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A random rayburst sampling (RRBS) framework was developed to detect the nucleus and cell membrane boundaries in three-dimensional (3-D) space. Raw images were acquired through a full-field optical coherence tomography system with submicron resolution—i.e., 0.8 ?? ? m in lateral and 0.9 ?? ? m in axial directions. The near-isometric resolution enables 3-D segmentation of a nucleus and cell membrane for determining the volumetric nuclear-to-cytoplasmic (N/C) ratio of a single cell. The RRBS framework was insensitive to the selection of seeds and image pixel noise. The robustness of the RRBS framework was verified through the convergence of the N/C ratio searching algorithm. The relative standard deviation of the N/C ratio between different randomly selected seed sets was only 2%. This technique is useful for various in vitro assays on single-cell analyses.

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.

Collagen shrinkage associated with denaturation from thermal treatment has a number of important clinical applications. However, individualized treatment is hindered by the lack of reliable noninvasive methods to monitor the process of collagen denaturation. We investigate the serial changes of collagen denaturation from thermal treatment of rat tail tendons at 58 degrees C by use of second harmonic generation (SHG) microscopy. We find that rat tail tendon shrinks progressively from 0 to 9 min of thermal treatment, and remains unchanged in length upon further thermal treatment. The SHG intensity also decreases from 0 to 9 min of thermal treatment and becomes barely detectable from further thermal treatment. Collagen shrinkage and the SHG intensity are well correlated in a linear model. In addition, SHG imaging reveals a tiger-tail-like pattern of collagen denaturation. The bands of denatured collagen progressively widen from increased thermal treatment and completely replace the adjacent bands of normal collagen after 9 min of thermal treatment. Our results show that collagen denaturation in rat tail tendon from thermal treatment is inhomogeneous, and that SHG intensity can be used to predict the degree of thermally induced collagen shrinkage. With additional development, this approach has the potential to be used in biomedical applications.