In this experiment, image processing techniques are applied to analyze the morphology of the stained cells by using the Shape Factor Determination Formula.

 

Objective:

 

To analyze the morphology of cells using an online image processing program.

 

Theory:

 

Morphological analysis is important to study the cellular organization and the physiological state of the cells, and thus it can be commonly used as a qualitative and quantitative measure of various biological assays. Analysis of cell morphology remains increasingly important, as the image analysis aids in the detailed examination of microscopic cells; study of cell behavior, and also provides quantitative measure of its curvature, area, perimeter, eccentricity and additional metrics of nuclear morphology for large populations of cells.  Analysis of the cells based on their morphological differences is applied to study the differentiation of stem cells, cancer cells, and in hematology. A wide variety of image analysis software packages have been developed that helps to convert to the microscopic images into more relatively quantitative measurements. In this lab, cell morphology of a stained embryo cell is analyzed using a public domain Java image processing program ImageJ. The basic steps involved in the cell analysis are as follows:

 

1. The embryo cell to be analyzed is imported into the software.
2. The basic measurements to analyze the cell morphology are then adjusted using the software menu bar.
3. A selection is made on an appropriate area of the embryo using the oval selection tool and the measurements are analyzed using the Shape Factor Determination Formula.
    

 

Staining:

 

Staining is usually done to enhance the contrast of a cell. Microscopic examination of the cells depends on proper specimen preparation prior to observation which helps to increase the visibility and clarity of the cell under study. Fixation is a process to preserve internal and external structures of a cell.  Cell should not undergo any change in its state during the process of staining process. To ensure this, it is necessary to toughen the cell and to stop any chemical reaction that might take place within the cell that is the cell should be fixed at its current state.  Based on the method of fixation, a microscopic slide might be used for longer or shorter period of time or used as temporary or permanent slides. Even when great care is taken to fix the cells properly, sometimes artifacts might be produced. Fixation step must therefore be standardized. Heat fixation and chemical fixation are the different methods used to fix a cell.  The procedure for fixation varies with the fixative and the type of tissue or cell under study.

 

For more details refer Cell Biology Virtual Lab I Cell Organization and Sub Cellular Structure Studies (Prokaryotic and Eukaryotic)

 

Image Analysis:

 

The microscopic image can be further used for the quantitative analysis of the cell such as its shape, area, perimeter, Feret’s diameter (describes the mean distance between pairs of parallel tangents to the projected outline of the selected cell) and other features of the cell using online software. The analysis of the cell morphology depends on the shape factor (S) determination factor. Shape factors are dimensionless quantities used in microscopy and image analysis techniques that numerically describe the shape of a particle, independent of cell size. It can be calculated from the measured dimensions such as diameter, perimeter, area and other dimensions of the selected cell.

 

Shape factor (S) is given by the equation

 

 

                        Where,

                                                        A = the area of the cell

                                                        P = the cell perimeter

                                                         = 3.14159
 

 

Shape factor value helps in the measure of the circularity or spreading of cells in the microscopic image. Shape factor values are often normalized and hence its value ranges from 0 to 1. Circular cells a shape factor equal to one and less than 1 or approaches to zero indicates an elongated cell.

While performing the morphology analysis of cell using image analysis software, it is critical to wash the stained cells properly to avoid most of the experimental errors.