**4. Results**

### **4.1 Illustrative bright-field and phase images of various cervical cell types**

In this section we begin by providing sample images of cervical cell nuclei that were obtained using our dual-mode DHM system. While quantitative information obtained in terms of morphological parameters is certainly important, a large number of clinical sites worldwide typically use visual examination of cells using a bright-field microscope for cell classification. The importance of changes in nucleus structure in cancer diagnosis is already well-known [32]. With the illustrative examples in this section, we wish to qualitatively describe the morphological features observed in quantitative phase images of cervical cell nuclei in various stages. The simultaneous presentation of bright-field images (that pathologists can correlate to) and the quantitative phase images as shown here is important in our opinion from the perspective of clinical users. It is important to note that our single-shot full resolution phase reconstruction technique allows us to observe the quantitative phase images with the same resolution as the bright-field images. The examples shown here also aim to illustrate that the information contained in the quantitative phase images is different in nature from that in the bright-field images. Quantitative analysis of the images using morphological parameters as described in **Table 2** will be provided in the following sections. In **Figure 2** we show illustrative examples of normal cells in the intraepithelial squamous layer. A progression from intermediate to superficial stages is shown in **Figure 2(a)**–**(j)** respectively. As a cell progresses from intermediate to superficial stage the chromatin in the cell nucleus is known to condense. The superficial cells are in the outermost layer of ecto-cervix and have

#### **Figure 2.**

*Maturation of cells in intra-epithelial squamous layer from intermediate to superficial is shown from (a)-(j) in both bright-field and phase modes. The phase maps correspond to the ROIs marked in the bright-field images. As the nuclei progress to superficial stage, the nucleus area gets smaller and the phase profile is seen to get taller by approximately a factor of 2.*

highly condensed pyknotic nucleus. While in the bright-field images the area of the nucleus is progressively decreasing and the color of the nucleus gets darker from intermediate to superficial stages, the accompanying phase map of the nucleus is seen to get taller by approximately a factor of 2. This change in the optical height profile cannot be inferred from the 2D bright-field images and the phase map is therefore seen to provide new morphological information. Further, from the phase profiles we also see that the evolution of the cells from intermediate to superficial stage happens via a continuous change. Next in **Figures 3**–**5** we examine the abnormal cell classes low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC) which progressively indicate higher grade abnormalities. The class LSIL consists of abnormal superficial and intermediate cells. Variable degrees of hyper-chromasia, nuclear size variation with coarsely granulated chromatin are identifiers of a typical LSIL cell. In the HSIL class, the degree of nuclear enlargement and hyper-chromasia is more than LSIL and the cells here are found in sheet-like aggregates. In both LSIL and HSIL cases the phase profiles of the cell nuclei are seen to have increased roughness or corrugations compared to the normal cells. In the SCC class which is considered to be a confirmed case of malignant cervical smear, the phase profile of the nucleus shows sharp narrow peaks with large phase values. It is once again important to note that the phase profile clearly provides new morphological information that is not readily available in the 2D bright-field images. Apart from the main classes above, the Bethesda system defines a class Atypical Squamous Cells

*Cytopathology Using High Resolution Digital Holographic Microscopy DOI: http://dx.doi.org/10.5772/intechopen.96459*

#### **Figure 3.**

*Illustration of LSIL cells, the three columns show the bright-field image, selected ROI and the phase image of the ROI.*

**Figure 4.** *Illustration of HSIL cells, the three columns show the bright-field image, selected ROI and the phase image of the ROI.*

**Figure 5.** *Illustration of SCC cells, the three columns show the bright-field image, selected ROI and the phase image of the ROI.*

ASC which include the samples that cannot be categorized as normal or abnormal typically. The nuclei in this class are typically larger in area, however, as seen in **Figure 6**, the average phase value in the nucleus is slightly lower compared to the LSIL, HSIL or SCC classes. In both ASC-US and ASC-H classes (**Figure 6**), the phase profile has undulations but the phase structure of ASC-H is flatter with lower average phase value in the nucleus as compared to that of the ASC-US cells. Further in some ASC nuclei, we observe local peaks in phase profile located near the nuclear boundary leading to a dip in the center. Finally we show rare abnormal cell cases in **Figure 7** that include inflamed, reactive, moon-type, virus-infected and koliocytotic classes. Just a few examples of these rare cells were present in our sample set. The phase profile for all these types appears corrugated with lower average phase values except for the virus infected cell type. While only a few representative images of each cell type have been shown simultaneously in bright-field and phase mode, we clearly observe that the phase profile offers distinctive morphological features that are not currently utilized in the clinical practice. This new information if incorporated in cyto-pathological examination, can be potentially valuable to clinicians.
