Studies have revealed that although uOCT with an axial resolution of or less is able to clearly delineate the subcellular details of xenopus laevis,1- 2 subcellular uOCT of scattering tissue such as mammalian epithelium—critical to cancer diagnosis—remains unsolved. To exclude the influence of relatively lower lateral resolution than that of optical coherence microscopy (OCM), we performed uOCT imaging of rat bladder epithelium by replacing the achromat (Obj) with a fully corrected microscopic objective (e.g., Nikon 16X/0.8NA water lens), but experiments concluded that no subcellular visualization although both axial and lateral resolutions were optimized, i.e., and , to be sufficient to resolve the epithelial membrane and nucleus. This result suggested that uOCT as an interference-based imaging technique is different from an intensity-based approach such as confocal microscopy,4- 5 whose spatial resolution is no longer determined by the focusing spot size (e.g., ultimately under the diffraction limit) but rather by speckle noise due to phase randomization among the local interfering scattering waves that may lead to severely degraded image contrast and reduced spatial resolution. In other words, the subcellular details may be uncovered if the speckle effects can be effectively reduced. For proof of concept, we applied various speckle reduction techniques, e.g., A-scan, frame, and composite NA averaging.6 We found that proper time-lapse frame averaging was a simple and effective approach that took advantage of dynamic micro motion in living biological tissue such as urinary bladder. Figure 3 shows an example of a fresh living rat bladder ex vivo. Panel (a) is a snapshot showing no visible cellular morphology in the urothelium due to speckle noise, whereas panel (b) is an average over four frames with optimized time lapse . It must be noted that the time lapse was crucial: If was too short, the motion-induced phase scrambling was insufficient to reduce speckle noise; if was too long, the motion artifact washed away the subcellular details. was determined experimentally based on spontaneous motion of the living bladder tissue under examination. As shown in Fig. 3, the difference was incredible—the seemingly impossible nuclear morphology of living epithelial cells was clearly uncovered. In contrast to the low-scattering large mesenchymal cells whose nuclei and cell membranes appeared highly backscattered in the uOCT image,1- 2 the membranes of the epithelial cells were submerged in highly backscattering cytoplasm but the epithelial nuclei appeared low backscattering, in agreement with OCM observations.4- 5 The fact of “dark” nuclei embedded in highly backscattering cytoplasm supported our hypothesis that speckles resulting from highly intracellular scattering (due to denser cytoplasm than in mesenchymal cells) ruined the subcellular details in an uOCT image of mammalian epithelial cells. According to Mie’s theory of scattering, small micro-organelles contribute favorably to backscattering (i.e., uOCT signal), whereas large nuclei favor forward scattering. The nuclear size of rat epithelial cells measured by uOCT, , compared well to that of the corresponding histology in panel (c), . More importantly, morphological details of the rat bladder at depths of below the bladder surface, including urothelium, lamina proporia, and upper muscularis, were well delineated without focus tracking and image fusion. Calibration of uOCT signal drop (using mirror reflectance) along the direction revealed that the depth of focus was ; nevertheless, the high scattering in lamina proporia and muscularis still allowed delineation of bladder structures down to below the urothelium at which the focus was centered.