Subcellular imaging of epithelium with time-lapse optical coherence tomography

Abstract

Abstract. We present the first experimental result of direct delineation of the nuclei of living rat bladder epithelium with ultrahigh-resolution optical coherence tomography ͑uOCT͒. We demonstrate that the cellular details embedded in the speckle noise in a uOCT image can be uncovered by time-lapse frame averaging that takes advantage of the micromotion in living biological tissue. The uOCT measurement of the nuclear size ͑7.9± 1.4 m͒ closely matches the histological evaluation ͑7.2± 0.8 m͒. Unlike optical coherence microscopy ͑OCM͒, which requires a sophisticated high-NA microscopic objective, this approach uses a commercial-grade single achromatic lens ͑f /10 mm, NA/0.25͒ and provides a cross-sectional image over 0.6 mm of depth without focus tracking, thus holding great promise of endoscopic optical biopsy for diagnosis and grading of flat epithelial cancer such as carcinoma in situ in vivo. Noninvasive in vivo imaging identification of pathogenesis at cellular resolutions is crucial to early clinical diagnosis of cancers.1 Technological advance in confocal microscopy and endoscopy has permitted noninvasive imaging of cellular morphology in intact tissue such as skin and bladder, colon, and cervical epthelia, but imaging depth is limited to 100 to 300 m and focus-tracking is required, which is difficult in many clinical applications. Unlike confocal microscopy, the lateral and axial resolutions of optical coherence tomography ͑OCT͒ are decoupled, 2 e.g., the axial resolution is determined by the source coherence length L c =2 ln 2/ 2 / ⌬, where and ⌬ are the source central wavelength and full-width half maximum ͑FWHM͒ spectral bandwidth. Therefore, the axial sectioning resolution of OCT can be substantially improved by employing broadband sources, and subcellular imaging of low-scattering tissue such as xenopus laevis ͑mesenchymal cells͒ in vivo has been reported. 1,2 However, for mammalian epithelial cells, the smaller cell size and thus denser microorganelle content results in increased cellular scattering, leading to phase randomization, i.e., speckle noise, that may drastically degrade the image contrast and resolution for imaging subcellular details. For instance, ultrahigh-resolution OCT ͑uOCT͒ with axial resolution exceeding 0.7 m has been reported, 2 but imaging of subcellular details of the epithelium remains unsolved, preventing this promising technique from becoming an optical biopsy tool for clinical diagnosis. In this letter, we demonstrate that the subcellular details of living rat urothelium can be uncovered after proper time-lapse dynamic averaging to minimize speckle noise in uOCT imaging. Figure 1 is a schematic of the time-domain uOCT setup used in this study. A Ti:Sapphire laser ͑⌬ = 128 nm͒ was used to illuminate a wavelength-flattened broadband fiberoptic Michelson interferometer. In the sample arm, light was collimated to 4.7 mm by a fiberoptic achromatic lens, scanned laterally by a 8-mm servo mirror, and focused onto biological tissue under examination by a commercial-grade achromati...

On the possibility of time-lapse ultrahigh-resolution optical coherence tomography for bladder cancer grading

“…Interestingly, time-domain ͑TD͒ time-lapse uOCT ͑TL-uOCT͒, taking advantage of cellular micromotion in fresh ex vivo tissue for effective speckle noise reduction, was recently demonstrated to uncover the subcellular details ͑i.e., nuclei͒ in bladder epithelium using a low-NA commercial achromatic lens ͑f 10 mm/NA 0.25͒. 6 In this letter, we present spectral-domain ͑SD͒ TL-uOCT to further enhance image contrast and resolution and enable 3-D subcellular imaging and provide new experimental data to evidence the possibility for epithelial cancer grading. Figure 1 illustrates an SD TL-uOCT setup for subcellular bladder imaging, in which an ultrafast Ti: Al 2 O 3 laser ͑ = 800 nm, ⌬ FWHM = 128 nm͒ was employed to illuminate a wavelength-flattened fiber-optic Michelson interferometer.…”

“…Optimizing the L c of uOCT was achieved by spectral reshaping using RSOD in the reference arm and fiberoptic polarization controllers ͑FPC͒ to maximize the bandwidth of the modulation cross-spectrum ͑e.g., ⌬ ജ 155 nm͒; this procedure was found much easier to implement than in previously reported TD uOCT. 6 Mismatch of dispersion between the two arms was coarsely adjusted by RSOD, wedge prisms, and FPC ͑for polarization-mode dispersion͒ and then fully compensated numerically, 7 which ensured an axial resolution approaching the transform limit, i.e., L c = 2.3 m or 1.7 m in bladder tissue ͑refractive index n Ϸ 1.37 is assumed͒. TL-uOCT, based on time-lapse frame averaging of dynamic cellular backscattering, has been shown to effectively reduce speckle noise and uncover subcellular details in fresh urothelium ex vivo.…”

“…TL-uOCT, based on time-lapse frame averaging of dynamic cellular backscattering, has been shown to effectively reduce speckle noise and uncover subcellular details in fresh urothelium ex vivo. 6 SD uOCT, owing to its improved image sensitivity and frame rate, can further enhance time-lapse phase scrambling ͑speckle removal͒ for subcellular delineation and thus potentially enable 3-D TL-uOCT. The detected TL-uOCT signal can be simplified by ensemble averaging of snapshots of uOCT signal I uOCT ͑L r ͒:…”

Polarization effect on the depth resolution of optical coherence tomography

“…[1][2][3] We all know that the depth resolution of an OCT system depends on the bandwidth and the center wavelength of the light source if the spectra and dispersion of the reference and sample arms in the interferometer are well balanced. 2,4,5 In biological tissues, scattering and birefringence can modify the polarization states of the incident sample light [6][7][8] in addition to the polarization modification by the single mode optical fibers in the sample and reference arms. Polarization controllers are usually used in a fiber-based OCT to optimize an OCT image by changing the amplitude and orientation of birefringence in the sample or reference fiber.…”

Speckle reduction in swept source optical coherence tomography images with slow-axis averaging

“…A number of methods have been used to reduce speckle noise in OCT, such as frame averaging [1][2][3][4][5][6][7] (or space compounding [8]), angular compounding, [9,10] frequency compounding, [11] strain compounding, [12] and single Bscan filtering. [13,14] Among them, frame averaging technique is widely used in clinical OCT systems, such as RTVue (Optovue, CA), Spectralis (Heidelberg Engineering, Heidelberg, Germany), Spectral OCT/SLO (OPKO/OTI, Miami, FL), Cirrus (Zeiss Meditec, CA) and 3D OCT-2000 (Topcon, Japan).…”