Label-free Quantitative Phase Imaging Research Articles

Abstract 4769: Mapping cellular metabolic activity within U87MR glioblastoma cells in response to chemical and environmental insult using label-free quantitative phase imaging

Abstract Introduction: One of our drug development target diseases is Glioblastoma multiforme (GBM), a serious neurological disease with a very poor prognosis for the patients due, in part, to the large number of metabolic pathways it can follow. Materials and Methods: We are using U87-MR cells as a model. We employ time-lapse quantitative phase imaging technology that quantifies the refractive index (RI) of cells via light interference. There is no need for labeling of the cells. The RI is an intrinsic physical property of the cells which lends itself to highly precise and accurate quantitative analysis. Our basic protocol involves seeding cells, providing atmosphere and temperature control. Time-lapse images are automatically obtained at intervals ranging from 10 seconds up to 5 minutes and over time periods from minutes to days. Cells are unlabeled, and can be untreated, or treated with drugs or components of multi-drug formulations. We used drugs that have known effects on the cell cycle, mitochondria and induce apoptosis. We also tested drugs such as temozolomide (TMZ) the first line regimen in GBM therapy protocols. Results: Our first notable finding relates to U87-MR motility. The cells self-assemble into a pattern of well separated cell clusters with tubular connections. These connections are initiated by mitochondrial tunneling and are stable over time. The tunnels are reinforced with actin, and mitochondrial fusion occurs at substrate contact foci. These connections between cells also are conduits for intercellular communication and the transport of objects such as mitochondria (MT). Many drugs that have nuclear targets, including demecolcine, topoisomerase inhibitors, present effects in the cytoplasm of the cells, including vacuoles and possible the remnants of DNA repair. MT contain their DNA. The necessary genes are needed to control their own replication to replace damaged MT and to initiate alternative energy sources, such as mitophagy where cellular protein digestion is utilized as a source of energy. There are numerous factors that cause a wide range of textures within cells, including MT fission, lipid droplets, and autophagy. In many treatments, we see a definite region forming with very active textural changes. Due to its proximity to the nuclear membrane as well as the endoplasmic reticulum, we hypothesize that this may be where mTOR1 and mTOR2C activities originate. In the TMZ studies, we found a strong correlation between the dosage, and the morphology of the cells with a transformation to mesenchymal morphologies at the higher dosages. Discussion: GBM cells adapt to and control a number of different metabolic pathways which are primarily characterized by biochemical analysis. We have vastly improved-time lapse imaging capabilities. We produced first evidence of successful characterization of the metabolic changes based on RI quantifications and visual effects. Citation Format: Ed Luther, Livia P. Mendes, Vladimir P. Torchilin. Mapping cellular metabolic activity within U87MR glioblastoma cells in response to chemical and environmental insult using label-free quantitative phase imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4769.

Abstract 6019: Identification of the neo-genesis of glioblastoma stem cells using label-free quantitative phase imaging

Abstract Introduction: Glioblastoma (GBM) is the most aggressive neurological cancer with a very poor prognosis due to its propensity to regenerate after medical interventions. Our experimental model is U87-MG cells, which contain subsets of cancer stem cells (CSC) and have the unusual property of spontaneously forming neurospheres in culture. We are investigating the origins of these stem cells. Materials and Methods: We employ recently developed time label-free, time-lapse quantitative phase imaging (QPI) technology that allows precise quantification of the refractive index (RI) distributions on a sub-cellular level. These measurements are stored in 2D or 3D arrays which can be presented as images or videos at various resolutions. Cells were seeded into sealable culture Petrie dishes and imaged while maintaining appropriate environmental controls. Results: We first noted CSCs in late stage cultures in exploratory experiments, where they presented as small cells (10-15 um) compared to the 50-100 um range of mature U8-MR cells. Their refractive index (RI) is 1.42, compared to about 1.40 for the cytoplasm of mature cells. QPI technology sensitivity enables almost complete separation between the two cell types in RI histograms. We exemplify a grouping of cells in an 80 um2 field of view, tracked in multiple time segments ranging from 20 minutes to 12 hours over a period of about 18 hours, containing a mature U87 which serves as a reference marker. Around this cell, there are 3 distinct pro-stem (PS) cells 12 to 16 um in size: PS-1 with numerous 0.5 um particles in the size range of mitochondria, PS-2 with a nucleus with nucleoli but no nuclear membrane, and PS-3 with a distinct nucleus, nucleoli, and cytosol. The cells are all connected in a network of nanotubes and tunnels. PS-1 and PS-2 are not very active in the early analysis, but they eventually move together and appear to fuse while still maintaining their individual shapes. PS-3 is very actively interacting with another cell on the border of the analysis window. It is extending and retracting a tube and is apparently moving materials both to and away from the other cell in a very regular and linear fashion, suggesting a primitive form of transcription. Discussion: The processes we observed are consistent with tumor stem cell generation by autophagy, mitophagy, and catabolization followed by reprograming of the remnants and formation of neoplastic stem cells. The novelty of our approach is determined by technical capabilities of our QPI platforms, which is non-invasive label-free approach coupled with ultra-high-resolution analysis, previously unobtainable in traditional time-lapse imaging systems. Citation Format: Ed Luther, Livia P. Mendes, Vladimir P. Torchilin. Identification of the neo-genesis of glioblastoma stem cells using label-free quantitative phase imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6019.

Classification of cell morphology with quantitative phase microscopy and machine learning.

We describe and compare two machine learning approaches for cell classification based on label-free quantitative phase imaging with transport of intensity equation methods. In one approach, we design a multilevel integrated machine learning classifier including various individual models such as artificial neural network, extreme learning machine and generalized logistic regression. In another approach, we apply a pretrained convolutional neural network using transfer learning for the classification. As a validation, we show the performances of both approaches on classification between macrophages cultured in normal gravity and microgravity with quantitative phase imaging. The multilevel integrated classifier achieves average accuracy 93.1%, which is comparable to the average accuracy 93.5% obtained by convolutional neural network. The presented quantitative phase imaging system with two classification approaches could be helpful to biomedical scientists for easy and accurate cell analysis.

Label-free biochemical quantitative phase imaging with mid-infrared photothermal effect

Label-free optical imaging is valuable in biology and medicine because of its non-destructive nature. Quantitative phase imaging (QPI) and molecular vibrational imaging (MVI) are the two most successful label-free methods, providing morphological and biochemical information, respectively. These techniques have enabled numerous applications as they have matured over the past few decades; however, their label-free contrasts are inherently complementary and difficult to integrate due to their reliance on different light–matter interactions. Here we present a unified imaging scheme with simultaneous and in situ acquisition of quantitative phase and molecular vibrational contrasts of single cells in the QPI framework using the mid-infrared photothermal effect. The robust integration of subcellular morphological and biochemical label-free measurements may enable new analyses, especially for studying complex and fragile biological phenomena such as drug delivery, cellular disease, and stem cell development, where long-time observation of unperturbed cells is needed under low phototoxicity.

Ptychographic modulation engine: a low-cost DIY microscope add-on for coherent super-resolution imaging

Imaging of biological cells and tissues often relies on fluorescent labels, which offer high contrast with molecular specificity. The use of exogenous labeling agents, however, may alter the normal physiology of the bio-specimens. Complementary to the established fluorescence microscopy, label-free quantitative phase imaging provides an objective morphological measurement tool for bio-specimens and is free of variability introduced by contrast agents. Here, we report a simple and low-cost microscope add-on, termed ptychographic modulation engine (PME), for super-resolution quantitative phase imaging. In this microscope add-on module, we attach a diffuser to a 3D-printed holder that can be mechanically moved to different x-y positions. We then use two vibrational motors to introduce random positional shifts to the diffuser. The add-on module can be placed between the objective lens and the specimen in most existing microscope platforms. Thanks to the diffuser modulation process, the otherwise inaccessible high-resolution object information can now be encoded into the captured images. In the ptychographic phase retrieval process, we jointly recover the complex object wavefront, the complex diffuser profile, and the unknown positional shifts of the diffuser. We demonstrate a four-fold resolution gain over the diffraction limit of the employed 2× objective lens. We also test our approach for in vitro cell imaging, where we are able to adjust the focus after the data has been captured. The reported microscope add-on provides a turnkey solution for super-resolution quantitative phase imaging. It may find applications in label-free bio-imaging where both large field-of-view and high resolution are needed.

Effects of substrate patterning on cellular spheroid growth and dynamics measured by gradient light interference microscopy (GLIM).

The development of three-dimensional (3D) cellular architectures during development and pathological processes involves intricate migratory patterns that are modulated by genetics and the surrounding microenvironment. The substrate composition of cell cultures has been demonstrated to influence growth, proliferation and migration in 2D. Here, we study the growth and dynamics of mouse embryonic fibroblast cultures patterned in a tissue sheet which then exhibits 3D growth. Using gradient light interference microscopy (GLIM), a label-free quantitative phase imaging approach, we explored the influence of geometry on cell growth patterns and rotational dynamics. We apply, for the first time to our knowledge, dispersion-relation phase spectroscopy (DPS) in polar coordinates to generate the radial and rotational cell mass-transport. Our data show that cells cultured on engineered substrates undergo rotational transport in a radially independent manner and exhibit faster vertical growth than the control, unpatterned cells. The use of GLIM and polar DPS provides a novel quantitative approach to studying the effects of spatially patterned substrates on cell motility and growth.

Quantitative phase imaging reveals matrix stiffness-dependent growth and migration of cancer cells

Cancer progression involves complex signals within the tumor microenvironment that orchestrate proliferation and invasive processes. The mechanical properties of the extracellular matrix (ECM) within this microenvironment has been demonstrated to influence growth and the migratory phenotype that precedes invasion. Here we present the integration of a label-free quantitative phase imaging technique, spatial light interference microscopy (SLIM)—with protein-conjugated hydrogel substrates—to explore how the stiffness of the ECM influences melanoma cells of varying metastatic potential. Melanoma cells of high metastatic potential demonstrate increased growth and velocity characteristics relative to cells of low metastatic potential. Cell velocity in the highly metastatic population shows a relative stability at higher matrix stiffness suggesting adoption of migratory routines that are independent of mechanics to facilitate invasion. The use of SLIM and engineered substrates provides a new approach to characterize the invasive properties of live cells as a function of microenvironment parameters. This work provides fundamental insight into the relationship between growth, migration and metastatic potential, and provides a new tool for profiling cancer cells for clinical grading and development of patient-specific therapeutic regimens.

Multi-modal chip-based fluorescence and quantitative phase microscopy for studying inflammation in macrophages.

Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.

Label-Free Optical Marker for Red-Blood-Cell Phenotyping of Inherited Anemias.

The gold-standard methods for anemia diagnosis are complete blood counts and peripheral-smear observations. However, these do not allow for a complete differential diagnosis as that requires biochemical assays, which are label-dependent techniques. On the other hand, recent studies focus on label-free quantitative phase imaging (QPI) of blood samples to investigate blood diseases by using video-based morphological methods. However, when sick cells are very similar to healthy ones in terms of morphometric features, identification of a blood disease becomes challenging even with QPI. Here, we introduce a label-free optical marker (LOM) to detect red-blood-cell (RBC) phenotypes, demonstrating that a single set of all-optical parameters can clearly identify a signature directly related to an erythrocyte disease through modeling each RBC as a biological lens. We tested this novel biophotonic analysis by proving that several inherited anemias, such as iron-deficiency anemia, thalassemia, hereditary spherocytosis, and congenital dyserythropoietic anemia, can be identified and sorted, thus opening a novel route for blood diagnosis on a completely different concept based on LOMs.

Quantifying collagen fiber orientation in breast cancer using quantitative phase imaging.

Tumor progression in breast cancer is significantly influenced by its interaction with the surrounding stromal tissue. Specifically, the composition, orientation, and alignment of collagen fibers in tumor-adjacent stroma affect tumor growth and metastasis. Most of the work done on measuring this prognostic marker has involved imaging of collagen fibers using second-harmonic generation microscopy (SHGM), which provides label-free specificity. Here, we show that spatial light interference microscopy (SLIM), a label-free quantitative phase imaging technique, is able to provide information on collagen-fiber orientation that is comparable to that provided by SHGM. Due to its wide-field geometry, the throughput of the SLIM system is much higher than that of SHGM and, because of the linear imaging, the equipment is simpler and significantly less expensive. Our results indicate that SLIM images can be used to extract important prognostic information from collagen fibers in breast tissue, potentially providing a convenient high throughput clinical tool for assessing patient prognosis.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion.