Hidenao Iwai

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.

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.

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.

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.

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.

Long-term measurement of spontaneous membrane fluctuations over a wide dynamic range in the living cell by low-coherent quantitative phase microscopy

Surface topography and its dynamic fluctuations in live cultured cells were obtained by low-coherent quantitative phase microscopy (LC-QPM), using a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low coherency of the light-source, only the light reflected at a specific sectioning height of the sample generates interference fringes on the CCD camera. Because the digital holographic technique enables us to quantitatively measure the intensity and phase of the optical field, a nanometer-scale surface profile of a living cell can be obtained by capturing the light reflected by the cell membrane. The lateral and the vertical spatial resolution was 0.56 microns and 0.93 microns, respectively, and the mechanical stability of the phase measurement was better than 2 nanometers. The measurements were made at fast (21 frames/sec) and slow (2 frames/sec, time-lapse) frame rates and the slow measurements were performed over a period of 10 minutes. The temporal fluctuations of the cell membrane were analyzed by the mean-square-displacement (MSD) as a function of the time-difference τ. By merging the fast and slow data, the MSDs from τ = 50 msec to τ = 300 sec were obtained and wide-dynamic-range measurements of the MSDs from 2 nm2 to over 100000 nm2 were demonstrated. The results show significant differences among different cell types under various conditions.

Measurement of topographic phase image of living cells by white-light phase-shifting microscope with active stabilization of optical path difference

We report a full-field, phase-shifting microscope with precise control of the optical path difference (OPD) and show topographic phase images of living cells. Our system is based on a Linnik interference microscope with Kohler illumination of a halogen lamp for the imaging lightsource. Phase-sensitive active-stabilization of the OPD is employed with an infrared laser whose optical path is the same as that of halogen light. We previously reported the results of cultured cell topography with this stabilization scheme; however the phase stability and the image quality were insufficient. We have improved the noise-cancellation system and achieved control of the OPD with stability down to 0.7 nm in the bandwidth of 500 Hz. Quarter wavelength phase-shifting was carried out with sub-nanometer accuracy, and clear topographic phase images of cultured single-layer cells were obtained. The Kohler illumination with the halogen lamp, whose coherence length is 2 &mgr;m, enables homogenous illumination and suppression of artifact signals arising from optical components not associated with the surface of interest.

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called ”membrane nanotubes” or “tunneling nanotubes.” However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

Tissue spectroscopy with a newly developed phase modulation system based on the microscopic Beer-Lambert law

We have developed a phase and intensity-modulated spectroscopy system (PMS) using a newly developed algorithm based on the microscopic Beer-Lambert law. Experiments with phantoms and the human body demonstrate the feasibility and reliability of the system as well as the new algorithm. Our goal is to develop compact, cost-effective, highly reliable, and user-friendly medical equipment for the quantitative monitoring of oxygen metabolism, and so on. The PMS system consists of three time-shared wavelength laser diodes with a 70MHz modulation frequency as sources and a 3mm diameter silicon PIN photodiode as a detector with an in-phase quadrature demodulator (IQD) for AC amplitude and phase detection. The PIN photodiode is operated at a low voltage and is durable against strong extraneous light. In addition, a specially designed low-noise amplifier is achieve a high S/N and reliable measurement. Our algorithm is independent of boundary conditions, exterior shape, scattering properties of the medium, and optode separation for measurement. We can therefore quantify the absolute concentration for oxy- deoxy-hemoglobin and hemoglobin saturation in living tissue of various shapes precisely.

Three-dimensional morphological imaging of human induced pluripotent stem cells by using low-coherence quantitative phase microscopy

There is an increasing need for non-invasive imaging techniques in the field of stem cell research. Label-free techniques are the best choice for assessment of stem cells because the cells remain intact after imaging and can be used for further studies such as differentiation induction. To develop a high-resolution label-free imaging system, we have been working on a low-coherence quantitative phase microscope (LC-QPM). LC-QPM is a Linnik-type interference microscope equipped with nanometer-resolution optical-path-length control and capable of obtaining three-dimensional volumetric images. The lateral and vertical resolutions of our system are respectively 0.5 and 0.93 μm and this performance allows capturing sub-cellular morphological features of live cells without labeling. Utilizing LC-QPM, we reported on three-dimensional imaging of membrane fluctuations, dynamics of filopodia, and motions of intracellular organelles. In this presentation, we report three-dimensional morphological imaging of human induced pluripotent stem cells (hiPS cells). Two groups of monolayer hiPS cell cultures were prepared so that one group was cultured in a suitable culture medium that kept the cells undifferentiated, and the other group was cultured in a medium supplemented with retinoic acid, which forces the stem cells to differentiate. The volumetric images of the 2 groups show distinctive differences, especially in surface roughness. We believe that our LC-QPM system will prove useful in assessing many other stem cell conditions.