New modified 2D Otsu thresholding method

• This method is extension of 2D Otsu method; the threshold values obtained from this method are
and the average grayscale threshold is optimized using this new method.
Approach to Optimization of average grayscale threshold:
• First, is determined using the logarithmic plot of pixel frequency distribution. is rough estimate
of the background class separating the image class.
• Using the and , is calculated as shown in Equation 1.
• The is optimized in an iterative way as shown in Equation 2 & 3.

(1)
(2)

(3)
New modified 2D Otsu thresholding method
Approach to Optimization of average grayscale threshold:
• Error is defined as the difference between the number of particles estimated from weighted
median and 2D Otsu method.
• Convergence is achieved when error becomes zero or is constant for three consecutive
iterations.
• The method returns , are final thresholds for the processed image.
• Morphological operations are done on the binary image to improve the image.

Fill Close

Figure: Morphological operations applied on the binary image.

Final approach for analysis
Image Imported to MATLAB from Florescence Microscope

De-noising Steps Median Filtering TV algorithm

Background Detection and Removal

Thresholding using Modified 2D Otsu Method

Morphological Operations Fill & Close

Data Extraction

Figure: Finalized image analysis approach for images of florescence microscope.