Plane Brightness Adjustment Plugin
Jan Michalek (michalek at biomed dot cas dot cz)
Martin Capek (capek at biomed dot cas dot cz)
Jiri Janacek (janacek at biomed dot cas dot cz)
Download Plane_Brightness_Adjustment.jar, change its name to PlaneBrightnessAdjustment.zip and uncompress to retrieve the source code.
Drag and drop Plane_Brightness_Adjustment.jar to the "ImageJ" window, when "Save Plugin..." dialog appears, we recommend to put the file in the plugins>Stack folder. Or download the .jar file to the ImageJ>plugins>Stack folder directly and use Help>Refresh Menus command. The plugin will then be in the Plugins>Stacks menu item.
In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image post-processing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover.
This plugin is designed to perform processing on 8-bit grayscale and RGB images, and is able to work with images in stacks.
Since a median filter of a large kernel is used in the algorithm, the processing of large images, especially RGB, may be time consuming.
Dialog box parameters:
Slope means the maximum allowed change (an 8-bit integer) of the Lipschitz filter between intensities of two neighboring pixels. Values from 1 to 5 often yield good results.
Threshold means from which pixel value the adjustment starts to work. The values below threshold are not corrected.
Maxfactor is a limit imposed on the factor with which the grayscale values are multiplied to increase brightness. If too many pixels get saturated by PlaneBrightnessAdjustment, decrease Maxfactor. If brightness is still inhomogenous after PlaneBrightnessAdjustment, increase Maxfactor. A value of Maxfactor=8 may be tried first.
 Michalek, J. - Capek, M. - Mao, X. W. - Kubinova, L. Application of morphology filters to compensation of lateral illumination inhomogeneities in confocal microscopy images. Analysis of biomedical signals and images. Brno : University of technology, 2010. p. 49-54. ISBN 978-80-214-4106-4. ISSN 1211-412X. [International EURASIP Conference Biosignal /20./. Brno (CZ), 27.06.2010-29.06.2010]
 Michalek, J. - Capek, M. - Kubinova, L. Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy. Microsc Res Tech. 74(9) 2011, pp. 831-8. DOI: 10.1002/jemt.20965
See also: Our plugin StackContrastAdjustment.
CLSM stack of images 7-microns apart from a rat brain specimen: (a) the original images, (b) the reciprocal of the Lipschitz-cover estimated gain, (c) images corrected after applying the upper Lipschitz cover morphological operator. HC PLAPO 20x water immersion objective (N.A. = 0.70), the excitation wavelength of 488 nm and emission wavelength range from 500 to 600 nm were used. The size of each image is 550 x 550 microns.