Cell death detection by quantitative three-dimensional single-cell tomography

Nai-Chia Cheng, Tsung-Hsun Hsieh, Yu-Ta Wang, Chien-Chih Lai, Chia-Kai Chang, Ming-Yi Lin, Ding-Wei Huang, Jeng-Wei Tjiu, Sheng-Lung Huang, Nai-Chia Cheng, Tsung-Hsun Hsieh, Yu-Ta Wang, Chien-Chih Lai, Chia-Kai Chang, Ming-Yi Lin, Ding-Wei Huang, Jeng-Wei Tjiu, Sheng-Lung Huang

Abstract

Ultrahigh-resolution optical coherence tomography (UR-OCT) has been used for the first time to our knowledge to study single-cell basal cell carcinoma (BCC) in vitro. This noninvasive, in situ, label-free technique with deep imaging depth enables three-dimensional analysis of scattering properties of single cells with cellular spatial resolution. From three-dimensional UR-OCT imaging, live and dead BCC cells can be easily identified based on morphological observation. We developed a novel method to automatically extract characteristic parameters of a single cell from data volume, and quantitative comparison and parametric analysis were performed. The results demonstrate the capability of UR-OCT to detect cell death at the cellular level.

Keywords: (100.2960) Image analysis; (170.1530) Cell analysis; (170.1870) Dermatology; (170.4500) Optical coherence tomography.

Figures

Fig. 1
Fig. 1
A schema of UR-OCT system. LD: laser diode; L1 and L2: 40X and 60X aspheric lenses; OL: 4X objective lens; LPF: long-pass filters; M: mirror; BS: beam splitter; LWDO: 10X long working distance objective; FM: flipper mirror; PZA: piezo actuator; S: sample; LS: 2D linear stage; EP: eyepiece; PD: photo diode.
Fig. 2
Fig. 2
For each B-scan, intensity image (a) was transform into binary image by applying a threshold (b) which was higher than noise level by 3 dB on every pixels (c). The corresponding image of cell region which was found by automatically boundary detecting method (d). Cellular density can also calculated by dividing the area above the threshold and the total area of the bell shape intensity distribution diagram (e).
Fig. 3
Fig. 3
Microscopic image of BCC cell line and UR-OCT/confocal microscopy images of live and dead BCC cells. BCC cell line was suspended in Matrigel and imaged by a bright-field microscope (a). Live and dead BCC cells were randomly selected from the sample and scanned by UR-OCT and confocal microscopy. Live cell were encoded in green (b) and dead cell which was were encoded in red (e), according to the calcein and propidium’s fluorescence spectrum. Two-dimensional cross-sectional imaging (c, f) were performed across the center of each cell. Three-dimensional imaging of the whole cell (d, g) were realized by combining several cross-sectional images whose covering range were slightly larger than the size of the cells.
Fig. 4
Fig. 4
Co-registered BCC cells image of UR-OCT and confocal microscopy. En face image of UR-OCT (a) with corresponding CFM and CRM images (b, c) of BCC cells. The CFM and CRM images were acquired before UR-OCT imaging since the fluorescence decayed rapidly after staining procedure. Red arrows, strong scattering from small organelles.
Fig. 5
Fig. 5
Different properties between live and dead BCC cells. (a) Signal average; (b) cellular density; (c) average dynamic range; (d) cell volume of BCC cells. White bar, normal group (n = 7); black group, apoptotic group (n = 7); **P

Fig. 6

Correlation of signal average, cellular…

Fig. 6

Correlation of signal average, cellular density, and average dynamic range between live and…

Fig. 6
Correlation of signal average, cellular density, and average dynamic range between live and dead BCC cells. Scatter plots of cellular density and signal average, average dynamic range and signal average of live (a, c) and dead group (b, d). Significant positive correlations of the three parameters were observed in dead group.
Fig. 6
Fig. 6
Correlation of signal average, cellular density, and average dynamic range between live and dead BCC cells. Scatter plots of cellular density and signal average, average dynamic range and signal average of live (a, c) and dead group (b, d). Significant positive correlations of the three parameters were observed in dead group.

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