Selection - Specify allows you to specify the size, shape and location of the ROI. All Selections: use the arrow keys to nudge selections one pixel at a time. Use the arrow keys with the alt key held down to change the width or height one pixel at a time. As a selection is created or resized, its location, width and height are displayed in the status bar. Selections can also be moved around the image by dragging. Selections can be copied, analysed, etc. using other menu commands. 4

5 Analyze-Tools-ROI Manager allows you to have multiple ROIs active. It will also allow you to move them, save them and measure them. Other Tools Lines: Allows you to draw straight, segmented or freehand lines. Double-click on the tool to change the line width. Go to Edit-Draw to make the line permanent or Ctr-D. Arrow: You can create arrows by using the arrow tool. Double-click on it to change the properties. Grid: You can also apply a grid to use with this tool by downloading the grid plugin ( )and installing it. You can specify the size of the grid and it can be shown as lines, crosses or points. Magic Wand: Allows you to trace the outline of an object by looking for the edges. It works best if the object has been thresholded and made binary (black/white) for maximum contrast. You can select multiple objects such as cells by holding the shift key down as you go. The tolerance can be set to include a greater range of grayscale. The selections can be added to the ROI Manager. Text: Double-click on the tool to change the options. If the image is grayscale, then text can only be black or white. If you want coloured text, you need to change the image to colour. The colour of the text will be whatever has been specified as foreground colour. Move it to where you want the text to appear and then go to Edit-Draw (Ctrl-D). Eyedropper: Allows you to choose the background and foreground colours by double-clicking on the eyedropper and choosing colours from the Colour Picker. Click on the tiny arrow to swap the foreground and background colours. The colour of the eyedropper then shows the foreground colour while the box around it shows the background colour. Magnifying glass: Use the Left mouse button to zoom in (magnify) and the right mouse button to zoom out (reduce). Next to the name in the title bar of the image, you will see the % of the image currently displayed. Hand: useful when viewing a magnified image or an image that is larger than the screen will allow to display. Click on the image and hold the mouse button down whilst dragging the mouse. The image will pan with the mouse. Colours Edit-Options-Colors: Allows you to set colours for background, foreground & selections. Image Properties Pixels and Grayscale: Digital images are made up of "pixels" (short for picture elements). Each pixel is a spot with a given intensity or greyscale value which is an integer, e.g. in the range 0 (black) to 255 (white) if it is 8 bit, (12bit), etc. 5

6 Bit depth: Generally 8, 12 or 16bits per pixel. Image-Show Info: displays a list of known information about the image. Image-Type: allows you to change the image mode. Some operations are designed to work with 8 bit grayscale or 8bit colour so if you have an RGB image, you may need to change it to 8 bit colour or grayscale. An alternative is to split the image into its component channels. RGB stands for Red, Blue and Green- each of these intensities can be an integer between 0 and 255. Image Color: allows you to merge images, change colours, etc. Lookup Tables (LUT) Images are displayed using a lookup table. This assigns a color to be used for each of 256 possible displayed pixel values (for 8bit images). Analyze-Show LUT: displays the current lookup table of the active image. Image-Lookup Tables: allows selection of a range of color palettes. Invert LUT: inverts the pixel values. LUTs are also useful for checking whether you have even or uneven illumination. 6

7 Spatial Calibration Analyze/Set Scale allows you to calibrate the image so that your results are in units such as microns rather than pixels. You know the pixel values from the Show Info window and you should know the aspect ratio and size of the field of view. If you click Global, ImageJ will apply this calibration to all open images. If you have a calibration bar on an image, you can use the line tool to draw along it and then go to Analyze Set Scale and type in the micron value. Or you can take an image of a micrometer slide with the same objective lens and magnification and use that to calibrate your images. Adding a scale bar 1. Go to Analyze-Tools-Scale Bar. 2. Choose the size, position, colour, etc. of the scale bar you want. 3. Choose the location. 4. You can choose to hide the text if you just want a bar. 5. If you want to draw the size of the bar you want or specify a different location on the image to the options listed, then use the line tool to draw a selection. Choose At Selection for the location. The bar will be drawn at that location and will be the same length as the selection. Edit Cut & Paste: works similarly to most other programs. You can make a ROI, copy it, move the selection around to where you want to paste it to, then click Edit-Paste. You can still move the selection box together with the pasted pixels around the image using the mouse and position it where you want it. When you are happy with the position, just click outside the selection to make it permanent. It is not in a layer anymore. 7

8 Undo: only goes back one step. Sometimes, you have to use File-Revert instead. Clear Inside/Clear Outside: Clears the area inside/outside an ROI, e.g. if it s an area you don t want to analyse or process. Crop: You can crop by drawing a selection and then going to Image-Crop. It will always crop out a rectangle/square. You can t crop out a circle, it will just crop a rectangle of the size that contains the circle boundary. Histogram Analyze histogram: displays the image histogram (frequency), and shows maximum and minimum grayscale values. The log scale shows more minor components. Image Adjust- Contrast and brightness: use to enhance images by dynamically changing the lookup table mapping. Click on the brightness slider and drag from side to side. You can also adjust the contrast setting independently. Brightness adds or subtracts a constant to each pixel shift in histogram along x axis but doesn t change the distribution. Contrast lower level set (e.g. to 0) and higher level set (e.g. to 255) and rest of pixel values adjusted proportionately. Window Level: adjustment of intensity levels (also known as histogram stretch ) 8

9 The examples below illustrate how these procedures alter pixel values. The image below is 8bit grayscale. The histogram to the RHS shows the pixel value distribution and some statistics including Maximum and Minimum values for pixels in the image. You can generate a list of the values by clicking on List. The list can be saved and opened in Excel. Shown below is the default window for Brightness & Contrast (B&C). The histogram is shown in the window with a scale from (8bit). 9

10 Now we apply changes to the LUT by moving the Brightness slider. As brightness is increased, the maximum value decreases and the line moves up. To implement the changes on the image, you need to click on Apply. Until then, only the display of the image is changed, the pixel values are still the same. Following the application of the changes, we can see that the histogram has changed. Maximum and minimum values have changed along with other data. 10

11 After moving the Contrast slider and applying those changes, we can see the altered histogram as below. This time the histogram has been stretched between 0 and 255. We now have some values out of detection range (see minimum and maximum values). 11

12 Window/Level (W&L) also changes pixel values. Shown below is the W&L window. Note the position of the histogram in the W&L window. Now that changes to the LUT have been applied, you can see that the histogram has moved over to the RHS, the image is not as black in appearance and the minimum and maximum values are closer together. In this case, the result is less contrast and apparently higher background in the image. Color balance: allows you to change the colour balance. An additional plugin from the MBF bundle (also called Colour balance) allows you to draw a ROI in a white area and then white balance the image (Plugins Colour Colour balance). 12

13 Threshold: for thresholding images for segmentation and analysis purposes. This will be covered in the follow-up seminar. Size: changes the image size. Canvas size: extends the area around the image. Analyze Tools Color histogram A useful tool to analyse the RGB values in order to adjust colour balance correctly. Or you can look at the values in the status bar. Background Removal Process-Subtract Background: (rolling ball) tries to remove anything on a scale larger than the set radius (good for removing continuously varying smooth backgrounds from gels and other images). You can also generate a background image from your image to remove uneven background using Calculator Plus. Change the rolling ball radius until no detail is visible. Process-Image-Calculator: allows you to do mathematical operations using 2 images. Calculator Plus: Enables you to subtract a background image from an image to remove uneven background or shadowing. Works for RGB images as well as grayscale. It is best to have a background image that you have acquired at the same time as the image of interest. 13

14 The example below shows a background image being created for this image using the rolling ball so that I can demonstrate the Calculator Plus plugin. Original image Has shadows in the corners due to the microscope not being set up correctly Original image with LUT applied Original image converted to 8bit grayscale and a LUT (16_colors) applied to emphasise the uneven illumination. This is a useful application of the LUTs to check illumination issues. 14

15 The background image is created by going to Process Subtract Background as below: Click on Preview to see the background image. This is also a useful way to see what radius you need for using the rolling ball. Once no detail is visible, click OK. To apply the rolling ball directly, just turn off the Create Background option. You can rename the background image if you want to. Original image Background image 15

16 The background image is then used to correct the background as shown below, i.e. dividing the original image by the background image and then multiplying by 255. Resulting image 16

17 Original and processed image as below: Image after background subtraction with LUT applied Result image converted to 8bit grayscale and a LUT (16_colors) applied. Note the uneven shading in the corners, shown on the original image below, has disappeared. Original image with LUT applied Original image converted to 8bit grayscale and a LUT (16_colors) applied. Note shading in corners. Note: You do need to take care that you don t lose detail through the correction process resulting in an image that looks too bleached of colour. 17

18 Process Menu The Process menu contains image processing filters and other operators used for enhancing images or segmenting features for image analysis. Filters Filters represent group processing rather than individual pixel operations. There are many things you can do to change (e.g. improve) the look of the image or make analysis/thresholding easier, eg. sharpen edges, reduce noise, subtract background, etc. Filters are not limited to rectangular selections. The entire image is filtered if no selection is active. They work best on greyscale images. (Remember that if you have an RGB image, you can split the channels, apply the filters you want, and then merge again). Most of these filters in ImageJ (with the notable exception of Rank Filters such as maximum, minimum, etc), are implemented using 3 x 3 spatial convolutions. In this procedure, the value of each pixel in the selection is replaced with the weighted average of its 3 x 3 neighbourhood. I have included some details on how the 3x3 tables differ for two of the filters. The 3 x 3 tables shown below are the coefficients (weighting factors) for the filters. The popularity of plus and minus one in these tables is due to the fact that multiplication by one is very efficient, i.e., it is unnecessary. Smooth - Blurs (softens) the selected area. It can be used to reduce noise in an image or to even out the greyscale in the area Sharpen - Increases contrast and accentuates detail in the selection, but may also accentuate noise Rank filters rank (sort) the nine pixels in each 3 x 3 neighborhood and replace the pixel with the median, minimum (lightest), or maximum (darkest) value. Use the Median filter to reduce noise. This removes high values for the target/central pixel which might be due to electronic noise. A lower value is inserted. This is repeated on the next pixel, etc. until the whole image is treated. It gets rid of noise without causing significant blurring of the image. Despeckle is also a median filter. It replaces each pixel with the median value in its 3 x 3 neighborhood. Median filters are good at removing salt and pepper noise. 18

19 The Minimum filter erodes (shrinks) objects in grayscale images similar to the way binary erosion shrinks objects in binary images. The Maximum filter dilates (expands) objects in grayscale images similar to the way binary dilation expands objects in binary images. Unsharp Mask: sharpens up the image, good if you need extra contrast, often better than Sharpen. Analyze Menu This menu has all the analysis functions including the Set Measurements window, where you select what measurements you want. This will be covered in more detail in the follow-up seminar. Image-Stacks Allows you to work with stacks, (e.g. z series or time series data). You need to have a series (time, z, x-y) of images which can be built into a stack. If you open the images as a numbered sequence, then they will automatically be put into a stack. ImageJ will also open stacks created in MetaMorph (stk files). Convert Images to Stack: converts a set of 2D images that you have already opened into a stack. Animate: animates the images in a stack at a rate up to 100 frames per second. Convert Stack to Images: splits the stack into individual images. Next Slice/Previous Slice: browsing images can be done using the > and < keys. The number of the current slice and the total number of slices are displayed in the title bar. You can also use the slider bar in the stack window. Make Montage: allows you to make a montage out of your stack images. Z Project: simple projection algorithms designed to render 3D images into 2D projections, allows volume rendering, useful for visualizing the internal structures of 3D images. 3D Project: Allows you to project the stack and then rotate it. Orthogonal view: provides an orthogonal (or section) view. Stacks - Movies Time stamper: allows you to add the time information onto your movie. Zoomify: allows you to make a movie where you zoom in on a particular region. 19