Atsushi Maruyama

Detection of ER stress in vivo by Raman spectroscopy

The endoplasmic reticulum (ER) is an organelle in which most membrane and secretory proteins are synthesized. If these proteins are not folded correctly, unfolded proteins accumulate in the ER lumen, causing a cellular situation known as ER stress. Recently, many studies on the relationship between ER stress and diseases have been reported. Thus, studies of ER stress in vivo should yield information that is useful in pathology. Model mice have been developed as a powerful tool to visualize ER stress in vivo, but this approach depends on transgenic technology. Here, we report on a method of detecting ER stress in vivo by Raman spectroscopy. Our experiments revealed that two specific Raman bands were reduced in both cultured cells and animal tissues in an ER stress dependent manner. This suggests that Raman spectroscopy could be a useful tool to detect ER stress in vivo without transgenic technology.

An optical biopsy system with miniaturized Raman and spectral imaging probes: in vivo animal and ex vivo clinical application studies

An optical biopsy system which equips miniaturized Raman probes, a miniaturized endoscope and a fluorescent image probe has been developed for in vivo studies of live experimental animals. The present report describes basic optical properties of the system and its application studies for in vivo cancer model animals and ex vivo human cancer tissues. It was developed two types of miniaturized Raman probes, micro Raman probe (MRP) made of optical fibers and ball lens hollow optical fiber Raman probe (BHRP) made of single hollow optical fiber (HOF) with a ball lens. The former has rather large working distance (WD), up to one millimeter. The latter has small WD (~300μm) which depends on the focal length of the ball lens. Use of multiple probes with different WD allows one to obtain detailed information of subsurface tissues in the totally noninvasive manner. The probe is enough narrow to be inserted into a biopsy needle (~19G), for observations of the lesion at deeper inside bodies. The miniaturized endoscope has been applied to observe progression of a stomach cancer in the same rat lesion. It was succeeded to visualize structure of non-stained cancer tissue in live model animals by the fluorescent image technique. The system was also applied to ex vivo studies of human breast and stomach cancers.

Masked plasma oxidation: simple micropatterning of extracellular matrix in a closed microchamber array

We present a simple micropatterning process to create a closed microchamber array with an extracellular matrix (ECM). The process includes homogeneous coating of the array with the ECM, micropatterned decomposition by plasma oxidation with physical masking, and subsequent sealing. These processes are sufficiently simple to enable scaled-up production of microchamber arrays coated with ECMs.

Application study of the optical biopsy system for small experimental animals

An optical biopsy system for small experimental animals has been developed. The system includes endoscope probe, portable probe and two kinds of miniaturized Raman probes. The micro Raman probe (MRP) is made of optical fibers and the ball lens hollow optical fiber Raman probe (BHRP) is made of hollow fiber. The former has large focal depth and suitable to measure average spectra of subsurface tissue. The latter has rather small focal depth and it is possible to control focal length by selecting ball lens attached at the probe head. It is suitable to survey materials at the fixed depth in the tissue. The system is applied to study various small animal cancer models, such as esophagus and stomach rat models and subcutaneous mouse models of pancreatic cancers. In the studies of subcutaneous tumor model mouse, it is suggested that protein conformational changes occur in the tumor tissue within few minutes after euthanasia of the mouse. No more change is observed for the following ten minutes. Any alterations in the molecular level are not observed in normal skin, muscle tissues. Since the change completes in such a short time, it is suggested that this phenomenon caused by termination of blood circulation.

Two-Dimensional Raman Correlation Analysis of Diseased Esophagus in a Rat

Generalized two-dimensional (2D) Raman correlation analysis effectively distinguished a benign tumor from normal tissue. Line profiling Raman spectra of a rat esophagus, including a benign tumor, were measured and the generalized 2D synchronous and asynchronous spectra were calculated. In the autocorrelation area of the amide I band of proteins in the asynchronous map, a cross-like pattern was observed. A simulation study indicated that the pattern was caused by a sharp band component in the amide I band region. We considered that the benign tumor corresponded to the sharp component.

Noninvasive subsurface analyzing technique using multiple miniaturized Raman probes

The optical property of the ball lens mounted hollow optical fiber Raman probe (BHRP) is studied in the present study. Since the ball lens has rather large aberration, the focus of the BHRP is dispersed and the spatial resolution in depth direction goes low. The spatial dispersion of the focal point was evaluated using model samples. The BHRP equipped a sapphire ball lens of 500 μm diameter was employed. Layered samples consisting of a polymethyl methacrylate (PMMA) substrate and various thicknesses of polyethylene (PE) films were measured with the BHRP. The relative band intensities of the upper and the lower layers appear at different rates in the obtained spectra, reflecting the optical properties of the probe. According to the spectra, the optical dispersion of the focal point is estimated. The result suggests that the spatial dispersion of the focus point fitted to Gaussian distribution. The working distance (WD) is 53 μm and the FWHM of the fitted Gauss distribution is 64 μm.