Toyohiko Yamauchi

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.

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.

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.

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 21u2009picometers/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.

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.