Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell–extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell–ECM interaction during angiogenesis, wound healing, and metastasis.
Research trends in endoscopy have been to reduce the dimension of the system for minimally invasive diagnostics and to improve spatial resolution to the microscopic level for the detailed investigation of specimens. In developing endoscopes that meet these needs, ultrathin imaging probes such as graded index lenses and fiber bundles have been widely used. And a single imaging probe is used for both illumination and detection to maintain the small diameter of the probe unit. However, this causes a fundamental problem, that is the back-reflection noise from the surface of the imaging probes. This back-reflection noise can overwhelm signals from target objects with weak contrast, which is the case for biological tissues, and degrade image contrast to such an extent that the objects remain unresolved.
Here, we present an endomicroscope free from back-reflection noise generated at an ultrathin imaging probe and yet guaranteeing microscopic spatial resolution. In our method, we send illumination through single individual core fibers in the image fiber bundle, and detect signal light by the other core fibers. By blocking the back-reflection occurring only at the core used for the illumination, we remove the back-reflection noise before it reaches the detector sensor. The transmission matrix of the fiber bundle is measured and used to reconstruct a pixelation-free and high-resolution image from the raw images captured by the other fibers, which are blurred and pixelated. We demonstrated that the proposed imaging method improved 3.2 times on the signal to noise ratio produced by the conventional illumination-detection scheme.
Graded-index (GRIN) lenses have been widely used for developing compact imaging devices due to the small dimensions and simple optics designs. GRIN lenses, however, have intrinsic aberration which causes a distortion of the image and thus are subject to limited resolution and blurred imaging quality. Here, we employ the high-precision wavefront measurement technique for compensation of the distortion of a GRIN lens to obtain a high-resolution and high-contrast image. In doing so, we demonstrate a high-resolution and ultra-thin endo-microscope using a GRIN. A reflection-type interferometric microscope through a GRIN lens was constructed using multiple lasers (473 nm, 532 nm, and 633 nm) as light sources. The characteristics of the aberration of the GRIN lens were measured using the digital holographic method. The distortion of the GRIN lens was removed by numerical image processing with the prior information from the pre-calibration. We apply this technique to a reflection image of biological tissues acquired by our custom-built GRIN lens probe. Consequently, a diffraction limited lateral resolution as well as improved axial resolution can be achieved. Our approach will facilitate the use of GRIN lenses for compact imaging devices without compromising optical resolution and image quality.
A graded-index (GRIN) lens is suitable for developing an ultra-thin endoscope due to its small diameter and simplicity for optics design. A GRIN lens, however, generates intrinsic optical aberration causing low resolution and poor imaging quality. Recently, wavefronts of light can be measured with very high precision and the optical aberration can be corrected in numerical ways even for the case of highly scattering media. In this study, based on the high precision wavefront sensing and numerical image processing techniques, we demonstrate a high-resolution and ultra-thin endo-microscope using a GRIN rod lens as a core imaging optics. We constructed a reflection-type interferometric microscope through a GRIN rod lens using a p-polarized Nd:YAG laser (532 nm) as a light source. By recording and processing blank transmission images as a function of various illumination states, the characteristics of the aberration generated by the GRIN lens were obtained. After this pre-calibration, we could numerically compensate the aberration induced onto a reflection image of an object. Consequently, a diffraction limited lateral resolution as well as improved axial resolution could be achieved. Our approach will fascinate the use of GRIN lenses for compact and high-resolution imaging devices including ultra-thin endo-microscopes.
Photothermal treatment (PTT) using nanoparticles has gained attention as a promising alternative therapy for malignant tumors. One strategy for increasing the selectivity of PTT is the use of macrophages as a cellular vector for delivering nanoparticles. The aim of the present study is to examine the use of macrophages as a cellular vector for efficient PTT and determine the appropriate irradiation power and time of a near-infrared (NIR) laser using real-time phase-contrast imaging. Thermally induced injury and death of cancer cells were found to begin at 44°C to 45°C, which was achieved using the PTT effect with gold nanoshells (NS) and irradiation with a NIR laser at a power of 2 W for 5 min. The peritoneal macrophage efficiently functioned as a cellular vector for the NS, and the cancer cells surrounding the NS-loaded macrophages selectively lost their cellular viability after being irradiated with the NIR laser.
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