Simultaneous fingerprint and high‐wavenumber fiber‐optic Raman spectroscopy enhances real‐time in vivo diagnosis of adenomatous polyps during colonoscopy

Colorectal cancer can be prevented if detected early (e.g., precancerous polyps‐adenoma). Endoscopic differential diagnosis of hyperplastic polyps (that have little or no risk of malignant transformation) and adenomas (that have prominent malignant latency) remains an unambiguous clinical challenge. Raman spectroscopy is an optical vibrational technique capable of probing biomolecular changes of tissue associated with neoplastic transformation. This work aims to apply a fiber‐optic simultaneous fingerprint (FP) and high wavenumber (HW) Raman spectroscopy technique for real‐time in vivo assessment of adenomatous polyps during clinical colonoscopy. We have developed a fiber‐optic Raman endoscopic technique capable of simultaneously acquiring both the FP (i.e., 800–1800 cm^–1) and HW (i.e., 2800–3600 cm^–1) Raman spectra from colorectal tissue subsurface (<200 µm) for real‐time assessment of colorectal carcinogenesis. In vivo FP/HW Raman spectra were acquired from 50 patients with 17 colorectal polyps during clinical colonoscopy. Prominent Raman spectral differences (p < 0.001) were found between hyperplastic (n = 118 spectra), adenoma (n = 184 spectra) that could be attributed to changes in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations. Simultaneous FP/HW Raman endoscopy provides a diagnostic sensitivity of 90.9% and specificity of 83.3% for differentiating adenoma from hyperplastic polyps, which is superior to either the FP or HW Raman technique alone. This study shows that simultaneous FP/HW Raman spectroscopy technique has the potential to be a clinically powerful tool for improving early diagnosis of adenomatous polyps in vivo during colonoscopic examination.

     

Real‐time endoscopic Raman spectroscopy for in vivo early lung cancer detection

Currently the most sensitive method for localizing lung cancers in central airways is autofluorescence bronchoscopy (AFB) in combination with white light bronchoscopy (WLB). The diagnostic accuracy of WLB + AFB for high grade dysplasia (HGD) and carcinoma in situ is variable depending on physician's experience. When WLB + AFB are operated at high diagnostic sensitivity, the associated diagnostic specificity is low. Raman spectroscopy probes molecular vibrations and gives highly specific, fingerprint‐like spectral features and has high accuracy for tissue pathology classification. In this study we present the use of a real‐time endoscopy Raman spectroscopy system to improve the specificity. A spectrum is acquired within 1 second and clinical data are obtained from 280 tissue sites (72 HGDs/malignant lesions, 208 benign lesions/normal sites) in 80 patients. Using multivariate analyses and waveband selection methods on the Raman spectra, we have demonstrated that HGD and malignant lung lesions can be detected with high sensitivity (90%) and good specificity (65%).

     

Development and in vivo testing of a high frequency endoscopic Raman spectroscopy system for potential applications in the detection of early colonic neoplasia

The objective of this study was to build and test an adjunct system to a colonoscope for in vivo measurement of Raman spectra from colon tissue for potentially improving the detection of early cancers. The novelty of this system was that low cost fibre optic probes were used, without the addition of expensive optical filters. Good quality in vivo Raman spectra were successfully obtained with a 1 s integration time in the high frequency (HF) range from normal tissue and polyps of patients during a colonoscopy. The polyps were subsequently removed, and their pathology determined. The acquired in vivo Raman spectra showed clear changes between tissue with normal and tubular adenoma pathology. Further clinical study with this low cost HF Raman probe is warranted to fully test its clinical utility.

Left: Raman probe orientated on a suspected polyp (indicated by arrow) under video surveillance during a colonoscopy. Right: average Raman spectra from 2800–3050 cm^–1 obtained from polyps at different stages of disease. The peak intensities are in arbitrary units.     

Biochemical characterization of breast tumors by in vivo and in vitro magnetic resonance spectroscopy (MRS)

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have evolved as sensitive tools for anatomic and metabolic evaluation of breast cancer. In vivo MRS studies have documented the presence of choline containing compounds (tCho) as a reliable biochemical marker of malignancy and also useful for monitoring the tumor response to therapy. Recent studies on the absolute quantification of tCho are expected to provide cut-off values for discrimination of various breast pathologies. Addition of MRS investigation was also reported to increase the specificity of MRI. Further, ex vivo and in vitro MRS studies of intact tissues and tissue extracts provided several metabolites that were not be detected in vivo and provided insight into underlying biochemistry of the disease processes. In this review, we present briefly the role of various ¹H MRS methods used in breast cancer research and their potential in relation to diagnosis, monitoring of therapeutic response and metabolism.     

Determination of inclusion depth in ex vivo animal tissues using surface enhanced deep Raman spectroscopy

This work presents recent developments in spatially offset and transmission Raman spectroscopy for noninvasive detection and depth prediction of a single SERS inclusion located deep inside ex vivo biological tissues. The concept exploits the differential attenuation of Raman bands brought about by their different absorption due to tissue constituents enabling to predict the inclusion depth. Four different calibration models are tested and evaluated to predict the depth of surface enhanced Raman scattering labelled nanoparticles, within an up to 40 mm slab of porcine tissue. An external measurement carried out in transmission mode, with a noninvasively built model on the analysed sample, is shown to be insensitive to variations of the overall thickness of the tissue yielding an average root‐mean‐square error of prediction of 6.7%. The results pave the way for future noninvasive deep Raman spectroscopy in vivo enabling to localise cancer biomarkers for an early diagnosis of multiple diseases.      

Near-infrared and two-dimensional fluorescence spectroscopy monitoring of monoclonal antibody fermentation media quality: Aged media decreases cell growth

In biomanufacturing processes, the influence of feedstock components on product yield and quality is considerable and often poorly understood. Here we describe the capabilities of near-infrared spectroscopy (NIRS) and two dimensional (2D)-fluorescence spectroscopy in detecting chemical changes over time in two types of culture media (one basal media and one feed media) used in the production of monoclonal antibodies (mAbs) by Chinese hamster ovary (CHO) cells. Both spectroscopies were able to detect compositional changes in basal media over storage period of 12 weeks. NIRS was more effective in detecting changes in feed medium composition. The impact of storage time in process performance was evaluated by using aged media components in mAb cultivations. The study suggests that basal media aging results in a decrease of the integral of viable cells (IVC) (cell growth over time), while product titer is not significantly affected. Feed media appears to be less sensitive to storage and no correlation between the age of the media and cell culture performance was detected. Results obtained provide a basis on which to further improve cell culture raw material quality assessment using vibrational (e.g. NIRS) and optical (e.g. 2D-fluorescence) spectroscopic methods.     

Dispersive Raman spectroscopy allows the identification and quantification of melanin types

Melanins are the most prevalent pigments in animals and are involved in visual communication by producing colored traits that often evolve as intraspecific signals of quality. Identifying and quantifying melanins are therefore essential to understand the function and evolution of melanin‐based signals. However, the analysis of melanins is difficult due to their insolubility and the lack of simple methods that allow the identification of their chemical forms. We recently proposed the use of Raman spectroscopy as a simple, noninvasive technique that can be used to identify and quantify melanins in feathers and hairs. Contrarily, other authors later stated that melanins are characterized by a lack of defined Raman signals. Here, we use confocal Raman microscopy to confirm previous analyses showing that the two main chemical forms of melanins (eumelanin and pheomelanin) exhibit distinct Raman signal and compare different excitation wavelengths to analyze synthetic pheomelanin and natural melanins in feathers of different species of birds. Our analyses indicate that only laser excitation wavelengths below 1064 nm are useful for the analysis of melanins by Raman spectroscopy, and only 780‐nm laser in the case of melanins in feathers. These findings show that the capacity of Raman spectroscopy to distinguish different chemical forms of melanins depends on laser power and integration time. As a consequence, Raman spectroscopy should be applied after preliminar analyses using a range of these parameters, especially in fragile biological tissues such as feathers.     

Investigating anticancer properties of the sesquiterpene ferutinin on colon carcinoma cells,in vitro and in vivo

Aims

In this research, ferutinin was evaluated for its possible cytotoxic and apoptotic inducing effects in vitro and in vivo.

Main methods

To determine IC₅₀ values of ferutinin, CT26, HT29 and NIH/3T3 cells were treated with different concentrations of ferutinin. In addition to morphological changes in cells, the DNA damage was studied using DAPI staining, comet assay and PI staining. Ferutinin was also tested for its in vivo activity.

Key findings

Analyses of cell survival by MTT assay showed that the IC₅₀ values of ferutinin on CT26 and HT29 cells were 26 and 29 μg/ml, respectively, while after treating nontumoural mouse cells even with 50 μg/ml ferutinin, 70% of cells was still surviving. The results of DAPI staining and comet assay revealed that ferutinin significantly induced DNA damage in treated cells. Induction of sub-G₁ peak after PI staining was also indicative of apoptotic effects of ferutinin in cancerous cells. In vivo studies showed a significant regression in tumour size in mice treated with ferutinin as compared to control groups. Its antitumour effects were very similar to the cisplatin treated group. Histological studies demonstrated that apoptosis rate in tumour cells was increased in comparison to tumour cells in control mice without ferutinin treatment. Interestingly, haematoxylin and eosin staining showed no damage in the spleen and liver of ferutinin treated mice.

Significance

As ferutinin showed less toxic effects in nontumoural cells, and induced its effects via apoptosis induction, it could be considered as an effective anticancer agent for future preclinical experiments.     

Investigating marine bio‐calcification mechanisms in a changing ocean with in vivo and high‐resolution ex vivo Raman spectroscopy

Ocean acidification poses a serious threat to marine calcifying organisms, yet experimental and field studies have found highly diverse responses among species and environments. Our understanding of the underlying drivers of differential responses to ocean acidification is currently limited by difficulties in directly observing and quantifying the mechanisms of bio‐calcification. Here, we present Raman spectroscopy techniques for characterizing the skeletal mineralogy and calcifying fluid chemistry of marine calcifying organisms such as corals, coralline algae, foraminifera, and fish (carbonate otoliths). First, our in vivo Raman technique is the ideal tool for investigating non‐classical mineralization pathways. This includes calcification by amorphous particle attachment, which has recently been controversially suggested as a mechanism by which corals resist the negative effects of ocean acidification. Second, high‐resolution ex vivo Raman mapping reveals complex banding structures in the mineralogy of marine calcifiers, and provides a tool to quantify calcification responses to environmental variability on various timescales from days to years. We describe the new insights into marine bio‐calcification that our techniques have already uncovered, and we consider the wide range of questions regarding calcifier responses to global change that can now be proposed and addressed with these new Raman spectroscopy tools.     

In vivo identification and characterization of binding sites for selective serotonin reuptake inhibitors in mouse brain

The present study was undertaken to identify and characterize in vivo binding sites of selective serotonin reuptake inhibitors (SSRIs) in the mouse brain by using [3H]paroxetine as radioligand. Relatively higher concentration of [3H]paroxetine was detected in the whole brain (minus cerebellum) than in the plasma of mice after the i.v. injection of the radioligand, and the half-life (t1/2) of elimination was much slower. The in vivo specific [3H]paroxetine binding in the mouse brain after the i.v. injection was defined as the difference of particulate-bound radioactivity between the whole brain and cerebellum, and it was dose-dependently attenuated by oral or intraperitoneal administration of fluoxetine (8.68-116 micromol/kg). Furthermore, oral administration of fluvoxamine, fluoxetine, paroxetine and sertraline at the pharmacologically relevant doses reduced significantly (25-94%) in vivo specific [3H]paroxetine binding in the cerebral cortex, striatum, hippocampus, thalamus and midbrain of mice, and their significant decreases were observed up to at least 8 h (fluvoxamine), 24 h (fluoxetine), and 12 h (paroxetine and sertraline) later. The value of area under the curve (AUC) for decrease in [3H]paroxetine binding vs. time in each brain region was largest for fluoxetine among these SSRIs, due to the relatively longer-lasting occupation of brain serotonin transporter. The AUC value in mouse brain after oral administration of each SSRI was 1.2-3.2 times greater in the thalamus and midbrain than in the cerebral cortex, striatum and hippocampus. Thus, the present study has revealed that [3H]paroxetine may be a suitable radioligand for in vivo characterization of brain binding sites and pharmacological effects of SSRIs.     

Portable spectroscopic system for in vivo skin neoplasms diagnostics by Raman and autofluorescence analysis

The present paper studies the applicability of a portable cost‐effective spectroscopic system for the optical screening of skin tumors. in vivo studies of Raman scattering and autofluorescence (AF) of skin tumors with the 785 nm excitation laser in the near‐infrared region included malignant melanoma, basal cell carcinoma and various types of benign neoplasms. The efficiency of the portable system was evaluated by comparison with a highly sensitive spectroscopic system and with the diagnosis accuracy of a human oncologist. Partial least square analysis of Raman and AF spectra was performed; specificity and sensitivity of various skin oncological pathologies detection varied from 78.9% to 100%. Hundred percent accuracy of benign and malignant skin tumors differentiation is possible only with a combined analysis of Raman and AF signals.      

An aid for identification of dermatoglyphic patterns in subjects difficult to fingerprint

The author's in vivo microscopic technique, initially developed for observation of capillaries in human skin, can be helpful for detecting fingerprint patterns in subjects in whom an abnormal skin surface makes it difficult to record or even observe these patterns by the usual techniques.     

An in vivo parameter identification method for arteries: numerical validation for the human abdominal aorta

A method for identifying mechanical properties of arterial tissue in vivo is proposed in this paper and it is numerically validated for the human abdominal aorta. Supplied with pressure-radius data, the method determines six parameters representing relevant mechanical properties of an artery. In order to validate the method, 22 finite element arteries are created using published data for the human abdominal aorta. With these in silico abdominal aortas, which serve as mock experiments with exactly known material properties and boundary conditions, pressure-radius data sets are generated and the mechanical properties are identified using the proposed parameter identification method. By comparing the identified and pre-defined parameters, the method is quantitatively validated. For healthy abdominal aortas, the parameters show good agreement for the material constant associated with elastin and the radius of the stress-free state over a large range of values. Slightly larger discrepancies occur for the material constants associated with collagen, and the largest relative difference is obtained for the in situ axial prestretch. For pathological abdominal aortas incorrect parameters are identified, but the identification method reveals the presence of diseased aortas. The numerical validation indicates that the proposed parameter identification method is able to identify adequate parameters for healthy abdominal aortas and reveals pathological aortas from in vivo-like data.     

A comparative Raman and CARS imaging study of colon tissue

An experimental evaluation of the information content of two complimentary techniques, linear Raman and coherent anti‐Stokes Raman scattering (CARS) microscopy, is presented. CARS is a nonlinear variant of Raman spectroscopy that enables rapid acquisition of images within seconds in combination with laser scanning microscopes. CARS images were recorded from thin colon tissue sections at 2850, 1660, 1450 and 1000 cm^–1 and compared with Raman images. Raman images were obtained from univariate and multivariate (k‐means clustering) methods, whereas all CARS images represent univariate results. Variances within tissue sections could be visualized in chemical maps of CARS and Raman images. However, identification of tissue types and characterization of variances between different tissue sections were only possible by analysis of cluster mean spectra, obtained from k‐means cluster analysis. This first comparison establishes the foundation for further development of the CARS technology to assess tissue. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Autofluorescence and white light imaging‐guided endoscopic Raman and diffuse reflectance spectroscopy for in vivo nasopharyngeal cancer detection

Nasopharyngeal cancer (NPC) is an endemic with high incidence in Southern China and Southeast Asia countries. Screening for NPC under conventional white light imaging (WLI) nasopharyngoscope examination remains a great clinical challenge due to its poor sensitivity. Here, we developed an integrated 4‐modality endoscopy system combining WLI, autofluorescence imaging (AFI), diffuse reflectance spectroscopy and Raman spectroscopy technologies for in vivo endoscopic cancer detection for the first time. A pilot clinical test of the system for NPC detection was conducted, in which 283 in vivo Raman and diffuse reflectance spectral data sets from 30 NPC patients and 30 healthy subjects were acquired under the guidance of AFI and WLI. Both high diagnostic sensitivity (98.6%) and high specificity (95.1%) for differentiating cancer from normal tissue sites were achieved using this system combined with principal component analysis‐linear discriminant analysis diagnostic algorithm, demonstrating great potential for improving real‐time, in vivo diagnosis of NPC at endoscopy.      

Development of a dual-wavelength fluorescent nanoprobe for in vivo and in vitro cell tracking consecutively

Many imaging probes have been developed for a wide variety of imaging modalities. However, no optical imaging probe could be utilized for both microscopic and whole animal imaging. To fill the gap, the dual-wavelength fluorescent imaging nanoprobe was developed to simultaneously carry both visible-range fluorescent dye and near-infrared (NIR) dye. Emission scan confirms that the nanoprobe exhibits two separate peaks with strong fluorescent intensity in both visible and NIR ranges. Furthermore, the dual-wavelength fluorescent nanoprobe has high photostability and colloidal stability, as well as long shelf-life. In vitro cell culture experiments show that the nanoprobe has the ability to label different types of cells (namely, esophageal, prostate, fibroblast and macrophage cell) for fluorescent microscope imaging. More importantly, cell tracking experiments confirm that cell migration and distribution in various organs can be tracked in real time using in vivo whole-body NIR imaging and in vitro microscopic imaging, respectively.     

In vivo synthesis of cholecystokinin in the rat cerebral cortex: identification of COOH-terminal peptides with labeled amino acids

The synthesis of the COOH-terminal octa- and tetrapeptides of cholecystokinin (CCK) has been studied in rat cerebral cortex after intraventricular administration of radioactive amino acids characteristic of the porcine COOH-terminal octapeptide of CCK, CCK-8. After immunosorption with a COOH-terminal directed antibody, cortical CCK was fractionated on Sephadex G-50 columns. The experiments demonstrated newly synthesized CCK forms which coeluted with porcine CCK-8 and CCK-4. Except for threonine the amino acids employed, methionine, tryptophan, aspartic acid, glycine and phenylalanine were incorporated. The sequence-specific radioimmunoassay, the incorporation of the employed labeled amino acids, and the elution pattern by gel filtration, suggest an almost identical structure of porcine and rat cortical CCK-8, and a concomitant synthesis of CCK-8 and CCK-4 in rat cerebral cortex.     

近红外反射光谱分析技术在植物育种与种质资源研究中的应用

近年来近红外反射光谱分析技术(NIRS)在植物育种与种质资源研究中的应用已成为一个活跃的研究领域,本从NIRS分析植物的种类和品质类型、种质资源品质分析和评价、加速品质育种进程等方面作一综述,并对NIRS在作物育种中的应用作了探讨。     

A comparative evaluation of diffuse reflectance and Raman spectroscopy in the detection of cervical cancer

Optical spectroscopic techniques show improved diagnostic accuracy for non‐invasive detection of cervical cancers. In this study, sensitivity and specificity of two in vivo modalities, i.e diffuse reflectance spectroscopy (DRS) and Raman spectroscopy (RS), were compared by utilizing spectra recorded from the same sites (67 tumor (T), 22 normal cervix (C), and 57 normal vagina (V)). Data was analysed using principal component – linear discriminant analysis (PC‐LDA), and validated using leave‐one‐out‐cross‐validation (LOOCV). Sensitivity, specificity, positive predictive value and negative predictive value for classification between normal (N) and tumor (T) sites were 91%, 96%, 95% and 93%, respectively for RS and 85%, 95%, 93% and 88%, respectively for DRS. Even though DRS revealed slightly lower diagnostic accuracies, owing to its lower cost and portability, it was found to be more suited for cervical cancer screening in low resource settings. On the other hand, RS based devices could be ideal for screening patients with centralised facilities in developing countries.