For each digitalized image a frequency histogram of fluorescence intensity was generated: in this histogram a threshold was established to eliminate non-specific fluorescence, pixels with fluorescence intensity above the threshold were considered specific for SERT-IR.
Each digitalized image included a millimetre measurement scale placed adjacent to the gonads to permit gonad size to be determined from the photograph.
Slides were pictured at 200 magnification using an Eclipse 800 microscope (Nikon, Champignysur- Marne, France) and a digital camera; labelling was then analyzed by quantitative digitalized image analysis using Lucia software (Nikon).
The digitalized image was obtained in a storm scanner (Storm 680, Molecular Dynamics, Inc .. Expression profiles were obtained with Atlas image 2.0 software (Clontech, Palo Alto, CA, USA) through comparison of normal vs. tumor tissues.
Cores were evaluated as blinded digitalized image files.
The image of the valleculae prior to swallowing is obtained by freezing the digitalized image on the computer screen.
The digitalized image was segmented manually using NDP Viewer (Hamamatsu, Japan) to identify the lumen contour, fibrous tissue, lipid and hemorrhage, etc.
(C ) Next, we calculated the orientation of each actin filament relative to the polarity field vector for each pixel in the digitalized image.
At a magnification of ×10, automated sequential digitalized images were taken and stored, then a mosaic picture was created including all the images with minimal field overlapping.
The gels of 2DE were recorded as digitalized images using a Calibrated Densitometer (GS-800, Bio-Rad, USA), and the search for differentially expressed proteins was carried out using the software of PDQuest Advanced 8.0.1.
