Raw data, phase and amplitude reconstructions, synthesized phase contrast and DIC images for various samples and magnifications: micro-lens array (4x 0.1 NA), wild-type c. elegans (10x 0.25 NA), HEK 293T cells (20× 0.4 NA), MCF7 cells (20× 0.4 NA).

<p>Raw data, phase and amplitude reconstructions, synthesized phase contrast and DIC images for various samples and magnifications: micro-lens array (4x 0.1 NA), wild-type c. elegans (10x 0.25 NA), HEK 293T cells (20× 0.4 NA), MCF7 cells (20× 0.4 NA).</p

Comparison of standard DIC and PhC images to their synthesized counterparts from cDPC.

<p>Ground truth DIC images were acquired using a 20x 0.75 NA objective and phase contrast images using a 20x 0.4 NA PhC objective. cDPC images were acquired using a 20x 0.4 NA objective and our filter insert.</p

Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC)

<div><p>We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification—an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various <i>in vitro</i> cell samples and c. elegans in a micro-fluidic channel.</p></div

Experimental comparison of single-shot cDPC with monochromatic DPC and through-focus phase retrieval methods.

<p>(Left) Source patterns. (Middle) Raw camera measurements. (Right) Recovered optical field. DPC methods (partially coherent) were acquired using a 20× 0.4 NA objective lens, while through-focus images (spatially coherent) were captured using 60× 0.8 NA, in order to ensure equal resolution in all cases.</p

Experimental demonstration of motion blur reduction with cDPC vs. conventional DPC.

<p>Our cDPC method results in significantly reduced motion blur artifacts due to its single-shot acquisition.</p

Single-shot color Differential Phase Contrast (cDPC) microscopy.

<p>a) Optical schematic of a brightfield microscope with a cDPC color filter placed at the back focal plane of the condenser in Köhler configuration. b) Installation in Nikon TE300 microscope condenser turret. c) Reconstruction: the captured color image is separated into its RGB components, which are then used to recover two unknowns (amplitude and phase) via a well-posed linear deconvolution. The sample is a micro-lens array (Fresnel Technologies 605). d) CAD model and image of fabricated cDPC insert.</p

Transfer functions for amplitude and phase contrast in each cDPC color channel.

<p>Left: spectral contribution of each illumination filter as captured by the camera’s Bayer pattern. The following columns show the components of the source represented in each image, and the amplitude and phase transfer functions in the spatial frequency domain. Bottom row: sum of each column, representing the calibrated and scaled source and the total coverage of amplitude and phase transfer functions, respectively.</p

Longitudinal Study of Mammary Epithelial and Fibroblast Co-Cultures Using Optical Coherence Tomography Reveals Morphological Hallmarks of Pre-Malignancy

<div><p>The human mammary gland is a complex and heterogeneous organ, where the interactions between mammary epithelial cells (MEC) and stromal fibroblasts are known to regulate normal biology and tumorigenesis. We aimed to longitudinally evaluate morphology and size of organoids in 3D co-cultures of normal (MCF10A) or pre-malignant (MCF10DCIS.com) MEC and hTERT-immortalized fibroblasts from reduction mammoplasty (RMF). This co-culture model, based on an isogenic panel of cell lines, can yield insights to understand breast cancer progression. However, 3D cultures pose challenges for quantitative assessment and imaging, especially when the goal is to measure the same organoid structures over time. Using optical coherence tomography (OCT) as a non-invasive method to longitudinally quantify morphological changes, we found that OCT provides excellent visualization of MEC-fibroblast co-cultures as they form ductal acini and remodel over time. Different concentrations of fibroblasts and MEC reflecting reported physiological ratios <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049148#pone.0049148-Sadlonova1">[1]</a> were evaluated, and we found that larger, hollower, and more aspherical acini were formed only by pre-malignant MEC (MCF10DCIS.com) in the presence of fibroblasts, whereas in comparable conditions, normal MEC (MCF10A) acini remained smaller and less aspherical. The ratio of fibroblast to MEC was also influential in determining organoid phenotypes, with higher concentrations of fibroblasts producing more aspherical structures in MCF10DCIS.com. These findings suggest that stromal-epithelial interactions between fibroblasts and MEC can be modeled <em>in vitro</em>, with OCT imaging as a convenient means of assaying time dependent changes, with the potential for yielding important biological insights about the differences between benign and pre-malignant cells.</p> </div

As phericity of acini.

<p>The minimum asphericity value of 1 indicates a perfect sphere, while less spherical acini have higher asphericity values. Acini comprised of MCF10DCIS.com cells are seen to become increasingly aspherical in the presence of fibroblasts.</p

3D-OCT image acquisition of the co-cultures, and analysis of the shape and size of acini.

<p>A. 3D-OCT image acquistion: the surface of the gel is aligned near the top of each image, and the depth-resolved light scattering from cells beneath the gel surface is apparent at depths up to ∼1 mm; segmentation of acini to characterize the overall size and the lumen is also shown. B. Temporal changes in acini and lumen sizes analyzed from 3D-OCT images of the co-cultures. C. An example isosurface rendering of an acinus from a 3D-OCT image-stack; slicing of the rendered volume clearly shows the lumen. D. An example 3D rendering of an aspherical acinus.</p