Digital holographic microscopy for quantitative and label-free oral cytology evaluation

Abstract. Conventional cytology is a rapid chair-side method for diagnosis, but it relies on laborious fixing and staining protocols. As cytology specimens are transparent, it is very hard to visualize them under a bright-field microscope without staining. Quantitative phase imaging techniques have opened up an interesting and potential diagnostic method for volumetric three-dimensional (3-D) visualization of the transparent specimens without any need for sample preparations. We explore the use of digital holographic microscopy in clinical application of oral cytology for the 3-D visualization of buccal cells with high contrast without any additional sample preparations. We also propose nuclear to cytoplasmic (N  /  C) volumes as a much more accurate parameter for identification of multinucleate and actively dividing cells. We quantify the cellular volumes, and N  /  C ratios for 203 buccal cells taken from five healthy volunteers to clinically validate the technique and compare them with the traditional N  /  C area ratios as well as the histology standards. The mean N  /  C area and volume ratios are found to be 0.0322  ±  0.0149 and 0.0648  ±  0.0286, respectively. Our approach highlights the dawn of a new method for a label-free/nondestructive volumetric oral cytology evaluation, with high potential for exploration of suspicious oral lesions, in subjects with chronic habits such as alcoholism and tobacco use.