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While developing a three dimensional measurement technique by the retardation-modulated differential interference contrast (RM-DIC) microscope, we found a problem. The problem is that the measurement range is restrict in λ because it applies weak phase approximation. To overcome this drawback, we propose a three dimensional (3-D) reconstruction method with z-axis scanning. This technique needs high optical sectioning like confocal microscopy. We investigate the characteristic of optical sectioning in DIC microscope, then, we confirm that DIC microscope has high optical sectioning, experimentally. We also confirm that RM-DIC microscope has higher one. Combining the optical sectioning of RM-DIC microscope and z-scanning, we develop a new 3-D reconstruction method. This novel technique overcomes the problem, the measurement range goes micron order.

While considering measurements of grating type samples with the retardation-modulated differential interference contrast (RM-DIC) microscope, we found a problem of phase objects. The problem is that smaller measured values than the actual phase distribution are obtained when phase objects are beyond the weak phase approximation range. We analyze the image characteristics of phase objects and show that the problem is caused by the effect of an image component which is proportional to the square of the phase distribution. To overcome this disadvantage, we propose a new analysis method named self phase-correction (SPC). The SPC method corrects the phase distribution of the first order approximation under the weak phase condition and obtains the actual phase distribution of an object's phase beyond the weak phase approximation range. We propose a two-image SPC method and a three-image SPC method for the RM-DIC microscope, and examine the suitability of these methods. We confirm that the three-image SPC method effectively overcomes the problem, and that the measurement range of the RM-DIC microscope can thus be extended to a half-wavelength phase difference.

Pluripotent stem cells (PSCs) have various degrees of pluripotency, which necessitates selection of PSCs with high pluripotency before their application to regenerative medicine. However, the quality control processes for PSCs are costly and time-consuming, and it is essential to develop inexpensive and less laborious selection methods for translation of PSCs into clinical applications. Here we developed an imaging system, termed Phase Distribution (PD) imaging system, which visualizes subcellular structures quantitatively in unstained and unlabeled cells. The PD image and its derived PD index reflected the mitochondrial content, enabling quantitative evaluation of the degrees of somatic cell reprogramming and PSC differentiation. Moreover, the PD index allowed unbiased grouping of PSC colonies into those with high or low pluripotency without the aid of invasive methods. Finally, the PD imaging system produced three-dimensional images of PSC colonies, providing further criteria to evaluate pluripotency of PSCs. Thus, the PD imaging system may be utilized for screening of live PSCs with potentially high pluripotency prior to more rigorous quality control processes.

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