Myopia, a global public health problem, is recognized by the World Health Organization as the leading cause of visual impairment in uncorrected people. At present, a large number of reports focus on the pathological manifestations of retinal level myopia. However, corneal histological changes that may be associated with myopia have not been thoroughly investigated. Raman spectroscopy is a rapid and non-destructive analytical technique with the advantages including label-free, non-invasive and highly specific, providing detailed information at the molecular level. Important components of all the human tissue (proteins, nucleic acids, lipids, etc.) have corresponding Raman spectral characteristic peaks, which contribute to the study of myopia at the molecular level. In this study, a microscopic confocal Raman system (MCRS) was built to collect Raman spectrum of corneal stromal samples, which was obtained through femtosecond laser small incision corneal stroma lens extraction (SMLIE). One hundred fifty-nine corneal stromal Raman spectrum data were collected (54 low myopia, 69 moderate myopia and 36 high myopia). Ten characteristic peaks and corresponding components were further identified. K-nearest neighbor (KNN) was used with principal component analysis (PCA) to classify the samples and the classifications were validated by k-fold cross-validation. Three types of samples with different degrees of myopia were differentiated under the PCA-KNN model with an accuracy of 93.1%. Two characteristic peaks (1099 cm-1 and 2940 cm-1) show greatly contribution to the classification results. The results provide that Raman spectroscopy combined with PCA-KNN analysis can effectively distinguish the degree of myopia and is expected to explore the potential causes of myopia.
KEYWORDS: Raman spectroscopy, Tomography, Optical tomography, Imaging systems, Signal detection, 3D image processing, 3D image reconstruction, Projection systems
Volumetric imaging enables rapid, quantitative and global measurements of cells, tissues or organisms to obtain their biomolecular information and has become a powerful tool for studying cellular metabolism, brain function and developmental biology. Optical projection tomography (OPT) plays an important role in whole-body imaging of cells, organs, embryos and organisms because it enables three-dimensional (3D) imaging with high spatial and temporal resolution of samples at the millimeter level. However, the OPT technique relies on fluorescent labels for chemical targeting, which can perturb the biological function of living system. As a label-free molecular imaging technique, widefield Raman imaging enables high-resolution analysis of large field-of-view samples. Its combination with projection tomographic strategy enables high-resolution 3D imaging of large-scale samples in a label-free manner. However, this technique was failure to determine the tissue microstructure and specific spatial distribution. Here, we proposed a concept of new label free volumetric imaging, dual-modality of optical-Raman projection tomography. In this concept, Raman projection tomography was assigned to achieve volumetric imaging of chemical composition and distribution in a 3D volume, and the OPT was used to obtain structural information of the 3D volume with micron-level spatial resolution. We further homebuilt a dual-modality imaging system for optical-Raman projection tomography and the feasibility of the system was validated by imaging polystyrene microspheres and dimethyl sulfoxide. Finally, we demonstrated the application potential by a series of bio-sample experiments.
Coherent anti-Stokes Raman scattering (CARS) microscopy enables the analysis of the chemical composition and distribution within living cells, biomolecules, or living organisms in a label-free manner. Compared with the traditional spontaneous Raman imaging technology, its advantages of high imaging sensitivity and resolution, fast imaging speed and strong signal intensity make it more popular in multiple disciplines. The available CARS microscopes are most adopted advanced crystal solid-state lasers, which are expensive, bulky, and sensitive to the environmental changes. Supercontinuum fiber lasers with a wide spectral tuning range are increasingly used in biomedical applications due to their low cost, small size, and low environmental impact. Here, we homebuilt a CARS microscope based on a supercontinuum fiber laser, a specially tailored laser with a dual-channel time-synchronous outputs. The influence factors were investigated including the objective numerical aperture, laser power, and sample concentration, etc. The feasibility of CARS microscope was then verified by imaging the polystyrene microspheres (PS) and polymethyl methacrylate microspheres (PMMA). Finally, we imaged the lipid droplet distribution of EC109 cell, which revealed the application potential of the supercontinuum fiber laser-based CARS microscope in biomedical applications.
KEYWORDS: Raman spectroscopy, Remote sensing, Gaussian beams, Chemical analysis, Bessel beams, Analytical research, Tablets, Stomach, Skin, Signal to noise ratio
To meet the diversity needs of diagnosis, treatment or prevention of diseases, different pharmaceutical dosage forms are designed and manufactured. The main role of each dosage form is drug carrier. However, changing forms might have some other different effects in clinical usages. For example, the capsule and tablets are absorbed by the intestine and stomach respectively, solutions and patches can act directly on the surface of skin etc. The quantity and quality analysis of the main drug in different form is a key issue in quality control. Therefore, it is a meaningful research of developing a facility method to detect the drug in different dosage forms. The traditional drug detection methods principally analyze and evaluate the performance through chemical reactions, photo-electricity or electrophoresis. However, these methods will cause damage to the samples. Owing to the non-invasive, non-destructive and label-free characteristics, Raman spectroscopy (RS) technique plays an important role in different fields. The current RS setup uses Gaussian beam as the excitation light, which can provide higher signal-to-noise in the thin or transparent sample. However, the Gaussian beam dispersed rapidly in the scattering medium, it is not conducive to in vivo or deep imaging. The Bessel beam having long focusing characteristics and self-reconstructing properties may provide solution to this problem. We here presented a new scheme for RS, which used a Bessel beam as the excitation light. The feasibility and effectiveness of the proposed scheme for detecting the drug in different pharmaceutical dosage forms were verified by series experiments.
Traditional drug detection technique is highly accurate but time consuming and labor intensive. Raman spectroscopy (RS) is a fast and non-destructive detection technique that provides detailed information on chemical composition, phase and morphology, crystallinity and molecular interaction of the sample. The current Raman spectrometer is mainly based on the use of Gaussian light, providing with good signal to noise ratio for a thin or transparent sample. However, owing to the scattering effect, the Gaussian beam will become diffuse in the scattering medium. This makes it not conducive to in vivo or deep imaging. Utilizing the long focusing characteristics and self-reconstructing properties of Bessel beam, we here presented a new scheme for RS, which used a Bessel beam as the excitation light. The Bessel beam-based RS was first verified with the standard samples, and then comparatively tested on several drugs. Taking the acetaminophen as the test sample, we compared the Bessel beam-based RS with the traditional Gaussian beam based one with or without a scattering medium. With the addition of a scattering medium, the signal-to-noise ratio of Raman spectra based on Bessel beam decreases less than that based on the Gaussian light, which demonstrated the great potential of the use of Bessel beam in in vivo or deep RS. This study provides great value for in vivo applications of Raman spectroscopy.
Optical projection tomography(OPT) provides an approach to recreating three-dimensional images of small biological specimens. Light traverses through a straight line to achieve a homogeneous illumination of the specimen. As the specimens in the conventional OPT could not survive or the survival time was too short, this paper proposes a new type of sample fixation method for OPT imaging. The specimen was anaesthetized in a petri dish, and the dish was fixed under the rotational stage of our homemade OPT system for imaging. This method can reduce the damage to the specimen and be more conducive to the continuous observation for in vivo OPT. However, the sample fixation causes the problem of insufficient sampling. To obtain optical projection tomographic image with insufficient samples, this paper uses the iterative reconstruction algorithm combining with the prior information to solve the inverse reconstruction problem.
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