Page 301 - Fundamentals of Light Microscopy and Electronic Imaging

284 DIGITAL IMAGE PROCESSING

1. Save and duplicate the raw image file. Use the duplicate for processing; the orig-
inal is always available in case you make a mistake.
2. Flat-field-correct the image to restore photometric accuracy of pixel values. As
a bonus, this procedure removes artifacts from microscope optics, the illumina-
tor, and the camera.
3. Adjust brightness and contrast with histogram stretching. This is important for
displaying all of the meaningful content of the image.
4. Adjust the gamma ( ) to allow exponential scaling for displaying bright and dim
features in the image.
5. Apply the median filter or a blurring filter to reduce speckles and noise.
6. Apply unsharp mask or other sharpening filters to increase the visibility of
details.

PRELIMINARIES: IMAGE DISPLAY AND DATA TYPES

Software programs display images on the computer monitor with the help of a look-up-
table (LUT), a conversion function that changes pixel input values into gray-level output
values ranging from 0 to 255, the 8 bit display range of a typical monitor. Manipulation
of the LUT changes the brightness and contrast of the image and is required for the dis-
play and printing of most microscope images. For some programs like IPLab (Scanalyt-
ics, Inc., Fairfax, Virginia) adjusting the LUT only changes how the image is displayed;
it does not alter the values of pixels in the image file. When an adjusted image is saved,
the LUT settings are appended to the image file, but the pixel values remain unchanged.
Some software programs show the LUT function superimposed on or next to the
image histogram, a display showing the number of all of the individual pixel values
comprising the image. This presentation is helpful in determining optimal LUT settings.
The default settings of the LUT usually assign the lowest pixel value an output value of
0 (black) and the highest pixel value an output value of 255 (white), with intermediate
values receiving output values corresponding to shades of gray. This method of display
guarantees that all acquired images will be displayed with visible gray values on the
monitor, but the operation can be deceiving, because images taken at different exposure
times can look nearly the same, even though their pixel values may be different. How-
ever, as will be pointed out later, images acquired with longer exposure times have
improved quality (better signal-to-noise ratio). The default LUT function is usually lin-
ear, but you can also select exponential, logarithmic, black-white inverted, and other
display functions.
Data types used for processing are organized in units of bytes (8 bits/byte), and are
utilized according to the number of gray levels in an image and the number of gray lev-
els used in image processing. The names, size, and purpose of some data types com-
monly encountered in microscope imaging are as follows:

8
• Byte files contain 8 bits (1 computer byte), giving 2 or 256 gray-level steps per
pixel. Although byte format is used for display and printing, large format data types
do not have to be saved in byte format in order to be printed. Conversion to byte for-
mat should only be performed on duplicated image files so that high-resolution
intensity values in the original image are not lost.