Katsumi Ishizaki

Variable phase-contrast fluorescence spectrometry for fluorescently stained cells

This letter describes the spectroscopic measurements of fluorescently stained cells. Variable phase-contrast fluorescence spectrometry was used on fluorescently stained cells to achieve high two-dimensional spatial resolution. Phase shift interferometry by autocorrelation interference made it possible to measure fluorescence spectra in the field of view without the separation of wavelengths, as in the case of a conventional dispersive spectrometer. In this letter, the authors describe the experimental characteristics of fluorescence spectra generated from fluorescently stained cells and verify that the fluorescence spectra of the stained area in the cells can be measured by our method.

A precise method for rotating single cells

A precise method to rotate single cells is reported. In this method, the light pressure in the optical axis direction is harnessed as a rotating torque. Two proximal points in each cell are illuminated from different directions using two beams, and a light pressure is created that acts as a rotating torque. Using this proposed method, we could control the rotational direction of a microsphere regardless of the refractive index distribution in a noncontact operation. The microsphere could be rotated using proximal two-beam optical tweezers, and the rotational velocity could be controlled by changing the light intensity.

Translational velocity measurement for single floating cell based on optical Fourier transform theory

This letter reports on translational velocity measurement, which is needed for tracking a low contrast cell. We propose a new optical spatial filtering method that is based on the optical Fourier transform theory. In this method, a pinhole is installed as a spatial filter on the optical Fourier transform plane. By means of this spatial filter, the arbitrary component of the spatial frequency is derived from the random refractive index distribution as the periodic light intensity distribution. By observing the changes of this light intensity, we can obtain the translational velocity of a low-contrast cell by means of a high-response photodiode.

Proposal of spectroscopy-tomography of single cell

Currently, diagnosis of cancer is performed by biopsy, whereby medical doctors observe a removed specimen, focusing their attention on morphological changes in the cell sequence and cell nuclei. For early cancer, the only effect is a slight increase in the size of the cell nuclei in comparison with normal cells. Based on medical knowledge, it is presumed that an extremely small amount of a specific protein may be contained in a cell nucleus. We propose spectroscopy-tomography of single cell to measure slight changes in this protein. This technology is composed of two elemental technologies, high spatial resolution spectrometry and a precise single cell rotating method. We propose variable phase-contrast spectrometry as the high spatial resolution spectrometry and proximal two-beam optical tweezers as the precise rotating method. By these methods, we can obtain a 3-dimensional distribution of the cell components to a high spatial resolution. We verified the accuracy of variable phase-contrast spectrometry by measuring the height of a diffraction grating. We confirmed that a microsphere can be rotated by proximal two-beam optical tweezers.

Three-dimensional phase-contrast imaging of single floating cells

A three-dimensional phase-contrast imaging technique that does not involve fluorescent labeling has been developed for observing floating cells. In this method, a single floating cell is made to rotate and images are acquired at several orientations of the cell using a phase-contrast microscope. From these two-dimensional phase-contrast images, three-dimensional cross-sectional images are obtained using the conventional computed tomography algorithm. This proposed method enabled successful rotation of a floating cell (a breast cancer cell line) and reconstruction of three-dimensional phase-contrast images. In these reconstructed three-dimensional images, the distribution of cell organelles is obtained and the cell nucleus is clearly distinguishable.

Technique for measuring the rotational velocity of a single cell

A method for measuring the rotational velocity of a cell is presented. In this method, a parallel pencil beam illuminates the cell and the reflected light intensity distribution from the cell surface is observed using an objective lens. In the Fourier transform plane of the lens, this light intensity distribution is translated by an amount that corresponds to the rotational velocity of the cell. This translational velocity can be converted into the rotational velocity. The authors confirmed that the rotational velocity of a cell could be measured using this method by rotating a cancer cell using light pressure.

6-DOF control of single living cells by proximal two-beam optical tweezers

We performed a spectroscopy-tomography study of a single living cell to obtain 3-dimensional distribution of proteins in high spatial resolution in real time. In this report, we mention the 6-DOF manipulation of a single living cell to achieve the high spatial resolution 3-dimentional spectrometry. We propose the proximal two- beam optical tweezers as rotational operation. We decided to illuminate the proximal two points in each from different directions using two beams. In this case, the light pressure generated by light absorption is made to act as rotating torque. Using this proposed method, we can operate the rotational velocity of a microsphere regardless of refractive index distribution by non-contact operation. In addition, rotational speed is controlled by optical PWM operation. This proposed optical PWM operation is that the received light intensity is changed by the illumination time. This method can be developed into the 6-DOF control of single-cell. And we propose the optical spatial filtering method, paying attention to the diffracted light that is generated from a sample, as translational velocity measurement. This measurement derives the arbitrary component of the spatial frequency from the random refracted index distribution as the periodic light intensity distribution. This periodic light intensity distribution changes in accordance with the translation of an object. Therefore, we can obtain the translational velocity of the non- labeled cell by high-response photodiode.

Displacement measurement of the depth migration of transparent cells

This letter reports a method for displacement measurement of the depth migration of transparent cells. This proposed optical spatial filtering method allows visualization of the transparent cells and determination of depth migration as a horizontal displacement positive or negative first order diffracted light on the detector surface. When the sample is displaced upward or downward from the focal plane, first and negative first order diffracted light form images at a different point as a light circle. The coordinates of these two light circles on the detector surface change places when the displacement of depth migration moves to the opposite direction.

Variable phase-contrast spectrometry for reconstructing the 3- dimensional distribution of components in single living cells

We propose the spectroscopy-tomography of single cells to improve the early detection and treatment of cancer. This technology can obtain the 3-dimensional distribution of components at a high spatial resolution. In this paper, we mention the analysis result of the cross-sectional images of the microsphere whose diameter is 10 μm. The distribution of the internal submicron-defect in the microsphere can be analyzed. To obtain the correct 3-dimensional absorption distribution, the axial runout can not be allowed. However, the center of rotation is displaced because the cells have complex refractive index distribution. Therefore we propose the image processing that uses the normalized correlation function as estimated value. The cross-sectional image of the microsphere is improved and the vague internal defect becomes to be distinguished by this proposed method. Moreover, based on this method, the 3-dimentional refractive index distribution in a single cell is estimated and the part which has a high refractive index in the cell is distinguished clearly. And we mention the proposed variable phase-contrast spectrometry as the 2-dimensional high spatial resolution spectrometry. This proposed method is a phase-shift interferometry between the 0th order diffracted light and the higher order diffracted light. We discuss the experimental results of the spectral characteristics using the proposed variable phase-contrast spectrometry. We measured the spectral characteristics at each pixel using the color filter of the liquid crystal and verified that the 2-dimensional spectral characteristics can be measured with good result.

Imaging technology of three-dimensional distribution for sugar chain on single living cell membrane

We study on the imaging technology of three-dimensional distribution for sugar chain on single living cell-membrane. This technology can observe the entire cell surface. To observe the cell surface, the local area image of cell-membrane is taken by TIRF (total internal reflection fluorescence) microscopy. And by scanning the whole cell surface area, we can obtain the image of the entire cell membrane. These observed local area images can be converted into an entire surface image by the pattern matching processing. For this scanning technology, we propose the proximal two beam optical tweezers to rotate the single floating cell. This proximal two beam optical tweezers can rotate the floating single cell in the nutrient medium by light pressure. Two beams illuminate the single cell at proximal two points from below and above. The cell is trapped at the center of these two focal points. At the same time, light pressures that are generated at two focal points are made to act as rotational torque. Conventionally TIRF microscope is well known as the observation technology for the cell-membrane using the evanescent light as the exciting light. We can observe the local area images of the fluorescently labeled sugar chain that binds the glycoprotein. Using the proposed optical system, we can obtain the fluorescent distribution images on the cell-membrane.