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SignificanceSpeckle variations induced by intracellular motion (IM) in the urothelium was observed in optical coherence tomography (OCT) images. It is feasible to use the IM as a dynamic contrast to segment the urothelium with only two sequential OCT images. This new method opens the possibility of tracking the distribution of the urothelial cells to identify the microinvasion of bladder tumors.ApproachWith fresh porcine bladder tissue, IM was analyzed by tracking speckle variations using autocorrelation function, then quantified with CONTINE algorism to identify the decorrelation time (DT) of the speckle variations. Variance analysis is conducted to show IM amplitude and distribution in the urothelium. The segmentation of the urothelium is demonstrated using tissue samples with and without significantly stretching.ResultsSignificant speckle variations induced by IM exists in the urothelium. However, the distribution of the IM is heterogeneous. The DTs are majorly distributed between 1ms to 30ms. Using the IM as a dynamic contrast, the urothelium can be accurately and exclusively segmented, even the layer structure of the bladder is invisible.ConclusionsWith fresh porcine bladders, we show that IM can be used as a dynamic contrast to exclusively segment the urothelium. This contrast may provide a new mechanism for OCT to diagnosis the invasion of urothelial cancerous cells for the better staging of bladder cancer.