Fabrication and validation of an LED array microscope for multimodal, quantitative imaging.

The combination of multiple imaging modalities in a single microscopy system can enable new insights into biological processes. In this work, we describe the construction and rigorous characterization of a custom microscope with multimodal imaging in a single, cost-effective system. Our design utilizes advances in LED technology, robotics, and open-source software, along with existing optical components and precision optomechanical parts to offer a modular and versatile design. This microscope is operated using software written in Arduino and Python and has the ability to run multi-day automated imaging experiments when placed inside of a cell culture incubator. Additionally, we provide and demonstrate methods to validate images taken in brightfield and darkfield, along with validation and optimization for differential phase contrast (DPC) quantitative phase imaging.

Multiparametric quantitative phase imaging for real-time, single cell, drug screening in breast cancer.

Quantitative phase imaging (QPI) measures the growth rate of individual cells by quantifying changes in mass versus time. Here, we use the breast cancer cell lines MCF-7, BT-474, and MDA-MB-231 to validate QPI as a multiparametric approach for determining response to single-agent therapies. Our method allows for rapid determination of drug sensitivity, cytotoxicity, heterogeneity, and time of response for up to 100,000 individual cells or small clusters in a single experiment. We find that QPI EC50 values are concordant with CellTiter-Glo (CTG), a gold standard metabolic endpoint assay. In addition, we apply multiparametric QPI to characterize cytostatic/cytotoxic and rapid/slow responses and track the emergence of resistant subpopulations. Thus, QPI reveals dynamic changes in response heterogeneity in addition to average population responses, a key advantage over endpoint viability or metabolic assays. Overall, multiparametric QPI reveals a rich picture of cell growth by capturing the dynamics of single-cell responses to candidate therapies.

Fabrication and Bonding of Refractive Index Matched Microfluidics for Precise Measurements of Cell Mass.

The optical properties of polymer materials used for microfluidic device fabrication can impact device performance when used for optical measurements. In particular, conventional polymer materials used for microfluidic devices have a large difference in refractive index relative to aqueous media generally used for biomedical applications. This can create artifacts when used for microscopy-based assays. Fluorination can reduce polymer refractive index, but at the cost of reduced adhesion, creating issues with device bonding. Here, we present a novel fabrication technique for bonding microfluidic devices made of NOA1348, which is a fluorinated, UV-curable polymer with a refractive index similar to that of water, to a glass substrate. This technique is compatible with soft lithography techniques, making this approach readily integrated into existing microfabrication workflows. We also demonstrate that this material is compatible with quantitative phase imaging, which we used to validate the refractive index of the material post-fabrication. Finally, we demonstrate the use of this material with a novel image processing approach to precisely quantify the mass of cells in the microchannel without the use of cell segmentation or tracking. The novel image processing approach combined with this low refractive index material eliminates an important source of error, allowing for high-precision measurements of cell mass with a coefficient of variance of 1%.

Cell viscoelasticity is linked to fluctuations in cell biomass distributions.

The viscoelastic properties of mammalian cells can vary with biological state, such as during the epithelial-to-mesenchymal (EMT) transition in cancer, and therefore may serve as a useful physical biomarker. To characterize stiffness, conventional techniques use cell contact or invasive probes and as a result are low throughput, labor intensive, and limited by probe placement. Here, we show that measurements of biomass fluctuations in cells using quantitative phase imaging (QPI) provides a probe-free, contact-free method for quantifying changes in cell viscoelasticity. In particular, QPI measurements reveal a characteristic underdamped response of changes in cell biomass distributions versus time. The effective stiffness and viscosity values extracted from these oscillations in cell biomass distributions correlate with effective cell stiffness and viscosity measured by atomic force microscopy (AFM). This result is consistent for multiple cell lines with varying degrees of cytoskeleton disruption and during the EMT. Overall, our study demonstrates that QPI can reproducibly quantify cell viscoelasticity.

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.