Christopher B. Raub

Accurate quantitative phase digital holographic microscopy with single- and multiple-wavelength telecentric and nontelecentric configurations.

In this work, we investigate, both theoretically and experimentally, single-wavelength and multiwavelength digital holographic microscopy (DHM) using telecentric and nontelecentric configurations in transmission and reflection modes. A single-wavelength telecentric imaging system in DHM was originally proposed to circumvent the residual parabolic phase distortion due to the microscope objective (MO) in standard nontelecentric DHM configurations. However, telecentric configurations cannot compensate for higher order phase aberrations. As an extension to the telecentric and nontelecentric arrangements in single-wavelength DHM (SW-DHM), we propose multiple-wavelength telecentric DHM (MW-TDHM) in reflection and transmission modes. The advantages of MW-TDHM configurations are to extend the vertical measurement range without phase ambiguity and optically remove the parabolic phase distortion caused by the MO in traditional MW-DHM. These configurations eliminate the need for a second reference hologram to subtract the two-phase maps and make digital automatic aberration compensation easier to apply compared to nontelecentric configurations. We also discuss a reconstruction algorithm that eliminates the zero-order and virtual images using spatial filtering and another algorithm that minimizes the intensity of fluctuations using apodization. In addition, we employ two polynomial models using 2D surface fitting to compensate digitally for chromatic aberration (in the multiwavelength case) and for higher order phase aberrations. A custom-developed user-friendly graphical user interface is employed to automate the reconstruction processes for all configurations. Finally, TDHM is used to visualize cells from the highly invasive MDA-MB-231 cultured breast cancer cells.

Development of a custom biological scaffold for investigating ultrasound-mediated intracellular delivery

In vitro investigations of ultrasound mediated, intracellular drug and gene delivery (i.e. sonoporation) are typically carried out in cells cultured in standard plastic well plates. This creates conditions that poorly resemble in vivo conditions, as well as generating unwanted ultrasound phenomena that may confound the interpretation of results. Here, we present our results in the development of a biological scaffold for sonoporation studies. The scaffolds were comprised of cellulose fibers coated with chitosan and gelatin. Scaffold formulation was optimized for adherence and proliferation of mouse fibroblasts in terms of the ratio and relative concentration of the two constituents. The scaffolds were also shown to significantly reduce ultrasound reflections compared to the plastic well plates. A custom treatment chamber was designed and built, and the occurrence of acoustic cavitation in the chamber during the ultrasound treatments was detected; a requirement for the process of sonoporation. Finally, experiments were carried out to optimize the ultrasound exposures to minimize cellular damage. Ultrasound exposure was then shown to enable the uptake of 100nm fluorescently labeled polystyrene nanoparticles in suspension into the cells seeded on scaffolds, compared to incubation of cell-seeded scaffolds with nanoparticles alone. These preliminary results set the basis for further development of this platform. They also provide motivation for the development of similar platforms for the controlled investigation of other ultrasound mediated cell and tissue therapies.

Quantitative assessment of cancer cell morphology and motility using telecentric digital holographic microscopy and machine learning: Holography of Cancer Cell Motile Phenotypes

The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two‐dimensional surfaces in vitro, MDA‐MB‐231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA‐MB‐231 cell line were cultured on glass, tissue‐culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F‐actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F‐actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA‐MB‐231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility.

Holography, machine learning, and cancer cells

COMMENTARY LIQUID biopsy to detect circulating solid tumor cells, cellfree DNA or tumor exosomes in peripheral blood holds great promise as a minimally invasive method to detect cancer at an early stage, plan treatments, and monitor disease response to therapy (1). Two great challenges to detecting cancer cells in whole blood are the low relative abundance of tumor cells in proportion to erythrocytes and leukocytes, and the need to introduce highly-specific exogenous labels to identify tumor biomarkers. A recently published article in Cytometry Part A (2) detects cancer cells using a rapid, label-free technique based on machine learning from quantitative phase/digital holographic microscopy datasets. The results demonstrate automated discrimination of normal and cancer cell types flowing in a microfluidic channel, pointing to the potential for high-throughput processing of circulating tumor cells in an imaging flow cytometer. The method is compatible with microfluidic approaches to sequester or enrich circulating tumor cells from normal blood cells and whole blood, respectively. Without staining, biological cells are transparent under bright field microscopy resulting in low contrast intensity images. In that article, Shaked and coauthors (2) utilize an internal contrast mechanism based on the index of refraction. Contrary to traditional qualitative phase contrast (PC) techniques, such as Zernike’s PC and differential interference contrast microscopy, this off-axis interferometric label-free approach can distinguish normal cells from cancer cell lines of different disease stages using quantitative textural and morphological parameters derived from the segmented optical thickness or optical path delay (OPD) of the cell on all spatial points. This OPD is the integral of the index of refraction at each pixel of a projection across the cells’ thickness. To use OPD parameters for sensitive cell screening, the phase signal must possess high fidelity and consistency across images and during the imaging session. The optical setup used in this study is a low-coherence off-axis interferometer with a Mach-Zehnder configuration. The source used is a supercontinuum laser coupled to an acousto-optical tunable filter with a 7 nm bandwidth. The low-coherence source is used to reduce speckle noise. Retroreflectors are used to adjust the sample and reference beam paths to enhance fringe visibility. Stationary aberrations and field curvature aberrations are compensated for using empty sample measurements. Phase unwrapping was performed using a standard unweighted least square algorithm to obtain the OPD maps of the cells. A normalized cut-edge detector algorithm, which is based on graph formulation, was used to separate cells from their background. This algorithm was followed by morphological image operations (opening, dilation, and thresholding) to connect the gradient lines and to remove background pixels erroneously detected. The cells from cultured cell lines, flowed through a microfluidic channel and imaged at high frame rates, appear as roundish, stippled objects in individual frames of phase reconstruction maps. The technique is sensitive to cell phase morphology and

Oral mucosa-on-a-chip to assess layer-specific responses to bacteria and dental materials

The human oral mucosa hosts a diverse microbiome and is exposed to potentially toxic biomaterials from dental restoratives. Mucosal health is partly determined by cell and tissue responses to challenges such as dental materials and pathogenic bacteria. An in vitro model to rapidly determine potential layer-specific responses would lead to a better understanding of mucosal homeostasis and pathology. Therefore, this study aimed to develop a co-cultured microfluidic mucosal model on-a-chip to rapidly assess mucosal remodeling and the responses of epithelial and subepithelial layers to challenges typically found in the oral environment. A gingival fibroblast-laden collagen hydrogel was assembled in the central channel of a three-channel microfluidic chamber with interconnecting pores, followed by a keratinocyte layer attached to the collagen exposed in the pores. This configuration produced apical and subepithelial side channels capable of sustaining flow. Keratinocyte, fibroblast, and collagen densities were optimized to create a co-culture tissue-like construct stable over one week. Cells were stained and imaged with epifluorescence microscopy to confirm layer characteristics. As proof-of-concept, the mucosal construct was exposed separately to a dental monomer, 2-hydroxylethyl methacrylate (HEMA), and the oral bacteria Streptococcus mutans. Exposure to HEMA lowered mucosal cell viability, while exposure to the bacteria lowered trans-epithelial electrical resistance. These findings suggest that the oral mucosa-on-a-chip is useful for studying oral mucosal interactions with bacteria and biomaterials with a histology-like view of the tissue layers.

Quantitative assessment of cancer cell morphology and movement using telecentric digital holographic microscopy

Digital holographic microscopy (DHM) provides label-free and real-time quantitative phase information relevant to the analysis of dynamic biological systems. A DHM based on telecentric configuration optically mitigates phase aberrations due to the microscope objective and linear high frequency fringes due to the reference beam thus minimizing digital aberration correction needed for distortion free 3D reconstruction. The purpose of this work is to quantitatively assess growth and migratory behavior of invasive cancer cells using a telecentric DHM system. Together, the height and lateral shape features of individual cells, determined from time-lapse series of phase reconstructions, should reveal aspects of cell migration, cell-matrix adhesion, and cell cycle phase transitions. To test this, MDA-MB-231 breast cancer cells were cultured on collagen-coated or un-coated glass, and 3D holograms were reconstructed over 2 hours. Cells on collagencoated glass had an average 14% larger spread area than cells on uncoated glass (n=18-22 cells/group). The spread area of cells on uncoated glass were 15-21% larger than cells seeded on collagen hydrogels (n=18-22 cells/group). Premitotic cell rounding was observed with average phase height increasing 57% over 10 minutes. Following cell division phase height decreased linearly (R2=0.94) to 58% of the original height pre-division. Phase objects consistent with lamellipodia were apparent from the reconstructions at the leading edge of migrating cells. These data demonstrate the ability to track quantitative phase parameters and relate them to cell morphology during cell migration and division on adherent substrates, using telecentric DHM. The technique enables future studies of cell-matrix interactions relevant to cancer.

Noninvasive assessment of articular cartilage surface damage using reflected polarized light microscopy

Abstract. Articular surface damage occurs to cartilage during normal aging, osteoarthritis, and in trauma. A noninvasive assessment of cartilage microstructural alterations is useful for studies involving cartilage explants. This study evaluates polarized reflectance microscopy as a tool to assess surface damage to cartilage explants caused by mechanical scraping and enzymatic degradation. Adult bovine articular cartilage explants were scraped, incubated in collagenase, or underwent scrape and collagenase treatments. In an additional experiment, cartilage explants were subject to scrapes at graduated levels of severity. Polarized reflectance parameters were compared with India ink surface staining, features of histological sections, changes in explant wet weight and thickness, and chondrocyte viability. The polarized reflectance signal was sensitive to surface scrape damage and revealed individual scrape features consistent with India ink marks. Following surface treatments, the reflectance contrast parameter was elevated and correlated with image area fraction of India ink. After extensive scraping, polarized reflectance contrast and chondrocyte viability were lower than that from untreated explants. As part of this work, a mathematical model was developed and confirmed the trend in the reflectance signal due to changes in surface scattering and subsurface birefringence. These results demonstrate the effectiveness of polarized reflectance microscopy to sensitively assess surface microstructural alterations in articular cartilage explants.

Magnetic nanoparticle-loaded alginate beads for local micro-actuation of in vitro tissue constructs

Magnetic nanoparticles (MNPs) self-align and transduce magnetic force, two properties which lead to promising applications in cell and tissue engineering. However, the toxicity of MNPs to cells which uptake them is a major impediment to applications in engineered tissue constructs. To address this problem, MNPs were embedded in millimeter-scale alginate beads, coated with glutaraldehyde cross-linked chitosan, and loaded in acellular and MDA-MB-231 cancer cell-seeded collagen hydrogels, providing local micro-actuation under an external magnetic field. Brightfield microscopy was used to assess nanoparticle diffusion from the bead. Phase contrast microscopy and digital image correlation were used to track collagen matrix displacement and estimate intratissue strain under magnetic actuation. Coating the magnetic alginate beads with glutaraldehyde-chitosan prevents bulk diffusion of nanoparticles into the surrounding microenvironment. Further, the beads exert force on the surrounding collagen gel and cells, resulting in intratissue strains of 0-10% tunable with bead dimensions, collagen density, and distance from the bead. Cells seeded adjacent to the embedded beads are subjected to strain gradients without loss of cell viability over two days culture. This study describes a simple way to fabricate crosslinked magnetic alginate beads to load in a collagen tissue construct without direct exposure of the construct to nanoparticles. The findings are significant to in vitro studies of mechanobiology in enabling precise control over dynamic mechanical loading of tissue constructs.

Noninvasive surface damage assessment of bovine articular cartilage expiants by reflected polarized light microscopy

Articular surface damage is a hallmark of cartilage degeneration. Noninvasive assessment of cartilage microstructural alterations has potential clinical value. In this study, we use bovine patellofemoral articular cartilage explants treated with mechanical scraping and collagenase to create cartilage surface disruption, and use polarized reflectance microscopy to quantify alterations to surface and sub-surface microstructure. Reflected polarized signal was sensitive to mild damage to the cartilage surface, and highlighted disruptive alterations. The results indicate the efficacy of reflected polarized light microscopy in assessing the microstructural status of superficial articular cartilage.Articular surface damage is a hallmark of cartilage degeneration. Noninvasive assessment of cartilage microstructural alterations has potential clinical value. In this study, we use bovine patellofemoral articular cartilage explants treated with mechanical scraping and collagenase to create cartilage surface disruption, and use polarized reflectance microscopy to quantify alterations to surface and sub-surface microstructure. Reflected polarized signal was sensitive to mild damage to the cartilage surface, and highlighted disruptive alterations. The results indicate the efficacy of reflected polarized light microscopy in assessing the microstructural status of superficial articular cartilage.

Abstract 2382: Novel integration of genomic and morphological information reveals tumor tissue heterogeneity

Cancer treatment decisions today are predominantly based upon decades-old histological methods, which more recently have been combined with simple phenotypic or molecular tests. To advance this paradigm, we have developed a digital molecular morphology platform that integrates histological images with next-generation sequencing (NGS) or PCR data. This allows us to visualize DNA mutations and RNA expression levels across a histological image, and to correlate mutational or expression status to cell morphological features. Thus, providing a novel approach to mapping tumor heterogeneity. The basic approach is to overlay a microstructure on top of HE 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2382.