Adding scale bars to images

Transcription

1 Adding scale bars to images ImageJ/FIJI IVision Nikon Nis- Elements Imaris 8 Imagej/FIJI 1. If you are viewing images acquired in Micro- Manager and you selected the lens before acquiring the images then the images should have the correct scaling, in which case jump to step 3. If not then read on. 2. To set the image calibration (µm/pixel value) go to Analyse>Set Scale Fill out the information in the Set Scale box, this will vary depending on the microscope and objective used: If you tick the Global option, it will set the scale for all images currently open 3. Once you have set the scale, you can then add a scale bar. Go to Analyse, Tools and Scale Bar The ScaleBar Plus dialogue will appear: Decide the size of scale bar you want, as well as its colour, location and whether or not you want text:

2 4. Once you are happy, remember to save your image File, Save As, Tiff A general note about the scale of your images The scale stays the same if you crop an image. So if 100µm = 103 pixels in A, 100µm = 103 pixels in B A B The scale will change if you resize an image (C) e.g. From 1024 x 1024 to 512 x 512 In this image your scale with be half as large. There are two approaches to this. Either note the factor by which you have reduced your image size and then alter the scale bar accordingly when you make it. Or have the scale bar on the image when your reduce it. (easier) C

3 Nikon Nis- Elements Images saved in Nikon nd2 format contain metadata which along with other things stores the image calibration (µm/pixel value). This means you merely need to add the scale bar. A scale bar can be added to any image by selecting the scale bar button on the right hand side of the window. If you select the down arrow next to it you can enter the Scale properties menu, here you can change the size/colour etc of the scale bar. Scale button If you create a snapshot of an image Edit>Create Full View Snapshot, the scale bar will be burned on and cannot be removed.

4 Imaris 8 Some image files such as Nikon nd2 image store Metadata along with the image. Part of this data is the image calibration (µm/pixel value). If your image is not already calibrated you can set the value in More>Edit>Image Properties. In the Geometry menu change the Voxel size as required for XYZ. In Imaris you can adjust the size, width/height/font by interacting with the scale bar itself using the mouse cursor. When the cursor becomes a crosshair this means you can move the scale bar around. If it is a double headed arrow you can adjust the scale bars size. To save a copy of the image with the scale bar added you can capture a Snapshot. Select the Snapshot icon from the top of the window. A snapshot preview will appear in the left margin. Here you can adjust various options and preview the snapshot before it is saved. See Imaris guide for more in depth information

5 IVision scale bar script Open the scale bar script from disk Script>Open script Select Go to shared scripts folder button Find the Scale bar script in the list and open The script window will open, there is a second window associated with the script which contains lots of buttons. Use the Save As button to save a copy of the script to your own folder. Enter the required values: Objective magnification- Magnification of the lens used to acquire the image. Zoom- Required when images were captured on a microscope with zoom optics (stereo/macroscope). Scaling factor- The magnification added by the tube lens if any. Binning- Defined when setting up the acquisition, also reported in the IVision status window. CCD pixel size- dimensions of the CCD s photodiodes (camera dependent) Coolsnap/Orca/Retiga (fluorescence) = 6.45µm Micropublisher (colour) = 3.4µm (5 MP), 3.45 (3MP) Length in microns- Enter the length of the scale bar you wish to appear on the image. Batch processing All options below the horizontal line in the script are to configure adding a scale bar to many open images. Ignore this section if only applying the scale to a single image. Open all the images that you wish to apply a scale bar (must be same magnification etc). Copy scale bar to all open images (required) Assumes all open images require a scale bar Relocate scale position on each image (optional) You will likely have to move the scale bar on each image so it doesn t obscure any important image features. If you do not select this option, the scale bar is placed in the same position on all images. Select Folder for saved images (required) The scale bars will be stamped on all images and saved as a separate copy and scale will be appended to the filename