GREY-SCALE TO BINARY IMAGE

A common requirement for a number of measurements with NIH IMAGE is a binary (black-on-white) object with a one-pixel-wide border.  The following is a way to obtain such binary images from grey scale by using NIH IMAGE.
It is important that the initial, grey-scale image have as high a contrast as possible, i.e., be a nearly-binary image to begin with.  This is primarily a matter of proper histological preparation and/or video digitization (1).


HOW TO GENERATE BINARY BORDER IMAGE WITH NIH IMAGE:

A.  Preliminaries
	1.  load NIH IMAGE program
	2.  open a gray-scale image, invert (if necessary, i.e., the image object should be dark on light background).
	3.  the image should be in TIFF format.  If not, save as TIFF and reopen this new image.  Close original image.

B.  To produce a binary image
---NOTE:  It may be advisable to make a duplicate (copy) of the image and work on the copy.  Then, one can check back to the original image as a reference for fidelity of operation.

	1.  select grey scale image object with rectangular box, if desirable for faster processing.  (Not absolutely necessary.)
	2.  pull down Enhance menu and choose Convolve... .
	3.  locate Convolution Kernel Folder and select (with double click) a Mexican hat kernel (named: HAT N, say, HAT11.)%
	4.  Following convolution operation, double click on the paint brush icon in the Tools list and choose an appropriate sized brush.  Then, isolate the object from non-object (background) with cuts (brush filled with white) and join the unconnected object parts and remove internal structures (with black-filled brush).  Use the eye dropper in Tools list to select white or black.
	5.  fill the object with paint bucket tool with medium gray color (gotten with eye dropper in LUT)
	5.  pull down Option, click on Threshold (or Density Slice) and make object red by adjusting red area in LUT. Then, pull down Enhance to Binary and select Make Binary from the sub-menu.  If the complete object was not obtained, then re-fill the object with black bucket and join missed part with a black brush.  Then repeat 5.
	6.  pull down Enhance to Binary and select Make Binary from sub-menu.
	7.  There may be some non-object still present.  If so, fill the object again with the paint bucket with medium gray.
	8.  pull down Option and select Threshold (or Density Slice) and make object red by adjusting red area in LUT
	9.  pull down Enhance to Binary and select Make Binary
	10. select object with rectangular box#
	11  pull down on Enhance to Binary and select Outline from the sub-menu to get one pixel wide border.
	12  save the binary, one-pixel-wide object as FILENAME.TIFF and use for IMAGEFRACTAL or other input.

# May not be necessary or desirable.
% The Kernel folder is part of the package obtained from zippy.  The kernels can be modified and saved with any text-only program.

(1)  T. G. Smith, Jr. et al., J. Neurosci. Methods, 26: 75-82, 1988.