8.4 Example: Image Analysis of Nulcei Using Hoechst33342

Figure 8.8: Stuctural Formual and Emission and Absorption Spectrum for Hoechst33342

The above graphic shows the structural formula and the emission and absorption spectrum for Hoechst33342.

8.4.1 Analyzing Nuclei

Figure 8.9: Nuclei of Cells in Medium Stained with Hoechst33342

The nuclei’s intensity is a result of the chromatin DNA’s and the background’s intensity.

Figure 8.10: Blur Filtration

Note that I_background represents the mean background intensity. If the background is constant (i.e., which is the case), I_background could be subtracted from the filtered image.

8.4.2 Segmentation and Generating ROIs

Figure 8.11: Segmentating and Generating ROIs for Hoechst33342-Stained Cells

Segmentation was done via trial and error.

While the ROIs could be determined automatically, they can be further visually inspected to remove bad ROIs - for instance, dirt particles or cells at the border of the image.

8.4.3 Analyzing Nuclei Number, Size, and DNA Content

Figure 8.12: Image Overlaid with ROIs

The following could be quantified from the above image:

1. Number of Nuclei

   This is the number of cells.

2. Area of Nuclei

   This indicates the size of the nucleus. It is denoted as: \(\displaystyle \text{number of pixels} \times \text{area of pixel in $\mu$m$^2$}\).

3. Mean Intensity per Nucleus (i.e., I_nucleus)

   This indicates chromatin density.

4. Total Intensity of Each Nucleus

   This is given by \(I_{nucleus} \times \text{number of pixels}\) and denotes the amount of chromatin.