Kung-Bin Sung

Digital holographic microtomography for high-resolution refractive index mapping of live cells

Quantification of three-dimensional (3D) refractive index (RI) with sub-cellular resolution is achieved by digital holographic microtomography (DHμT) using quantitative phase images measured at multiple illumination angles. The DHμT system achieves sensitive and fast phase measurements based on iterative phase extraction algorithm and asynchronous phase shifting interferometry without any phase monitoring or active control mechanism. A reconstruction algorithm, optical diffraction tomography with projection on convex sets and total variation minimization, is implemented to substantially reduce the number of angular scattered fields needed for reconstruction without sacrificing the accuracy and quality of the reconstructed 3D RI distribution. Tomogram of a living CA9-22 cell is presented to demonstrate the performance of the method. Further, a statistical analysis of the average RI of the nucleoli, the nucleus excluding the nucleoli and the cytoplasm of twenty CA9-22 cells is performed.

Tomographic diffractive microscopy of living cells based on a common-path configuration

We demonstrate a common-path tomographic diffractive microscopy technique for three-dimensional (3D) refractive-index (RI) imaging of unstained living cells. A diffraction grating is utilized to generate a reference beam that traverses a blank region of the sample in a common-path off-axis interferometry setup. Single-shot phase images captured at multiple illumination angles are used for 3D RI reconstruction based on optical diffraction tomography. The common-path configuration shows lower temporal phase fluctuations and better RI resolution than a Mach-Zehnder configuration. 3D subcellular RI distributions of live HeLa cells are quantified.

Quantification of the optical properties of two-layered turbid media by simultaneously analyzing the spectral and spatial information of steady-state diffuse reflectance spectroscopy.

We applied hyperspectral imaging to measure spatially-resolved diffuse reflectance spectra in the visible range and an iterative inversion method based on forward Monte Carlo modeling to quantify optical properties of two-layered tissue models. We validated the inversion method using spectra experimentally measured from liquid tissue mimicking phantoms with known optical properties. Results of fitting simulated data showed that simultaneously considering the spatial and spectral information in the inversion process improves the accuracies of estimating the optical properties and the top layer thickness in comparison to methods fitting reflectance spectra measured with a single source-detector separation or fitting spatially-resolved reflectance at a single wavelength. Further development of the method could improve noninvasive assessment of physiological status and pathological conditions of stratified squamous epithelium and superficial stroma.

High-throughput detection of immobilized plasmonic nanoparticles by a hyperspectral imaging system based on fourier transform spectrometry

To facilitate the application of plasmonic nanoparticles (PNPs) in high-throughput detection, we develop a hyperspectral imaging system (HSIS) combining dark-filed microscopy and imaging Fourier transform spectrometry to measure scattering spectra from immobilized PNPs. The current setup has acquisition time of 5 seconds and spectral resolution of 21.4 nm at 532.1 nm. We demonstrate the applicability of the HSIS in conjunction with spectral data analysis to quantify multiple types of PNPs and detect small changes in localized surface plasmon resonance wavelengths of PNPs due to changes in the environmental refractive index.

Accurate extraction of optical properties and top layer thickness of two-layered mucosal tissue phantoms from spatially resolved reflectance spectra

Abstract. We are reporting on an experimental investigation of a movable diffuse reflectance spectroscopy system to extract diagnostically relevant optical properties of two-layered tissue phantoms simulating mucosae that are covered with stratified squamous epithelium. The reflectance spectra were measured at multiple source-detector separations using two imaging fiber bundles in contact with the phantoms, one with its optical axis perpendicular to the sample surface (perpendicular probe) and the other with its distal end beveled and optical axis tilted at 45 deg (oblique probe). Polystyrene microspheres and purified human hemoglobin were used to make tissue phantoms whose scattering and absorption properties could be well controlled and theoretically predicted. Monte Carlo simulations were used to predict the reflectance spectra for system calibration and an iterative curve fitting that simultaneously extracted the top layer reduced scattering coefficient, thickness, bottom layer reduced scattering coefficient, and hemoglobin concentration of the phantoms. The errors of the recovered parameters ranged from 7% to 20%. The oblique probe showed higher accuracy in the extracted top layer reduced scattering coefficient and thickness than the perpendicular probe. The developed system and data analysis methods provide a feasible tool to quantify the optical properties in vivo.

Enhancing the sensitivity to scattering coefficient of the epithelium in a two-layered tissue model by oblique optical fibers: Monte Carlo study

Abstract. Diffuse reflectance spectroscopy has been applied to detect tissue absorption and scattering properties associated with dysplasia, which is a potential precursor of epithelial cancers. The ability of DRS techniques to detect dysplasia could be improved by enhancing the detection of optical properties of the thin epithelial layer where dysplasia occurs. We propose a beveled fiber bundle probe consisting of a source fiber and multiple detection fibers parallel to each other and oriented obliquely to the tissue surface and investigate the sensitivity of reflectance measured with the probe to optical properties of a two-layered normal oral mucosa model. A scalable Monte Carlo method is employed to speed up analyzing spatially resolved reflectance spectra. Results reveal that the oblique probe is more sensitive to epithelial scattering and less sensitive to both stromal absorption and scattering than conventional perpendicular fiber configuration. The clinical relevance of the enhanced sensitivity to epithelial scattering by the proposed probe is demonstrated by quantifying optical properties of the two-layered tissue model from simulated data. The average error of extracted epithelial scattering coefficient is 1.5% and 32% using the oblique and perpendicular probe, respectively. The errors in other optical properties are all below 10% using the oblique probe.

Investigating the spectral characteristics of backscattering from heterogeneous spherical nuclei using broadband finite-difference time-domain simulations

Reflectance spectra measured from epithelial tissue have been used to extract size distribution and refractive index of cell nuclei for noninvasive detection of precancerous changes. Despite many in vitro and in vivo experimental results, the underlying mechanism of sizing nuclei based on modeling nuclei as homogeneous spheres and fitting the measured data with Mie theory has not been fully explored. We describe the implementation of a three-dimensional finite-difference time-domain (FDTD) simulation tool using a Gaussian pulse as the light source to investigate the wavelength-dependent characteristics of backscattered light from a nuclear model consisting of a nucleolus and clumps of chromatin embedded in homogeneous nucleoplasm. The results show that small-sized heterogeneities within the nuclei generate about five times higher backscattering than homogeneous spheres. More interestingly, backscattering spectra from heterogeneous spherical nuclei show periodic oscillations similar to those from homogeneous spheres, leading to high accuracy of estimating the nuclear diameter by comparison with Mie theory. In addition to the application in light scattering spectroscopy, the reported FDTD method could be adapted to study the relations between measured spectral data and nuclear structures in other optical imaging and spectroscopic techniques for in vivo diagnosis.

Precancerous esophageal epithelia are associated with significantly increased scattering coefficients.

The progression of epithelial precancers into cancer is accompanied by changes of tissue and cellular structures in the epithelium. Correlations between the structural changes and scattering coefficients of esophageal epithelia were investigated using quantitative phase images and the scattering-phase theorem. An ex vivo study of 14 patients demonstrated that the average scattering coefficient of precancerous epithelia was 37.8% higher than that of normal epithelia from the same patient. The scattering coefficients were highly correlated with morphological features including the cell density and the nuclear-to-cytoplasmic ratio. A high interpatient variability in scattering coefficients was observed and suggests identifying precancerous lesions based on the relative change in scattering coefficients.

Investigating the backscattering characteristics of individual normal and cancerous cells based on experimentally determined three-dimensional refractive index distributions

The progression of epithelial dysplasia is accompanied by changes of sub-cellular structures which alter light scattering, particularly backscattering, properties of epithelial cells. In this study, we quantified the refractive index (RI) distributions of normal and cancerous epithelial cells of skin and oral cavity using digital holographic microtomography and investigated the backscattering characteristics of the cells using finite-difference time-domain simulations. The results show that cancerous cells present higher average values of nuclear and nucleolar RI and a higher standard deviation of cytoplasmic RI than normal cells. Both the total scattering and backscattering cross-sections of the cancerous cells are significantly higher than those of the normal cells.

Investigation of influences of the paraformaldehyde fixation and paraffin embedding removal process on refractive indices and scattering properties of epithelial cells

Abstract. The scattering properties and refractive indices (RI) of tissue are important parameters in tissue optics. These parameters can be determined from quantitative phase images of thin slices of tissue blocks. However, the changes in RI and structure of cells due to fixation and paraffin embedding might result in inaccuracies in the estimation of the scattering properties of tissue. In this study, three-dimensional RI distributions of cells were measured using digital holographic microtomography to obtain total scattering cross sections (TSCS) of the cells based on the first-order Born approximation. We investigated the slight loss of dry mass and drastic shrinkage of cells due to paraformaldehyde fixation and paraffin embedding removal processes. We propose a method to compensate for the correlated changes in volume and RI of cells. The results demonstrate that the TSCS of live cells can be estimated using restored cells. The percentage deviation of the TSCS between restored cells and live cells was only −8%. Spatially resolved RI and scattering coefficients of unprocessed oral epithelium ranged from 1.35 to 1.39 and from 100 to 450  cm−1, respectively, estimated from paraffin-embedded oral epithelial tissue after restoration of RI and volume.