NIH Image Tutorial

NIH Image: An Introduction and Tutorial

This on-line tutorial is based on the paper "NIH Image: A Public Domain Image Processing Program for the Macintosh" by Wayne Rasband and David Bright, published in Microbeam Analysis, 4 (1995).

NIH Image can acquire, display, edit, enhance, analyze, print and animate images. It reads and writes TIFF, PICT, PICS and MacPaint files, providing compatibility with many other applications, including programs for scanning, processing, editing, publishing and analyzing images. It supports many standard image processing functions, including contrast enhancement, density profiling, smoothing, sharpening, edge detection, median filtering, and spatial convolution with user defined kernels. A series of images can be animated or viewed in a stack. Particles can be counted and sized. NIH Image can be customized using a Pascal-like macro programming language and through use of plug-in code modules.

NIH Image comes with many other files: a user manual ("NIH Image Manual"), a programmer's guide ("Inside NIH Image"), a folder of convolution kernels, and a folder of macros. In addition, the NIH Image FTP site (zippy.nimh.nih.gov) has numerous example images including those used in this paper (via anonymous FTP from zippy.nimh.nih.gov, in the /pub/nih-image/images and the /pub/nih-image/stacks directories), and it has macros and plug-ins, as well as several enhanced versions of NIH Image.

MacLispix, described in an accompanying article in this issue, is another public domain image processing program for the Macintosh, which does some of the same things but in different ways. MacLispix is not as polished a Macintosh application as is NIH Image, but is intended for more special purpose analyses such as three dimensional scatter diagrams, principal components and chemical phase analysis, and for instances where more numerical precision than 8 bits per pixel is needed.

Features

NIH Image conforms well to the Macintosh user interface standard, and is visually and graphically oriented, making it easy to use with little experience. For example, NIH Image has a palette of tools for drawing, measuring and examining images which are fully described in "About NIH Image". A variety of measurements can be made on user-specif ied regions of interest and results exported to a spread sheet or plotting package. The "LUT" (color look up table) and "Map" windows allow control of the video lookup table, providing flexible contrast enhancement and false color. The "Info" window displays cursor position, pixel values, selection size, line length, etc. Images, look-up tables, macros and convolution kernels can be opened by dragging them to the NIH Image icon.

System requirements

NIH Image requires a Mac II or later with 8 MB or more of RAM. (Running NIH Image on a 4 MB Mac is a struggle.) System 7 or later is also required for versions 1.56 and later (because of the plug-ins and 24-bit to 8-bit color conversion), and for many of these examples. A Power PC native version is available as well as a non-FPU version for Macs without a floating-point co-processor.

NIH Image Users

There is an active electronic mailing list on the Internet with over 1000 subscribers and a dozen messages or so a day covering topics such as a) news of the latest versions of NIH Image b) special purpose macros c) image processing tips using NIH Image d) hardware - frame grabbers e) bugs, wish list items. Subscribe to the list by sending the one line message "subscribe nih-image <first name> <last name>" to listproc@soils.umn.edu.

The user base for NIH Image is large: over two thousand copies of v1.55 alone have been downloaded from the NIH Image FTP site.

Examples

( The actual steps begin with bullets.)

These examples are meant to be cook-book type lists that can be followed by a new user to get started. Menu commands are shown in italics, for example, File - Open is the Open command in the File menu.

Starting up and configuring

Select the Monitors control panel in the Control Panels folder (from the Apple - Control Panels menu) and set the display to 256 colors. (Versions of NIH Image 1.55 and later will work with other monitor settings, but the appearance of the images may differ from those described here, and the performance of NIH Image may be degraded.
In the Finder, click once on the NIH Image icon and use the File - Get Info command

to set the preferred size to 4000K. Leave NIH Image in a folder that also contains the macros folder and plug-ins folder. Make an alias of NIH Image and move it to either the Apple Menu items folder (in the System folder) or the desktop. To start up, do one of the following actions to either the NIH Image icon or its alias: double click it, select it in the Apple Menu, drag and drop one or more selected images onto it, or double click on an image that is an NIH Image document, such as the "TEM filter sample.tiff" (Steel, 1993) image discussed below. Which application (such as NIH Image) 'owns' the file can be displayed by going to the finder, selecting the file by clicking on it, and using the File - Get Info command or pressing the command-I keys.
In the event of the error message that there was not enough room for various buffers, use the Options - Preferences command in NIH Image to set the Undo and Clipboard buffers to 300K. (The Clipboard and Undo buffers don't need to be larger than the largest example image. You may have obtained a preferences file along with NIH Image, which might have preferences different from those recommended for these examples. ) Also make sure that the Invert Pixel Values box is checked so that black = 0 and white = 255. Save the preferences using File - Record Preferences , Quit and restart NIH Image.

Reading in an image

TIFF, PICT, PICS (for a stack of images) and a few other file formats are read and displayed using the File - Open command. Files can also be opened in groups either by selecting them in the Finder and dragging and dropping them on the icon of the NIH Image application, or, if a folder contains images of interest only, by selecting the Open All button in the Open command dialog. The Open command will also read IBM PC ".TIF" files and text files. Raw data, that is images without any encoding or header information, can be also be read with the File - Import command. Although several file formats such as TIFF are becoming widely accepted, the raw format with one byte (8 bits) per pixel is still useful for moving images between different computer systems, but one must remember to keep track of the image dimensions when the image is originally recorded, e.g. 480x640 pixels.
Open the image file TEM Filter Sample.tiff by using File - Open , by dragging and dropping the file onto NIH Image's icon or by double clicking on the file. The image is a transmission electron micrograph of a particulate sample deposited on a polycarbonate filter.
Note that the image is displayed in its own window with the name of the file in the title bar. The LUT (color Look Up Table), Tools, Map and Info (not shown in figure) windows are also displayed. (Fig. 1) When the cursor is run over the image window, the coordinates and pixel value for the cursor position are displayed in the Info window. The LUT window displays the current gray level or color look-up table. Move the cursor over the LUT window and observe that the pixel values are displayed in the Info window (255 or black at the bottom, and 0 or white at the top as the default, or 0 or black at the bottom and 255 or white at the top with the pixel values inverted - see above).
Click on the zoom tool - the magnifying glass at the upper left of the Tools window. The cursor will change to a magnifying glass. Move this cursor to a particle of interest and click on it once. The particle should be magnified by a factor of two, and centered in the window. At this point, if your screen is large enough, the image window can be expanded by dragging the grow icon in the lower right corner of the window. Clicking and holding on the hand in the Tools window allows panning of the image (with the hand cursor) by dragging any part of the image in the direction you wish it to go. The image can be zoomed as much as desired with more clicks on the magnifying glass tool, until a single pixel fills the window. Note that the zoom factor appears to the right of the file name in the title bar. A 3:1 zoom will make the individual pixels visible as small squares.

The image can be modified with the drawing tools - the pencil, eraser, brush, line drawing tool, paint bucket and spray can - as explained in the manual "About NIH Image". It is of interest here to note that when the image has been zoomed so that the individual pixels are visible, single pixels can easily be modified one by one with the pencil tool. The shade of gray applied by the pencil tool is the shade inside the paintbrush, which can be changed by clicking on the eyedropper tool, then clicking on the desired shade either in the color bar or in the image.

Depressing the option key will change the "+" to "-" inside the magnifying glass tool, which will reverse the effect of the zooming. Restore the image to its original state with the option key and zoom tool. If you have done any drawing on the image, also use the File - Revert To Saved command to return to the unmodified image. To avoid undesired drawing on the image when proceeding, select the rectangular selection tool by clicking on its icon at the upper right of the Tools window.

Display and Contrast Enhancement
Problem: This image has low contrast except for the dark particles (Fig. 1). When images are not pre-scaled for display, the contrast and brightness need to be adjusted, much as on any electron microscope. This can be done in a variety of ways, such as dragging the dots or sliders in the Map window, or invoking the Process - Enhance Contrast or Process - Equalize commands. (Note that the Process menu was named Enhance in versions of NIH Image earlier than 1.56.) Invoke the Enhance Contrast command, which works on the front image window (there is only one image window activated so far in this session) by selecting the Enhance Contrast command from the Process menu and letting up on the mouse. The result is Fig. 2 -- the ordinary linear contrast enhancement, which uses the full available brightness range (0 - 255) to display the image. Note that when the enhancement was done, the LUT and Map windows changed also.

Contrast enhancement can also be done by using the mouse to move the B (brightness) and C (contrast) sliders in the Map window, by moving any of the three dots on the graph in the Map window, or by selecting the LUT tool and manipulating the color bar in the LUT window.
The actual pixel values of the image have not been changed at this point - only the LUT values which are used to display them. The other option is to change the pixel values, and use the original linear LUT. This can be done using the Process - Apply LUT command and is necessary if the image is to be combined with another image with a different LUT. The appearance of the image will not change after using Apply LUT , but the color bar in the LUT window will change back to the original ramp, and the graph in the Map window will change back to the diagonal straight line.
A more severe enhancement is performed using the Process - Equalize

command. This can be done immediately (if you have not just used Process - Apply LUT ) because the image pixels are unaffected, and only the look up table is changed. It is not necessary to revert to the original image (with the Revert To Saved command). Equalization divides the pixels of the image as evenly as possible among all the brightness intervals. In other words, each gray level is used to display about the same number of pixels. This has the effect of bringing out detail in the background or larger areas of the image, while loosing contrast in the small features (Fig. 3). Now the thinner particles are evident, the pores of the filter are obvious, but detail is lost inside the particles. Note from the Map window that this enhancement (for this particular image) can be approximated with linear contrast enhancement by noting the position of the curve, clicking in the Map window, and then moving the line to about the same position.
The LUT can be shaped by hand with NIH Image - hold down the option key and move the cursor around the Map window, holding the mouse button down. A example is shown in Fig. 4, where the image was first contrast enhanced with the Process - Enhance Contrast and the Process - Apply LUT commands. The option key was then held down while drawing the curve in the Map window with the mouse.
To restore the image, use either File - Revert to Saved , which will read in the original file again, completely restoring the image, or Options - Gray Scale , which resets the LUT only .
Another enhancement procedure that works well on this image is to take the logarithm of it using the Process - Arithmetic - Log command, then switch to a false color LUT using Options - Color Tables - Fire-1 command. The Log operation does affect the image pixels, which is what is desired because the Fire-1 LUT is applied next to the new values. This LUT is also called a thermal scale (Fig. 5), and is one of the most intuitive false color scales.

The changes in the pixel values can be seen easily by looking at the histogram before (Fig. 6a) and after (Fig. 6b) the log operation, using Analyze - Show Histogram . The changes in pixel values will also be seen in the Info window, as the cursor is moved around the image, but they will be less obvious because the logarithms have been rescaled to integer values (actually 8 bit byte values) between 0 and 255, which is the same value range that the original image had.
An additional comment on color scales: after switching to the finder or another application, especially with the 'Load System LUT when Switching' preference checked, the LUT might look as it does in Fig. 7. This LUT, the system LUT, renders the current image meaningless, but it is used to display color photographs and drawings with images designed to use it. Generally, the limitation of 256 colors means that images are displayed with proper colors, or the finder icons and menus are displayed with proper colors, but not both. This behavior is affected by the "Load System LUT when Switching" box which is accessed by the Options - Preferences... command. When the box is checked, then the image and color bar will assume crazy colors (due to the system palette) when in the finder, but the icons of the finder will appear normally. If the box is not checked, then the image will appear correctly, the LUT may have a few scattered colors, and the finder icons will be black and white. The image and color bar are restored to their original appearance when switching from the finder back to NIH Image. When the monitor is set to other color settings, such as millions of colors, the image and color bar may not be automatically restored. If the grayscale image is not restored on switching back to Image, or if you have modified the color scale in some way, do it manually with the Options - Grayscale command.

Intensity Profile
An often used analytical tool for micrographs is the intensity profile. In NIH Image, the profiles are obtained using the profile plots tool (the graph icon in the Tools window), which is controlled by the line width tool (the lines of varying width in the lower right of the tools window). Working with the log of the sample image, Fig. 8, shows two profiles across a pore, one showing single pixel values along the line and the other the averages of several pixels along the fat line. To observe the profiles, one after the other, click on the thinnest line of the line width tool to select this thickness - a check mark will appear to the left of this line. Then click on the line profile tool. Finally, move the cursor to the position in the image for one end of the line to profile, and holding down the mouse button, drag the cursor to the other end of the line and release the mouse button. The 'rubber band line' will show the location for the line of the profile while the mouse button is down, and then will be replaced by a 'marching ants' line and a plot window upon release of the mouse button. To see the averaged profile, leave the marching ants line in the window, click on the fat line (bottom one in the line width tool) and then invoke the Analyze - Plot Profile command again. The former plot profile will be replaced by a new one reflecting the averaging of pixels across the width of the fat line. The 'marching ants' line will also be replaced by a 'marching ants' rectangle outlining the area of the image represented in the plot. Note that moving the cursor around the Plot window will display the position and amplitude of the plot in the Info window. The marching ants always represent a selection area in NIH Image, which can be erased by selecting a selection tool (the rectangular one for example), and clicking anywhere within the image.

Thresholding, Smoothing and Counting Particles
Discrete objects such as particles, cells, filter pores, etc. can be sized and counted with NIH Image. Start with the original test image, or restore it with the Revert to Saved command. To count and size the pores in the filter, enable the bi-level thresholding mode using Options - Density Slice (or double-click on the LUT tool) and set the threshold limits in the LUT window with the LUT tool to 243-254. Do this by double clicking on the LUT tool. A red band will appear in the color bar, and the corresponding pixels in the image will appear red. Drag the top of the red band in the color bar with the LUT tool to the top of the LUT window. Practically all of the image will appear red. Drag the bottom of the red band with the LUT tool to value 243 - read the values in the Info window. Now the red areas are associated mostly with the pores of the filter. (Fig. 9)

Note that intensity levels of 0 and 255 are not accessable for thresholding. I.e., zero and 255 can't be within the density slice because those entries in the lookup table can never be colored red. 0 is always white and 255 is always black. This is intentional, to prevent loosing the mouse, tools, etc. due to thresholding. If an image shows saturation, either in the black or white, then pixels with values of 0 or 255 might likely appear within the areas of interest, but not be selectable via thresholding. If the image has values of 0 that need to be selected, for example, they may be changed to value 1 using the Process - Arithmetic command twice: 1) Arithmetic - subtract , then use 1 as the constant to subtract. NIH Image clips results of arithmetic so that they lie within the range 0-255, so this subtraction results in all pixels being lowered by one intensity unit, except for the zero valued pixels which are still zero. Note that this affects the internal pixel values - if the Invert Pixel Values box is checked, then the values to read are in the parentheses to the right in the Value: line of the Info window. 2). Arithmetic - add , then use 1 as the constant to add. This results in all pixel values being restored, except for the old zero valued pixels, which now have the value 1, along with the pixels which used to have the value 1. Use the reverse arithmetic sequence to lower pixels with value 255 down to 254. (Note 1. This can also be done using Process - Fix Colors .) Noise in the image causes lots of little red selected areas and some holes in the red dots representing the pores. Most of the noise in this case can be ignored by the Analyze - Analyze Particles command by setting the 'Min Particle Size' to something greater than the area of the small red dots (such as 10 pixels), and by checking the 'Include Interior Holes' box. Alternatively, smoothing the image will get rid of a lot of the dots (Fig. 10). Do this with the Process - Smooth command.
When using the LUT tool, holding the option key down will temporarily turn the red highlights off, allowing for closer inspection of what is being selected. Double-clicking the LUT tool will toggle the red highlights on and off. The zoom tool (the magnifying glass) and the scroll tool (the hand) can also aid inspection.
To count the holes, first choose the parameters to be measured by clicking the appropriate boxes shown in the Analyze - Options dialog box if desired. The defaults - Area, Mean Density, X-Y Center, and Include Interior Holes were used here. Invoking the Analyze - Analyze Particles

command and then checking all of its boxes, results in Fig. 11. The numbers by the pores correspond to the numbers in the table of results, viewed using the Analyze - Show Results command (Fig. 12). There are various ways to save this table, using the File - _Export... command (see About NIH Image), for example it can be exported as a text file for importing into other applications or incorporating into a report.
Figure 13 shows the thresholded particles rather than the pores. The threshold levels were set at 1 and 216. Fig. 14 shows the outlined particles. This time, the 'Label Particles' box was not checked.

Size calibration and photographing

A version of the TEM filter image, rendered for publication with a micrometer marker and with a black background, is shown in Fig. 15. This figure was made using the calibration feature of NIH Image, the Text tool, the line drawing tool, and the Photo Mode command:
Get a version of the image with suitable contrast using the Revert to Saved command, the Process - Enhance Contrast command, and the Process - Apply LUT command.
The image field of view is 15 m wide. Click on the line selection tool. Make a line selection across the entire width of the image. Invoke the Analyze - Set Scale command. Select Micrometers for the units, then type in 15 for the known distance. Click 'OK'.
Click on the widest line in the line width tool. Then click on the Draw Lines tool (just below the paintbrush). Draw a horizontal line with the mouse in an uncluttered area of the image, keeping an eye on the length in the Info window. When the line is as close as possible to 1.0 m long, release the mouse button and the rubber band line will be replaced with a fat line or rectangle for the marker. If this line is not the correct color, IMMEDIATELY use the Edit - Undo Editing command to erase the line. Select the appropriate color as indicated by the color of the paintbrush (see above), and draw the line again. (Note 2. This could also be done by creating a 1 �m wide rectangular selection and using Edit - Fill .) Next, click on the Text tool ('A' in the Tool window), and click this tool to the right of the micrometer bar. Any time up to and including this point, use the Options - Size - 18 command and the Options - Style - Bold command, to make the type suitably large on the image. Then type "1 option-m m " to write "1 m" directly on the image to annotate it. Use the File - Save As... command to save this file under a different name, if desired, as this has permanently modified the image.
Select a background color by clicking on the erase tool, then clicking inside the LUT window - where the erase tool has temporarily changed to the eye dropper tool (the paintbrush tool will work in a similar fashion, but it is the color of the eraser that affects the background color.) The color of the eraser will change to the color clicked on with the eye dropper tool. Black was selected for this figure. Move the image to the desired position on the screen by dragging the title bar of the image window. Invoke the Special - Photo Mode command. The entire screen will turn black, except for the image, which appears without the title bar. The screen can be photographed directly, or using a slide copy camera. Alternatively, the image can be cut and pasted into a larger blank window of the desired color using the File - New command - the new window will have the color of the eraser. This larger window can be printed using the File - Print Image command.

Illusions

A couple of illusions of the visual system are illustrated well with NIH Image: Mach Bands, and the brightness vs. background illusion. Both of these affect how we see our data if the data is represented as images. The first can simulate a gradient (of intensity, concentration or whatever) where there is none, and the second can alter the interpretation of numerical values from gray levels using annotated scale bars.

Mach Bands
To generate Mach bands, first open a blank window using the File - New command. Then select the entire window using Edit - Select All , and fill it with a gray level ramp using Edit - Draw Scale . After setting the number of gray levels to a number, such as 6, with Options - LUT Options , the ramp will appear as a series of stripes, which appear slightly shaded like Venetian Blinds (Fig. 16). The bars are actually uniform, as can be seen by covering up all but one bar, by using the line profile tool, by inspecting the Info window while moving the cursor over the bars, or by doing a mesh plot using Analyze - Surface Plot . Notice that it is necessary to use Process - Apply LUT before profile plots and mesh plots show the bands. The Mach bands are caused by the abrupt intensity change at the edges, which the eye further enhances.

Foreground/Background

Often, annotated gray scale wedges or color bars are used to infer numerical values from images using the gray scales or colors. In some instances, in particular when a more natural or smooth color scale is used, this can be prone to error.
To show the illusion, fill a window with a gray level ramp as with the Mach Band illusion. If the 'crawling ants' border is still there, get rid of it either with Edit - Deselect All or by clicking on the image with any tool except the four to the upper right. Then select the rectangular selection tool, and click and drag it to create a small rectangular selection. Copy the selection and paste it back where it was using Edit - Copy and Edit - Paste .
The selection can now be moved about the window by dragging it with the mouse - do not click outside this region until you are finished. The selection will appear to change in brightness as it is moved across the ramp. If it is pasted in various places on the ramp, it will appear to have different gray levels, which in fact it does not. (Fig. 17). The illusion persists when using natural color scales such as the thermal scale (Fig. 18) which is applied using the Options - Color Tables - Fire 1 command. The illusion does not persist, however, when using color scales such as the spectrum color scale (Fig. 19), applied using the Options - Color Tables - Spectrum command that the eye perceives as 'op art' rather than as a shaded image.

If our perception of gray levels is faulty here, then our perception is also likely to be faulty when 'matching' gray levels in an image to a gray ramp or color bar.

Multiple images (stacks)

NIH Image has the capability of handling stacks of images, such as those making up a three dimensional data set. 'Boron stack.pics' is an example of a stack of 50 Boron images (Gillen, 1994) of grain boundaries in a steel sample. The images were taken at equal time intervals with an ion microscope, which etches away the surface at a more or less constant rate. The images thus approximate serial sections of the steel sample. (The stacks can be saved in two formats - PICS and TIFF. The boron stack requires 618K in PICS format and 4232K in TIFF format. The TIFF format is more widely used, but the PICS format was used for this example to save space.)
Load the stack using the file - open command. (This will require more memory than the 4000K recommended for all the preceding steps. With 4000K of memory assigned to NIH Image, only 13 of the 50 slices will be loaded. The following steps can be performed on this abbreviated stack, however.) The images will be displayed one by one as they are loaded, giving a view going down through the sample. When loaded, the stack is displayed with the Fire-1 color table, or thermal scale (Fig. 20). Note the �(1/50)� in the title bar indicating that the image shown is the first of fifty images in the stack. (Note 3. The white diamond in the title bar indicates that the image is density calibrated, in this case, because �Invert Pixel Values� was selected.) The > and < keys or the Stacks - Next Slice and Stacks - Last Slice can be used to view other images in the stack. The options - grayscale command can be used to view the images as they were originally acquired. Under some conditions, such as after switching back from the finder with the 'Load System LUT on switching' option set with the options - preferences command, the system palette, which is useless for this data set, will be in effect. The gray scale or thermal scale can be restored using the either the options - grayscale command or the options - color tables - Fire-1 command.
The stack can be viewed in a variety of ways. The stacks - animate command cycles through the images, showing them as a movie. The stacks - make montage command shows images taken in equal intervals throughout the stack as a composite (Fig. 21).
Stacks can be viewed from other perspectives, treating the layers of the stack as another spatial dimension. When the number of images in the stack is much less than the number of pixels along the side of one of the images, the images need to be replicated to keep the volume as a whole from looking too thin. This can be done by loading the stacks macro file (in the Macros folder) with the Special - Load Macros command. (The macro capability give a powerful way to automate NIH Image. To read the macro files, open them with NIH Image as a text file (with File - Open) , or peruse them with another text editor. Programming details can be found in "About NIH Image" and "Inside Image".) The figures that follow were made with the stack after using a replication factor of 3 with the newly loaded Special - Replicate Slices command. This triples the amount of memory required (17 MB assigned in the File - Get Info dialog box after selecting NIH Image in the finder), but thickens the stack so that the slice - slice distance approximates the pixel-pixel distance within an image. The replication of the slices renders the grain structure with the correct scaling when viewed from the side or from oblique angles. If that amount of memory is not available, the following steps can still be performed. The aspect ratio of the flattened images can be corrected using the Edit - Scale and Rotate... command.
Using the Stacks - Reslice command, the stack can be viewed in a plane perpendicular to the plane of the images as determined by the line selection tool (Fig. 22). The resulting image is shown in Fig. 23.
The stack can also be rendered as a synthetic volume using the Stacks - Project command, which shows the images as if they were printed on clear plastic, and stacked on one another in order to show the three dimensional object as embedded in a plastic cube. The command generates a new stack of images, each image being a synthesized view of the 'plastic cube' as viewed from a particular angle. The angles are chosen at equal intervals of rotation about the x, y or z axis so that when the new Projection stack is animated, the 'plastic cube' appears to rotate, giving a strong illusion of three-dimensionally. There are a variety of options to this command, that affect the opacity of the 'plastic' and the structures in the image, all of which are explained in 'About NIH Image'. Fig. 24 shows the first image, generated with the default settings, except that 'Brightest point' rather than 'Nearest Point' was used. Fig. 25 shows a montage of this stack. This operation takes about 2.5 minutes per frame on a Mac IIfx or about 22 seconds per frame on a PowerMac 6100/60. This operation also requires more memory, however there is an automatic option to store the projected stack on to the disk as separate images when sufficient RAM is not available. They can then be recombined later into a stack, and animated to view the grain structure in 'simulated 3-D'.

Conclusion

Many capabilities of NIH image could not be covered in a paper of this length. Among these are image arithmetic, background subtraction, operation of frame grabber cards, and using the rapidly increasing number of plug-ins. Use of these is covered in 'About NIH Image'.

This paper is to give the microscopist hands-on experience with some of the tools provided by NIH Image for the inspection and analysis of digital images. Many laboratories use it on a daily basis for acquiring images, sharing data and writing papers, as well as for analyzing images and enhancing them.

Published research assisted by the use of NIH Image should use the following quote in the materials and methods section "... analysis performed on a Macintosh <model> computer using the public domain NIH Image program (written by Wayne Rasband at the US. National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nih.gov.

NIH Image is an indispensable tool for the analysis and sharing of digital micrographs.

References

Gillen 1994, Image stack provided by Dr. Greg Gillen, Surface and Microanalysis Science Division, NIST. (personal communication)

O'Neill, R.R., Mitchell, L.G., Merril, C.R., Rasband, W.S. (1989) "Use of image analysis to quantitate changes in form of mitochondrial DNA after x-irradiation", Applied and Theoretical Electrophoresis 1: 163-167.

Steel, 1993, TEM Filter image provided by Dr. Eric Steel, Surface and Microanalysis Science Division, NIST. (personal communication)

Figure captions

Fig. 1-- Portion of screen containing image "TEM filter sample.tiff" as loaded, along with the menu bar and the LUT, Tools and Map windows of NIH Image. Note overall low contrast, but appropriate contrast for detail in the more dense particles. (Figures that follow do not include the menu bar, which is included here to show the appearance of the entire upper left corner of the Mac screen.

Fig. 2 -- TEM filter sample image in Fig. 1 with linear contrast enhancement as done by the Process - Enhance Contrast command. Note that the slope of the line in the Map window has increased, the black to white region in the color bar (LUT window) is shortened, and that more of the particles with less contrast and more of the filter substrate can be seen.

Fig. 3. -- Filter sample image with histogram equalization.

Fig. 4. -- Filter sample image with hand drawn LUT.

Fig. 5. -- Thermal scale enhancement. Logarithm of filter image (Process -Arithmetic - Log command) false colored with the thermal scale (Options - Color Tables - Fire-1 command).

Fig. 6. -- a) Histogram of original image. Observe using the Revert To Saved command and then the Analyze - Show Histogram command. b) Histogram of Logarithm image. Observe using the Process -Arithmetic - Log command and then the Analyze - Show Histogram command.

Fig. 7. -- Filter image shown with system LUT. This is rarely useful for analysis, but sometimes occurs upon switching to and from the Finder.

Fig. 8. -- Illustration of the line profile tool. -- plot across the filter, a pore and a particle. Left inset - plot of single pixel values along line as shown. Right inset - plot of averaged pixel values along a 'fat' line (not shown), with width of bottom line in the line width tool.

Fig. 9. -- Filter image with pores selected using thresholding (see text).

Fig. 10. -- Pores in smoothed TEM Filter image selected by thresholding.
Fig. 11. -- Result of Analyze Particles command. Pores are outlined and numbered.

Fig. 12. -- Table of results corresponding to Fig. 11 (only first 12 rows shown), shown with Show Results command.

Fig. 13. -- Particles in smoothed TEM Filter image selected by thresholding.

Fig. 14. -- Result of Analyze Particles command. Particles are outlined, except for the one touching the edge at the lower right.

Fig. 15. -- Contrast enhanced version of the Filter image suitable for publication, with micrometer bar. Black background not shown. (The figures were made by 'capturing' the screen (shift-command-3), a function provided by the Macintosh operating system, or by pieces of software such as 'Capture'*. Capturing does not work in the photo mode, so only the image window is shown in this figure.)

Fig. 16. -- Mach Band Illusion, generated with NIH Image (see text). Bands appear darker on the left and brighter on the right although they are of uniform color.

Fig. 17. -- Foreground/Background illusion (see text). The small rectangles all have the same shade of gray, although they appear to have differing shades of gray.
Fig. 18. -- Foreground/Background illusion persists when a thermal scale is applied to the image.

Fig. 19. -- Foreground/Background illusion does not persist with 'unnatural' or 'false color' type color scales. This scale applied to the image with Options - Color Tables - Spectrum command.

Fig. 20. -- The stack of images 'Boron Stack.pics' as it appears after loading. First image in the stack of 50 is displayed, with a thermal scale. Image dimension - 100 m.
Fig. 21. -- Montage of the Boron Stack, with an Increment of 2 in the Stacks - Make Montage... dialog box.

Fig. 22. -- Line selection for viewing stack along plane perpendicular to the images - see next figure.
Fig. 23. -- Boron stack viewed perpendicular to images in plane intersecting dotted line, Fig. 22.

Fig. 24. -- Synthetic volume rendering of Boron Stack, done with the Stacks - Project command (see text). First frame - top view, gray scale.

Fig. 25. -- Volume rendering of boron stack - montage of every other frame showing different rotations, gray scale. The three-dimensional structure of these grain boundaries is more apparent upon viewing the animation of this stack using the Stacks - Animate command.

NIH Image: An Introduction and Tutorial

Conclusion

References

Figure captions