Obtaining Densitometric Parameters
In this study, fluorescent images were not invesigated, which limits the amount of analysis that can be conducted on densitometry.
This study proposes a method of deriving percentage coverage, cell coverage area, number density, and number of cells in a given area, through the use of densitometric (gray value) calculations (this method was constructed after learning the range of capabilites of the ImageJ software in analysing morphometric parameters).
Similar to the methods explained in the section of Morphometry, the process of threshold, watershed and fill holes were used (as shown in Figure 6). However, in the analysis stage (Analyse > Set Measurements and Analyse > Analyse Particles), only Area and Mean Gray Value are needed in the calculation.
The goal is to obtain the area and mean gray value of a selected region within the image (as shown in Figure 6). This can be done using the Rectangular Selection on ImageJ's toolbar. After a region is selected, inAnalyse > Analyse Particles, select Mask to derive mean gray value and area of the rectangular selection.
Before continuing this method, I will explain the concepts key to understanding the method and its limitations. Firstly, in the selected region (as shown in Figure 6), the mean gray value denotes the amount of black pixels in that selection. This is because there is only two pixel values in that selection: black (255 intensity) and white (0 intensity). So for example, if half of the selection is covered with black pixels, the mean gray value would be 255/2=127.5. Hence, the ratio between mean gray value and maximum gray value (255), denotes the percentage coverage of black pixels in that selection, which is the percentage coverage of red blood cells.
Figure 6. Example of analysis image used to calculate percentage coverage, cell areal concentration, number of cells in a given area. Rectangular selection highlighted in yellow.
Area of selection is 5046um², mean gray value of selection is 141.748.
1. Introduction
2. Equations
To continue this method are a list of equations with explanations.
2.1 Obtaining Percentage Coverage, Cell Coverage Area, and Cell Number Density
As explained before, percentage coverage is the ratio
between mean gray value and maximum gray (255).
The Cell Coverage Area, which is the actual area covered by cells in the selection, is calculated as shown.
The number of cells (N) in the selection is approximately 25 (Figure 6).
The areal number density of red blood cells (shown in the equation as: n followed by subscript A)is the degree of concentration of countable objects: red blood cells.
In a given amount of cell coverage area, it is reasonable to assume there is a translatable approximate number of red blood cells, since the value of the area of red blood cells are relatively homogenous. However, it is crucial that there is no significant amount of overlapping between cells as this will result in the cell coverage area to be significantly lower than the total area of cells within that coverage. In this method, a constant k is created that denotes the ratio between number of cells and cell coverage area.
Using the recorded values of number of cells and cell coverage area, k can be calculated. It is important that the selected region is large enough so to take into consideration the range of red blood cell area, and avoid solely using cells that are clearly outliers in terms of their area when conducting the calculation of k.
After obtaining the value of k, a tranferable equation that denotes the areal number density of red blood cells can be derived.
The equation can be further simplified since their is also a constant in the calculation of percentage coverage - the denominator is 255 (which is maximum gray value).
2.2 Deriving a Constant
2.3 Finalising the Constant and Transferable Equations
