Inside Cover: Raman ChemLighter: Fiber optic Raman probe imaging in combination with augmented chemical reality (J. Biophotonics 7/2019)

The newly developed Raman ChemLighter allows the real‐time acquisition of spectroscopic data using a handheld probe. By intelligently combining the fiber‐based imaging approach with computational modeling, we can directly extract molecular information of a sample provide augmented chemical reality to visualize chemistry. Further details can be found in the article by Wei Yang, Abdullah S. Mondol, Clara Stiebing, Laura Marcu, Jürgen Popp, Iwan W. Schie ( e201800447 ).

     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Depth‐resolved multimodal imaging: Wavelength modulated spatially offset Raman spectroscopy with optical coherence tomography

A major challenge in biophotonics is multimodal imaging to obtain both morphological and molecular information at depth. We demonstrate a hybrid approach integrating optical coherence tomography (OCT) with wavelength modulated spatially offset Raman spectroscopy (WM‐SORS). With depth colocalization obtained from the OCT, we can penetrate 1.2‐mm deep into strong scattering media (lard) to acquire up to a 14‐fold enhancement of a Raman signal from a hidden target (polystyrene) with a spatial offset. Our approach is capable of detecting both Raman and OCT signals for pharmaceutical particles embedded in turbid media and revealing the white matter at depth within a 0.6‐mm thick brain tissue layer. This depth resolved label‐free multimodal approach is a powerful route to analyze complex biomedical samples.      

Interpreting CARS images of tissue within the C–H‐stretching region

Single band coherent anti‐Stokes Raman scattering (CARS) microscopy is one of the fastest implementation of nonlinear vibrational imaging allowing for video‐rate image acquisition of tissue. This is due to the large Raman signal in the C—H‐stretching region. However, the chemical specificity of such images is conventionally assumed to be low. Nonetheless, CARS imaging within the C—H‐stretching region enables detection of single cells and nuclei, which allows for histopathologic grading of tissue. Relevant information such as nucleus to cytoplasm ratio, cell density, nucleus size and shape is extracted from CARS images by innovative image processing procedures. In this contribution CARS image contrast within the C—H‐stretching region is interpreted by direct comparison with Raman imaging and correlated to the tissue composition justifying the use of CARS imaging in this wavenumber region for biomedical applications. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.      

Distinguishing unfolding and functional conformational transitions of calmodulin using ultraviolet resonance Raman spectroscopy

Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium‐binding protein exhibits a broad range of structural transitions, including domain opening and folding–unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well‐known, it has been challenging to examine the dynamics of transition between these structures in real‐time, due to a lack of suitable spectroscopic probes of CaM structure. In this article, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C‐terminal domain in response to Ca²⁺ binding. This spectral difference is entirely due to differences in tertiary contacts at the interdomain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation‐sensitive tyrosine residues, opening a path to time‐resolved studies of CaM dynamics using Raman spectroscopy.     

Real‐time in vivo cancer diagnosis using raman spectroscopy

Raman spectroscopy has becoming a practical tool for rapid in vivo tissue diagnosis. This paper provides an overview on the latest development of real‐time in vivo Raman systems for cancer detection. Instrumentation, data handling, as well as oncology applications of Raman techniques were covered. Optic fiber probes designs for Raman spectroscopy were discussed. Spectral data pre‐processing, feature extraction, and classification between normal/benign and malignant tissues were surveyed. Applications of Raman techniques for clinical diagnosis for different types of cancers, including skin cancer, lung cancer, stomach cancer, oesophageal cancer, colorectal cancer, cervical cancer, and breast cancer, were summarized.

Schematic of a real‐time Raman spectrometer for skin cancer detection. Without correction, the image captured on CCD camera for a straight entrance slit has a curvature. By arranging the optic fiber array in reverse orientation, the curvature could be effectively corrected.     

Raman tags: Novel optical probes for intracellular sensing and imaging

Optical labels are needed for probing specific target molecules in complex biological systems. As a newly emerging category of tags for molecular imaging in live cells, the Raman label attracts much attention because of the rich information obtained from targeted and untargeted molecules by detecting molecular vibrations. Here, we list three types of Raman probes based on different mechanisms: Surface Enhanced Raman Scattering (SERS) probes, bioorthogonal Raman probes, and Resonance Raman (RR) probes. We review how these Raman probes work for detecting and imaging proteins, nucleic acids, lipids, and other biomolecules in vitro, within cells, or in vivo. We also summarize recent noteworthy studies, expound on the construction of every type of Raman probe and operating principle, sum up in tables typically targeting molecules for specific binding, and provide merits, drawbacks, and future prospects for the three Raman probes.     

Polysaccharide-polynucleotide complexes VIII. Cation-induced complex formation between polyuridylic acid and schizophyllan

Raman spectroscopy has recently been applied ex vivo and in vivo to address various biomedical issues such as the early detection of cancers, monitoring of the effect of various agents on the skin, determination of atherosclerotic plaque composition, and rapid identification of pathogenic microorganisms. This leap in the number of applications and the number of groups active in this field has been facilitated by several technological advancements in lasers, CCD detectors, and fiber-optic probes. However, most of the studies are still at the proof of concept stage. We present a discussion on the status of the field today, as well as the problems and issues that still need to be resolved to bring this technology to hospital settings (i.e., the medical laboratory, surgical suites, or clinics). Taken from the viewpoint of clinicians and medical analysts, the potential of Raman spectroscopic techniques as new tools for biomedical applications is discussed and a path is proposed for the clinical implementation of these techniques.     

Inverse spatially‐offset Raman spectroscopy using optical fibers: An axicon lens‐free approach

Inverse spatially offset Raman spectroscopy (I‐SORS) seeks to interrogate deep inside a Raman‐active, layered, diffusely scattering sample. It makes a collimated laser beam incident onto the sample surface in the form of concentric illumination rings (of varying radii) from whose center the back‐scattered Raman signal is collected for detection. Since formation of illumination rings of different sizes requires an axicon to be moved along the axis of the collimated laser beam and axicons below a certain minimum size (~1 inch) are not readily available, this classical configuration incorporating an axicon cannot be used for designing a compact I‐SORS probe of narrower diameter. We report a novel scheme of implementing I‐SORS which overcomes this limitation by implementing ring illumination and point collection using two multi‐mode optical fibers. An important advantage of the proposed scheme is that unlike the previously reported inverse SORS configurations, it does not require physical movement of any of the optical components for generating spatial offsets needed for probing sub‐surface depths. Another advantage is its fiber‐optic configuration which is ideally suited for designing a compact and pencil‐sized I‐SORS probe, often desired in many practical situations for carrying out depth‐sensitive Raman measurements in situ from a layered turbid sample.      

Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy

Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data.     

Identification and characterization of bladder cancer by low‐resolution fiber‐optic Raman spectroscopy

Raman spectroscopy has been proved to be a promising diagnostic technique for various cancers detection. A major drawback for its clinical translation is the intrinsic weakness of Raman effects. Highly sensitive equipment and optimal measurement conditions are generally applied to overcome this drawback. However, these equipment are usually bulky, expensive and may also be easily influenced by surrounding environment. In this preliminary work, a low‐resolution fiber‐optic Raman sensing system is applied to evaluate the diagnostic potential of Raman spectroscopy to identify different bladder pathologies ex vivo. A total number of 262 spectra taken from 32 bladder specimens are included in this study. These spectra are categorized into 3 groups by histopathological analysis, namely normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors. Principal component analysis fed artificial neural network are used to train a classification model for the spectral data with 10‐fold cross‐validation and an overall prediction accuracy of 93.1% is obtained. The sensitivities and specificities for normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors are 88.5% and 95.1%, 90.3% and 98%, and 97.5% and 96.4%, respectively. These results demonstrate the potential of using a low‐resolution fiber‐optic Raman system for in vivo bladder cancer diagnosis.      

Surface‐enhanced Raman scattering‐active photonic crystal fiber probe: Towards next generation liquid biopsy sensor with ultra high sensitivity

The tremendous enhancement factors that surface‐enhanced Raman scattering (SERS) possesses coupled with the flexibility of photonic crystal fibers (PCFs) pave the way to a new generation of ultrasensitive biosensors. Thanks to the unique structure of PCFs, which allows direct incorporation of an analyte into the axially aligned air channels, interaction between the analyte and excitation light could be increased many folds leading to flexible, reliable and sensitive probes that can be used in preclinical or clinical biosensing. SERS‐active PCF probes provide unique opportunity to develop an opto‐fluidic liquid biopsy needle sensor that enables one‐step integrated sample collection and testing for disease diagnosis. Specificity being a key parameter to biosensors, the PCF inside the biopsy needle could be functionalized with targeting moieties to detect specific biomarkers. In this review article, we present some of the most promising recent biosensors based on PCFs including hollow‐core PCFs, suspended‐core PCFs and side‐channel PCFs. We provide a wide range of applications of such platform using Raman spectroscopy, label free SERS or labeled SERS detection and analyze some of the main challenges to be addressed for translating it to a clinically viable next generation sensitive biopsy needle sensing probe.     

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.

     

激光喇曼光谱技术在食品科学中的应用

激光喇曼光谱技术是一种非侵入、非弹性的光散射技术,它能够无损地提供丰富的分子结构和物质成分的信息。近来它在食品工业领域表现出很大的应用潜力。本文综述了激光喇曼光谱技术在食品科学中的应用及其新进展。主要包括果蔬农药残留的检测、肉类产品质量检测、伪劣食品鉴定、食物蛋白的研究以及食品加工监控等方面的应用。并对喇曼光谱技术在这些方面的应用前景作了进一步的展望。     

Superpixel Raman spectroscopy for rapid skin cancer margin assessment

Spontaneous Raman micro‐spectroscopy has been demonstrated great potential in delineating tumor margins; however, it is limited by slow acquisition speed. We describe a superpixel acquisition approach that can expedite acquisition between ~×100 and ×10 000, as compared to point‐by‐point scanning by trading off spatial resolution. We present the first demonstration of superpixel acquisition on rapid discrimination of basal cell carcinoma tumor from eight patients undergoing Mohs micrographic surgery. Results have been demonstrated high discriminant power for tumor vs normal skin based on the biochemical differences between nucleus, collagen, keratin and ceramide. We further perform raster‐scanned superpixel Raman imaging on positive and negative margin samples. Our results indicate superpixel acquisition can facilitate the use of Raman microspectroscopy as a rapid and specific tool for tumor margin assessment.     

Ultra‐low background Raman sensing using a negative‐curvature fibre and no distal optics

Measuring Raman spectra through an optical fibre is usually complicated by the high intrinsic Raman scatter of the fibre material. Common solutions such as the use of multiple fibres and distal optics are complex and bulky. We demonstrate the use of single novel hollow‐core negative‐curvature fibres (NCFs) for Raman and surface‐enhanced Raman spectroscopy (SERS) sensing using no distal optics. The background Raman emission from the silica in the NCF was at least 1000× smaller than in a conventional solid fibre, while maintaining the same collection efficiency. We transmitted pump light from a 785‐nm laser through the NCF, and we collected back the weak Raman spectra of different distal samples, demonstrating the fibre probe can be used for measurements of weak Raman and SERS signals that would otherwise overlap spectrally with the silica background. The lack of distal optics and consequent small probe diameter (<0.25 mm) enable applications that were not previously possible.      

Real‐time Raman and SRS imaging of living human macrophages reveals cell‐to‐cell heterogeneity and dynamics of lipid uptake

Monitoring living cells in real‐time is important in order to unravel complex dynamic processes in life sciences. In particular the dynamics of initiation and progression of degenerative diseases is intensely studied. In atherosclerosis the thickening of arterial walls is related to high lipid levels in the blood stream, which trigger the lipid uptake and formation of droplets as neutral lipid reservoirs in macrophages in the arterial wall. Unregulated lipid uptake finally results in foam cell formation, which is a hallmark of atherosclerosis. In previous studies, the uptake and storage of different fatty acids was monitored by measuring fixed cells. Commonly employed fluorescence staining protocols are often error prone because of cytotoxicity and unspecific fluorescence backgrounds. By following living cells with Raman spectroscopic imaging, lipid uptake of macrophages was studied with real‐time data acquisition. Isotopic labeling using deuterated palmitic acid has been combined with spontaneous and stimulated Raman imaging to investigate the dynamic process of fatty acid storage in human macrophages for incubation times from 45 min to 37 h. Striking heterogeneity in the uptake rate and the total concentration of deuterated palmitic acid covering two orders of magnitude is detected in single as well as ensembles of cultured human macrophages.

SRS signal of deuterated palmitic acid measured at the CD vibration band after incorporation into living macrophages.     

Raman spectroscopy investigation of biochemical changes in tumor spheroids with aging and after treatment with staurosporine

There has been increasing use of in vitro cell culture models that more realistically replicate the three‐dimensional (3D) environment found in vivo. Multicellular tumor spheroids (MTS) using cell lines or patient‐derived organoids have become an important in vitro drug development tool, where cells are grown in a 3D “sphere” that exhibits many of the characteristics found in vivo. Significantly, MTS develop gradients in nutrients and oxygen, commonly found in tumors that contribute to therapy resistance. While MTS show promise as a more realistic in vitro culture model, there is a massive need to improve imaging technologies for assessing biochemical characteristics and drug response in such models to maximize their translation into useful applications such as high throughput screening (HTS). In this study, we investigate the potential for Raman spectroscopy to unveil biochemical information in MTS and have investigated how spheroid age influences drug response, shedding light on increased therapy resistance in developing tumors. The wealth of molecular level information delivered by Raman spectroscopy in a noninvasive manner, could aid translation of these 3D models into HTS applications.