# Analysis of Compound Stained Cervical Cell Images Using Multi-Spectral Imaging
## 3 Experiments and Results  
### Pseudo-Color Image Synthesizing and Absorbance Unmixing  
The process of multi-spectral imaging is as follows: (1) Turn off the light source and obtain an image of the dark background. The dark background image contains the dark-current noise and the response of ambient light of the CMOS camera. (2) Place a blank slide on the microscope stage, turn on the light sources and switch the four LEDs, obtain multi-spectral images of blank background. (3) Replace the blank slide with a cervical smear, control the light source as mentioned above and obtain multi-spectral images of the cervical cells.  
Figure 3 shows the original images of the compound-stained cervical smear in monochromatic lights with the wavelengths of 620 nm, 520 nm, 460 nm and 570 nm. It can be observed that the problem of DNA absorbance interference caused by compound staining is serious. The cytoplasmic background in Figure 3 will affect the image processing. It is not feasible to carry out DNA quantitative analysis by using the original image. In our study, the proposed system adopts a grayscale camera (monochrome camera), which captures the image under a single band and then performs the peeling calculation. The grayscale change value is 0-255, and the pixel area is 2592 \(\times\) 1944 pixels.  
Figure 4 is the image after absorbance unmixing. It can be seen that the image of the cytoplasm has been removed; only the clear nuclei can be seen after the absorbance unmixing, which could be effective for a personal computer (PC) to conduct quantitative DNA analysis. This image reflects the true absorbance of DNA at a wavelength of 570 nm.  
Figure 4: The image after absorbance unmixing.  
Figure 3: Original images obtained under monochromatic light sources; (**a**) the image of the wavelength of 620 nm, (**b**) the image of the wavelength of 520 nm, (**c**) the image of the wavelength of 460 nm, (**d**) the image of the wavelength of 570 nm.  
sized after calculating each band's weight, based on the absorbance-unmixing model, as expressed in Equation (4).  
It can be observed that the color of this image is bright, the nuclei in the image are clearly visible and the edge of the cytoplasm is explicit. The pseudo-color image was sent to pathological doctors who, after reviewing, concluded that, compared with Papanicolaou smear, there was no significant difference in the background and color of the cytoplasm; the color of nuclei in the pseudo-color image was darker because the nuclei in this smear were Fuelgen-stained. However, this would not cause an interference or be an obstacle to TBS classification.  
The microspectral images used to reflect absorbance-unmixing results are illustrated in Figure 6. Figure 6(1a-7a) shows the images of co-stained cytoplasms and nuclei, while Figure 6(1b-7b) shows the grayscale images of cell nuclei after absorbance unmixing. The value under each figure is the corresponding DI index of the cell. It can be seen that the morphology of the entire cell is clear, providing a promising way to analyze the relative size of the cytoplasm and the nucleus. According to the observed grayscale images, the DNA content could be further analyzed through using the nucleus after unmixing. DNA content in the image represents the integrated optical density. The DNA index is obtained through using the ratio of the integrated optical density of a single cell to the reference integrated optical density. It should be known that DNA index is a significant indicator for representing the content of the cell nucleus, further representing the number of ploidy in the process of cell proliferation, and reflecting the CV of nuclear content in the process of cell classification.