The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm-1 which assigned to PO43- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.
Colorectal adenoma (CA) is a disease caused by various factors (such as genetic factors or environmental exposures). The appearance of colon polyp (CP) within colorectal might indicate the hint of CA development. Ball-lens hollow fiber Raman probe (BHRP) may has a high capability for detection of CA in living experimental animal and have already tested to rat’s CP in this study, which was designed to collaborate between BHRP with mini-endoscopy to observe the biochemical alteration within normal colon tissue and rat’s colon polyps in real time. BHRP and mini-endoscopy can distinguish the differences in their finger print spectra and make pictures the control and CP in the real time. At the first step, the real situation of normal colon and Rat’s CP were washed by saline and observed with mini-endoscopy. BHRP was introduced to Dextran sodium sulphate (DSS)-induced Rat's CP to detect some of biochemical alteration. The main purpose of this study was to introduce mini-endoscopy to guide the BHRP for diagnosing of CP in real time and to compare it with spectra of normal colon (control group) in living rat. As the result, BHRP can provide the differences in band of control and CP group, which can inform that the biochemical of normal and CP has changed. As a major parameter to distinct normal and CP tissue were phosphatidylinositol, phosphodiester group, lipid, and collagen. Mini endoscopy and BHRP is very sensitive devices for diagnosing of CP in real time.
The present study demonstrates that Raman spectroscopy is a powerful tool for the detection of virus-infected cells. Adenovirus infection of human embryonic kidney 293 cells was successfully detected at 12, 24, and 48 h after initiating the infection. The score plot of principal component analysis discriminated the spectra of the infected cells from those of the control cells. The viral infection was confirmed by the conventional immunostaining method performed 24 h after the infection. The newly developed method provides a fast and label-free means for the detection of virus-infected cells.
Raman spectroscopy is a promising tool for detection of virus infection in live cells. In the present study, we
demonstrate its feasibility to observe dynamic reaction of the live cell infected by virus. The Raman spectra of the
adenovirus infected live cell (293 HEK) are analyzed by comparing with those of control cells. Principal
component analysis (PCA) is employed also to analyze the spectra in detail. A band at 1650 cm-1 increases its
intensity in the spectra measured at 24 hours after the virus infection. The infection of the virus is also examined
by immune-staining and transmission electron microscope (TEM), and the virus infection is confirmed with these
method also. It should be noted that the present technique does not require specifying the type of virus in
advance.
Live subcutaneous tumor grown in nude mouse is studied in situ with hyperspectral autofluorescence imaging and Raman spectroscopy. The purpose of the study is to develop methods for characterization of biochemical changing and of histological type of tumor without labeling. The results show that there are site depending variation in the fluorescence and Raman spectra. At the spot in which calcification is in process, Raman spectra showed a strong and specific band at 957 cm-1 due to PO4 species. The autofluosescence image can prove the histological changes based on the NADH and FAD which are major fluorophores in biological tissues. The hyperspectral image is analyzed with principal component analysis and the reconstructed images successfully depicts a different between necrotic and viable part within living subcutaneous tumor.
The esophageal cancer has a tendency to transfer to another part of the body and the surgical operation itself sometimes gives high risk in vital function because many delicate organs exist near the esophagus. So the esophageal cancer is a disease with a high mortality. So, in order to lead a higher survival rate five years after the cancer’s treatment, the investigation of the diagnosis methods or techniques of the cancer in an early stage and support the therapy are required. In this study, we performed the ex vivo experiments to obtain the Raman spectra from normal and early-stage tumor (stage-0) human esophageal sample by using Raman spectroscopy. The Raman spectra are collected by the homemade Raman spectrometer with the wavelength of 785 nm and Raman probe with 600-um-diameter. The principal component analysis (PCA) is performed after collection of spectra to recognize which materials changed in normal part and cancerous pert. After that, the linear discriminant analysis (LDA) is performed to predict the tissue type. The result of PCA indicates that the tumor tissue is associated with a decrease in tryptophan concentration. Furthermore, we can predict the tissue type with 80% accuracy by LDA which model is made by tryptophan bands.
The aim of the present study is to evaluate the capability of a miniaturized Raman endoscope (mRE) system to monitor the advancement of colorectal tumors in live model mice. The endoscope is narrow enough to observe the inside of the mouse colon under anesthesia. The mRE system allows to observe the tissues and to apply a miniaturized Raman probe for the measurement at any targeted point within the colon. Raman spectroscopy allows obtaining information about molecular composition without damaging the tissue (i.e., noninvasively). Continuous monitoring of the same tumor is carried out to study molecular alterations along with its advancement. The Raman spectra measured before and after the anticancer drug (5-FU) treatment indicated spectral changes in the tumor tissue. It suggests that the tumor is not cured but supposedly transformed to another tumor type after the treatment.
Our Raman probe that is called as ball-lens hollow fiber Raman probe (BHRP) had been proved
possessing capability to detect the biochemical alteration within biological tissue. Whether BHRP
has high capability and sensitivity in diagnosing the biochemical changing of tissue or not, mouse's
normal rectal and anorectal prolapse (AP) were decided to be used as a model for this non invasive
method. This AP is azoxymethane and DSS-induced mouse’s anorectal prolapse. Main outcome of
BHRP will be potential for non-invasive method in tumor diagnosing. BHRP spectra obtained were
a high quality and allowed analysis of their differences between normal rectal (control group) and
AP. After spectral acquisition and comparison with corresponding images of hematoxylin/eosinstained
section observation used to make the histopathologic diagnosing, BHRP detected some
differences within the region of moiety of DNA, protein (i.e. collagen) and lipid, then following with
the alteration of symmetric P=O stretching vibration compared with the normal rectal tissue. BHRP
discriminate normal tissue and AP in the real-time.
The esophageal cancer is a disease with a high mortality. In order to lead a higher survival rate five years after the
cancer’s treatment, we inevitably need a method to diagnose the cancer in an early stage and support the therapy. Raman spectroscopy is one of the most powerful techniques for the purpose. In the present study, we apply Raman spectroscopy to obtain ex vivo spectra of normal and early tumor human esophageal sample. The result of principal component analysis indicates that the tumor tissue is associated with a decrease in tryptophan concentration. Furthermore, we can predict the tissue type with 80% accuracy by linear discriminant analysis which model is made by tryptophan bands.
Problem of viruses is very actual for nowadays. Some viruses, which are responsible for human of all tumors, are about
15 %. Main purposes this study, early detection virus in live cell without labeling and in the real time by Raman
spectroscopy. Micro Raman spectroscopy (mRs) is a technique that uses a Raman spectrometer to measure the spectra of microscopic samples. According to the Raman spectroscopy, it becomes possible to study the metabolites of a live
cultured cell without labeling. We used mRs to detect the virus via HEK 293 cell line-infected adenovirus. We obtained
raman specters of lives cells with viruses in 24 hours and 7 days after the infection. As the result, there is some
biochemical changing after the treatment of cell with virus. One of biochemical alteration is at 1081 cm-1. For the clarification result, we use confocal fluorescent microscopy and transmission electron microscopy (TEM).
Ball-lens hollow fiber Raman Probe (BHRP) and FTIR spectroscopy were main tools in this study. Thus, both of
equipments detected the alteration of antisymmetric and symmetric P=O stretching vibration within our mice colorectal
tumor models. Some differences of spectra due to randomly the edge of each BHRP and FTIR attached the surface of
tumor during measurements. Meanwhile, the application of FTIR potentially differentiates the grade levels of non-clinic
samples colorectal tumor models at four different grades (normal, grade 1, grade 2 and grade 3). Detailed investigations
were assignable to wave numbers that publicized to represent biochemical alteration. The whole of investigated spectra
in the fingerprint region revealed some different peaks and shoulders, most of which were assignable to wave numbers
that exposed to represent biochemical alteration within the tissue. Differences in peak heights and peak ratio indicated
differences in biochemical composition of cancer from different grade level. However, all collected colorectal tumor
model at different peak was distinguishable, where antisymmetric and symmetric P=O stretching vibration was imaged
and mapped clearly by both equipments. Therefore, BHRP were comfortable for in vivo studies. Meanwhile FTIR
spectral analysis in combination with calibration curve might be used to distinguish cancer grade within colorectal tumor
model tissue for ex vivo study.
One of suspect environmental endocrine disruptors that affect mouse male reproduction by altering the morphology of Sertoli cells and spermatogenic cells is phthalate. The effects of mono(2-ethylhexyl)phthalate (MEHP), one of metabolites of di(2-ethylhexyl)phthalate , on immature mouse testes in vivo were examined. We have recently shown
that MEHP induced Sertoli cells necrosis and spermatogenic cells apoptosis in mice by TUNEL method, F-actin staining,
and ultrastructural study, but there is no data for biochemical changing of testes due to those methods could not explore.
To verify in detail of it, we conducted Raman spectroscopy study with 785 nm wavelength laser line, 50mW of laser power and 3 minutes of exposure time to analysis the MEHP-treated testicular tissue, which has been fixatived by 4% paraformaldehyde (PFA). Five weeks old (5 w.o) male mice were used in this experiment. As the results, the alterations were observed by Raman spectroscopy that there are significantly differences of DNA, actin filament, type IV collagen and amide I between control group (0 μM MEHP) and treatment group (100 μM MEHP). These results significantly support histology staining observation (such as the apoptotic spermatogenic cells which is associated with DNA fragmentation and F-actin disruption) and ultrastructural observation (such as mitochondria rupture and disintegration of nucleus membrane). Raman spectroscopy can be used for 4% PFA-fixatived tissue observation. However, we recommend that Raman spectroscopy may be able to be expanded as an armamentarium not just for the clarification of histology staining and ultrastructural study, but furthermore, it may be as a non-invasion assessment for screening animal tissue toxicity of chemical in future.
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.
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.
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