Combining quantitative phase microscopy and laser-induced shockwave for the study of cell injury.

In this paper, we propose a new system for studying cellular injury. The system is a biophotonic work station that can generate Laser-Induced Shockwave (LIS) in the cell culture medium combined with a Quantitative Phase Microscope (QPM), enabling the real-time measurement of intracellular dynamics and quantitative changes in cellular thickness during the damage and recovery processes. In addition, the system is capable of Phase Contrast (PhC) and Differential Interference Contrast (DIC) microscopy. Our studies showed that QPM allows us to discern changes that otherwise would be unnoticeable or difficult to detect using phase or DIC imaging. As one application, this system enables the study of traumatic brain injury in vitro. Astrocytes are the most numerous cells in the central nervous system (CNS) and have been shown to play a role in the repair of damaged neuronal tissue. In this study, we use LIS to create a precise mechanical force in the culture medium at a controlled distance from astrocytes and measure the quantitative changes, in order of nanometers, in cell thickness. Experiments were performed in different cell culture media in order to evaluate the reproducibility of the experimental method.

Cephalopod-inspired optical engineering of human cells.

Although many animals have evolved intrinsic transparency for the purpose of concealment, the development of dynamic, that is, controllable and reversible, transparency for living human cells and tissues has remained elusive to date. Here, by drawing inspiration from the structures and functionalities of adaptive cephalopod skin cells, we design and engineer human cells that contain reconfigurable protein-based photonic architectures and, as a result, possess tunable transparency-changing and light-scattering capabilities. Our findings may lead to the development of unique biophotonic tools for applications in materials science and bioengineering and may also facilitate an improved understanding of a wide range of biological systems.

Label-free imaging flow cytometer for analyzing large cell populations by line-field quantitative phase microscopy with digital refocusing.

We propose a line-field quantitative phase-imaging flow cytometer for analyzing large populations of label-free cells. Hydrodynamical focusing brings cells into the focus plane of an optical system while diluting the cell suspension, resulting in decreased throughput rate. To overcome the trade-off between throughput rate and in-focus imaging, our cytometer involves digitally extending the depth-of-focus on loosely hydrodynamically focusing cell suspensions. The cells outside the depth-of-focus range in the 70-µm diameter of the core flow were automatically digitally refocused after image acquisition. We verified that refocusing was successful with our cytometer through statistical analysis of image quality before and after digital refocusing.

Label-free classification of cells based on supervised machine learning of subcellular structures.

It is demonstrated that cells can be classified by pattern recognition of the subcellular structure of non-stained live cells, and the pattern recognition was performed by machine learning. Human white blood cells and five types of cancer cell lines were imaged by quantitative phase microscopy, which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. The built classifier successfully classified WBCs from cell lines (area under ROC curve = 0.996). This label-free, non-cytotoxic cell classification based on the subcellular structure of QPM images has the potential to serve as an automated diagnosis of single cells.

Dynamic changes in the cell membrane on three dimensional low coherent quantitative phase microscopy (3D LC-QPM) after treatment with the near infrared photoimmunotherapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that relies on the binding of a near-infrared antibody photoabsorber conjugate (APC) to a cancer cell. Subsequent exposure to NIR light selectively induces rapid necrotic cell death on target-expressing cells with minimal off-target effects. When treated with NIR-PIT, targeted cells become swollen, develop blebs and burst within minutes of light exposure. Detailed spatial and temporal morphological changes of the cellular membrane of targeted cells treated with NIR-PIT have not been fully explored with state-of-the-art microscopic methods. In this study, we investigated the morphologic and kinetic effects of PIT on two types of cells, a spindle-shaped 3T3/Her cell and a spheric-shaped MDA-MB468 cell, after NIR-PIT using three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM). Adhesive cells treated with NIR-PIT demonstrated region-specific cell membrane rupture occurring first on the distal free edge of the cell near the site of adhesion, in a process that was independent of cell shape. The results show that the peripheral portions of the cell membrane near the site of adhesion are particularly vulnerable to the effects of NIR-PIT, likely because these sites exhibit higher baseline surface tension.

Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation.

Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.

Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity.

Immunogenic cell death (ICD) is a form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Cancer cells killed via ICD can elicit antitumor immunity. ICD is efficiently induced by near-infrared photo-immunotherapy (NIR-PIT) that selectively kills target-cells on which antibody-photoabsorber conjugates bind and are activated by NIR light exposure. Advanced live cell microscopies showed that NIR-PIT caused rapid and irreversible damage to the cell membrane function leading to swelling and bursting, releasing intracellular components due to the influx of water into the cell. The process also induces relocation of ICD bio markers including calreticulin, Hsp70 and Hsp90 to the cell surface and the rapid release of immunogenic signals including ATP and HMGB1 followed by maturation of immature dendritic cells. Thus, NIR-PIT is a therapy that kills tumor cells by ICD, eliciting a host immune response against tumor.

Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy.

Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT.

Digital optical phase conjugation for delivering two-dimensional images through turbid media.

Optical transmission through complex media such as biological tissue is fundamentally limited by multiple light scattering. Precise control of the optical wavefield potentially holds the key to advancing a broad range of light-based techniques and applications for imaging or optical delivery. We present a simple and robust digital optical phase conjugation (DOPC) implementation for suppressing multiple light scattering. Utilizing wavefront shaping via a spatial light modulator (SLM), we demonstrate its turbidity-suppression capability by reconstructing the image of a complex two-dimensional wide-field target through a highly scattering medium. Employing an interferometer with a Sagnac-like ring design, we successfully overcome the challenging alignment and wavefront-matching constraints in DOPC, reflecting the requirement that the forward- and reverse-propagation paths through the turbid medium be identical. By measuring the output response to digital distortion of the SLM write pattern, we validate the sub-wavelength sensitivity of the system.

Label-free characterization of living human induced pluripotent stem cells by subcellular topographic imaging technique using full-field quantitative phase microscopy coupled with interference reflection microscopy.

There is a need for a noninvasive technique to monitor living pluripotent stem cell condition without any labeling. We present an optical imaging technique that is able to capture information about optical path difference through the cell and cell adhesion properties simultaneously using a combination of quantitative phase microscopy (QPM) and interference reflection microscopy (IRM) techniques. As a novel application of QPM and IRM, this multimodal imaging technique demonstrated its ability to distinguish the undifferentiated status of human induced pluripotent stem (hiPS) cells quantitatively based on the variation of optical path difference between the nucleus and cytoplasm as well as hiPS cell-specific cell adhesion properties.

Single-shot full-field reflection phase microscopy.

We present a full-field reflection phase microscope that combines low-coherence interferometry and off-axis digital holographic microscopy (DHM). The reflection-based DHM provides highly sensitive and a single-shot imaging of cellular dynamics while the use of low coherence source provides a depth-selective measurement. The setup uniquely uses a diffraction grating in the reference arm to generate an interference image of uniform contrast over the entire field-of-view albeit low-coherence light source. We have measured the path-length sensitivity of our instrument to be approximately 21 picometers/Hz that makes it suitable for nanometer-scale full-field measurement of membrane dynamics in live cells.

Label-free imaging of intracellular motility by low-coherent quantitative phase microscopy.

The subject study demonstrates the imaging of cell activity by quantitatively assessing the motion of intracellular organelles and cell plasma membranes without any contrast agent. The low-coherent interferometric technique and phase-referenced phase shifting technique were integrated to reveal the depth-resolved distribution of intracellular motility. The transversal and vertical spatial resolutions were 0.56 µm and 0.93 µm, respectively, and the mechanical stability of the system was 1.2 nm. The motility of the cell was assessed by mean squared displacement (MSD) and we have compensated for the MSD by applying statistical noise analysis. Thus we show the significant change of intracellular motility after paraformaldehyde treatment in non-labeled cells.

Quantitative DIC microscopy using an off-axis self-interference approach.

Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

Spectroscopic phase microscopy for quantifying hemoglobin concentrations in intact red blood cells.

We report a practical method for label-free quantification of specific molecules using spectroscopic imaging of sample-induced phase shifts. Diffraction phase microscopy equipped with various wavelengths of light source is used to record wavelength-dependent phase images. We first perform dispersion measurements on pure solutions of single molecular species present in the cells, such as albumin and hemoglobin (Hb). With this prior calibration of molecular specific dispersion, we demonstrate the extraction of Hb concentration from individual human red blood cells. The end point of this study is noninvasive monitoring of physiological states of intact living cells.

Low-coherent quantitative phase microscope for nanometer-scale measurement of living cells morphology.

We have developed a Linnik-type interference microscope provided with a low-coherent light source to obtain topographic images of an intact cellular membrane on a nanometer scale. Our technique is based on measurement of the interference between light reflected from the cell surface and a reference beam. The results show full field surface topography of cultured cells and reveal an intrinsic membrane motion of tens of nanometers.

Full-field quantitative phase imaging by white-light interferometry with active phase stabilization and its application to biological samples.

We report a Koehler-illumination-based full-field, actively stabilized, low-coherence phase-shifting interferometer, which is built on a white-light Michelson interferometer. By using a phase-stepping technique we can obtain full-field phase images of the sample. An actively stabilized phase-lock circuit is employed in the system to reduce phase noise. An application to human epithelial cells (HeLa cells) is achieved in our experiment. The advancement of this technique rests in its ability to take images of unstained biological samples quantitatively and on a nanometer scale.