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This paper summarizes the progress achieved over the past fifteen years in applying vibrational (Raman and IR) spectroscopy to problems of medical diagnostics and cellular biology. During this time, a number of research groups have verified the enormous information content of vibrational spectra; in fact, genomic, proteomic, and metabolomic informa...
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... have-hopefully once and forever-answered these issues in a recently published paper which demonstrates that even prolonged exposure to fixative and fixation by different methods (drying, formalin and Surepath fixation) cause changes in spectral features that are significantly less than those caused by disease. This is shown via the PCA plot depicted in Figure 6. In the past, enormous spectral changes of cells and tissues upon fixation had been reported [45], which we believe were mostly due to morphological changes upon fixation. ...Similar publications
Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unacco...
Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bi...
In the past two decades, several molecular targeted inhibitors have been developed and evaluated clinically to improve the survival of patients with cancer. Molecular targeted inhibitors inhibit the activities of pathogenic tyrosine kinases. Particularly, aberrant receptor tyrosine kinase (RTK) activation is a potential therapeutic target. An incre...
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... Trehalose-water-protein interactions have been previously and extensively studied using spectroscopic methods. In particular, Raman spectroscopy has been used because of the advantage of label-free detection in biological samples [25][26][27]. RAMAN spectra analysis was therefore performed on different freeze-dried beads; completely pure trehalose beads, and some of the trehalose-LAMP mixture beads, and the spectra are given (Fig. 3). In the completely pure trehalose bead, the vibrations in the C-O-C skeletal structure produced strong C-C bond stretching peaks in the range of 400 to 1800 cm −1 , which is considered to be a fingerprint region of trehalose. ...
In many ways, globalization is beneficial, but in one way, it promotes the spread of alien (invasive) species through international trade and transport. In different habitats, Esox lucius (northern pike) can be considered a regionally alien species, and this fish tends to establish a higher density population than desired in fresh water. Early identification of such invasive species using sensitive and quick methods is important to be able to take immediate measures and avoid environmental problems. Loop-mediated isothermal amplification (LAMP) has emerged as the best DNA/RNA detection technique, without any expensive equipment and could be used to detect environmental DNA (eDNA). However, the reagents for amplification are not stable at ambient temperature for field applications. Therefore, this work aims to lyophilize the entire reaction mixture as a single microbead, with enzyme, and LAMP primers towards the detection of mitochondrial cytochrome B (Cyt B), a housekeeping gene in Esox lucius. Analytical and molecular techniques were performed to characterize and validate the lyophilized beads, respectively. The lyophilized beads were stored at two different temperatures, at 20 °C and 4 °C, and tested for biological activity after different time intervals. The result shows that lyophilized beads are bioactive for almost 30 days when stored at 20 °C, while beads at 4 °C did not lose their bioactivity after storage for up to one year. This study will be particularly useful for conducting on-site LAMP analyses in the field, where resources for freezing and storage are limited.
Graphical Abstract
... A promising twist in the liquid biopsy analysis might be brought by the implementation of methods of vibrational spectroscopy (VS) such as surface-enhanced Raman spectroscopy (SERS) or attenuated total reflection Fouriertransformed infrared spectroscopy (ATR-FTIR) [3,12]. VS was confirmed to be an excellent tool for the characterization of malignant tissue's chemical structure and composition [13][14][15][16]. Due to the fingerprint-like character of resulting data acquired from VS, all information about the studied sample is ready for interpretation, making VS a universal technique of molecular characterization. ...
... RHM is a molecular spectroscopy technique that utilizes multiple RS measurements of adjacent parts of the studied sample, followed by plotting the combined results as a tissue map image. The use of Raman spectroscopy (RS) to investigate cancer tissue samples is not new [14,15]; however, only a few studies utilized RHM for the cognitive selection of areas of analysis [24,25]. Contrary to RS random blind spot measurements [41] or rare grid mapping [42], this approach prevents mixing the results with areas of necrosis, inflammation, fibrosis, or colloid [3]. ...
The lack of specific and sensitive early diagnostic options for pancreatic cancer (PC) results in patients being largely diagnosed with late-stage disease, thus inoperable and burdened with high mortality. Molecular spectroscopic methodologies, such as Raman or infrared spectroscopies, show promise in becoming a leader in screening for early-stage cancer diseases, including PC. However, should such technology be introduced, the identification of differentiating spectral features between various cancer types is required. This would not be possible without the precise extraction of spectra without the contamination by necrosis, inflammation, desmoplasia, or extracellular fluids such as mucous that surround tumor cells. Moreover, an efficient methodology for their interpretation has not been well defined. In this study, we compared different methods of spectral analysis to find the best for investigating the biomolecular composition of PC cells cytoplasm and nuclei separately. Sixteen PC tissue samples of main PC subtypes (ductal adenocarcinoma, intraductal papillary mucinous carcinoma, and ampulla of Vater carcinoma) were collected with Raman hyperspectral mapping, resulting in 191,355 Raman spectra and analyzed with comparative methodologies, specifically, hierarchical cluster analysis, non-negative matrix factorization, T-distributed stochastic neighbor embedding, principal components analysis (PCA), and convolutional neural networks (CNN). As a result, we propose an innovative approach to spectra classification by CNN, combined with PCA for molecular characterization. The CNN-based spectra classification achieved over 98% successful validation rate. Subsequent analyses of spectral features revealed differences among PC subtypes and between the cytoplasm and nuclei of their cells. Our study establishes an optimal methodology for cancer tissue spectral data classification and interpretation that allows precise and cognitive studies of cancer cells and their subcellular components, without mixing the results with cancer-surrounding tissue. As a proof of concept, we describe findings that add to the spectroscopic understanding of PC.
... In addition to characterization of liquid biological samples, MIR spectroscopy has shown great potential in cellular studies. MIR spectroscopy-based cytology based on the quantification of vibrational fingerprints of cellular molecular constituents (e.g., proteins, lipids, carbohydrates, and nucleic acids) provides an excellent tool for rapid differentiation between different cell types [16][17][18] , as well as the detection of cellular responses (e.g., apoptosis under cytotoxic conditions) under various influences (e.g., environmental changes, drugs, and other stimuli 19 ). For FTIR spectroscopy of live cells, the use of attenuated total reflection -FTIR (ATR-FTIR), 13,15,[20][21][22][23] and transmission measurements using thin microfluidic devices 24,25 have been reported. ...
Water absorption of mid-infrared (MIR) radiation severely limits the options for vibrational spectroscopy of the analytes - including live biological cells - that must be probed in aqueous environments. While internal reflection elements, such as attenuated total reflection prisms and metasurfaces, partially overcome this limitation, such devices have their own limitations: high cost, incompatibility with standard cell culture workflows, limited spectral range, and small penetration depth into the analyte. In this work, we introduce an alternative live cell biosensing platform based on metallic nanogratings fabricated atop elevated dielectric pillars. For the MIR wavelengths that are significantly longer than the grating period, reflection-based spectroscopy enables broadband sensing of the analytes inside the trenches separating the dielectric pillars. Because the depth of the analyte twice-traversed by the MIR light excludes the highly absorbing thick water layer above the grating, we refer to the technique as Inverted Transflection Spectroscopy (ITS). We demonstrate the analytic power of ITS by measuring protein concentrations in solution. The ability of ITS to interrogate live cells that naturally wrap themselves around the grating is also exploited to characterize their adhesion kinetics.
... It has been validated in ex vivo studies. [16][17][18][19] Raman spectroscopy is a powerful technique that provides sub-micron spatial resolution and has the specificity to capture tissue compositional variation at the molecular level along tissue dimension length ranges. Raman mapping and imaging provide a high spatial resolution map of compositional tissue properties, including protein and lipid, which is impossible with conventional molecular biology techniques. ...
A tissue preparation method will inevitably alter the tissue content. This study aims to evaluate how different common sample preparation methods will affect the tissue morphology, biomechanical properties, and chemical composition of samples. The study focuses on intervertebral disc (IVD) tissue; however, it can be applied to other soft tissues. Raman spectroscopy synchronized with nanoindentation instrumentation was employed to investigate the compositional changes of IVD, specifically, nucleus pulposus (NP) and annulus fibrosus (AF), together with their biomechanical properties of IVD. These properties were examined through the following histological specimen types: fresh cryosection (control), fixed cryosection, and paraffin-embedded. The IVD tissue could be located using an optical microscope under three different preparation methods. Paraffin-embedded samples showed the most explicit details where the lamellae structure of AF could be identified. In terms of biomechanical properties, there was no significant difference between the fresh and fixed cryosection (p > 0.05). In contrast, the fresh cryosection and paraffin-embedded samples showed a significant difference (p < 0.05). It was also found that the tissue preparations affected the chemical content of the tissues and structure of the tissue, which are expected to contribute to biomechanical properties changes. Fresh cryosection and fixed cryosection samples are more promising to work with for biomechanical assessment in histological tissues. The findings fill essential gaps in the literature by providing valuable insight into the characteristics of IVD at the microscale. This study can also become a reference for a better approach to assessing the mechanical properties and chemical content of soft tissues at the microscale.
... Different methods of molecular vibrational spectroscopy (VS) were shown to be usable for characterizing the chemical structure of malignant tissues [11,12]. Raman spectroscopy (RS) can become an efficient tool supporting the early diagnosis of pancreatic malignancy [5,13]. ...
Purpose: Knowledge about pancreatic cancer (PC) biology has been growing rapidly in recent decades. Nevertheless, the
survival of PC patients has not greatly improved. The development of a novel methodology suitable for deep investigation
of the nature of PC tumors is of great importance. Molecular imaging techniques, such as Fourier transform infrared (FTIR)
spectroscopy and Raman hyperspectral mapping (RHM) combined with advanced multivariate data analysis, were useful in
studying the biochemical composition of PC tissue.
Methods: Here, we evaluated the potential of molecular imaging in diferentiating three groups of PC tumors, which originate from diferent precursor lesions. Specifcally, we comprehensively investigated adenocarcinomas (ACs): conventional
ductal AC, intraductal papillary mucinous carcinoma, and ampulla of Vater AC. FTIR microspectroscopy and RHM maps
of 24 PC tissue slides were obtained, and comprehensive advanced statistical analyses, such as hierarchical clustering and
nonnegative matrix factorization, were performed on a total of 211,355 Raman spectra. Additionally, we employed deep
learning technology for the same task of PC subtyping to enable automation. The so-called convolutional neural network
(CNN) was trained to recognize spectra specifc to each PC group and then employed to generate CNN-prediction-based
tissue maps. To identify the DNA methylation spectral markers, we used diferently methylated, isolated DNA and compared
the observed spectral diferences with the results obtained from cellular nuclei regions of PC tissues.
Results: The results showed signifcant diferences among cancer tissues of the studied PC groups. The main fndings are
the varying content of β-sheet-rich proteins within the PC cells and alterations in the relative DNA methylation level. Our
CNN model efciently diferentiated PC groups with 94% accuracy. The usage of CNN in the classifcation task did not
require Raman spectral data preprocessing and eliminated the need for extensive knowledge of statistical methodologies.
Conclusions: Molecular spectroscopy combined with CNN technology is a powerful tool for PC detection and subtyping.
The molecular fngerprint of DNA methylation and β-sheet cytoplasmic proteins established by our results is diferent for
the main PC groups and allowed the subtyping of pancreatic tumors, which can improve patient management and increase
their survival. Our observations are of key importance in understanding the variability of PC and allow translation of the
methodology into clinical practice by utilizing liquid biopsy testing.
... In addition to this, tissue microdissection can be performed, followed by analysis by RNA sequencing (RNA-seq) to obtain spatially resolved maps of transcriptome [40]. A detailed review of each method individually is outside of scope for this review; for detailed reviews of these different methods, see references [41] (FISH and RNA-seq), [42] (Raman and IR), [43] (multiplex IHC), [44] (IMC), and [45] (MSI). In addition to molecular imaging methods, there are other methods that can measure other properties of the tissue such as optical scattering using optical coherent tomography (OCT) [46], or speed of sound attenuation through photoacoustic imaging [47]. ...
Digital pathology is revolutionising the analysis of histological features and is becoming
more and more widespread in both the clinic and research. Molecular pathology extends the tissue
morphology information provided by conventional histopathology by providing spatially resolved
molecular information to complement the structural information provided by histopathology. The
multidimensional nature of the molecular data poses significant challenge for data processing, mining,
and analysis. One of the key challenges faced by new and existing pathology practitioners is how
to choose the most suitable molecular pathology technique for a given diagnosis. By providing
a comparison of different methods, this narrative review aims to introduce the field of molecular
pathology, providing a high-level overview of many different methods. Since each pixel of an image
contains a wealth of molecular information, data processing in molecular pathology is more complex.
The key data processing steps and variables, and their effect on the data, are also discussed.
... A growing number of scientific articles confirm an important role of molecular spectroscopy in the characteristics of the chemical structure and composition of various malignant tissues [246][247][248][249]s Due to high chemical selectivity, both Raman and infrared spectroscopies can become efficient tools supporting the molecular screening of pancreatic tissue sections. This methodology provides information about the content of various biologically significant molecules and functional groups, including phospholipids and triglycerides, proteins, nucleic acids, phosphates, and carbohydrates. ...
Simple Summary
Pancreatic cancer is a very aggressive and lethal malignant neoplasm with overall 5-year survival rates below 10%. The field of pancreatic cancer research is rapidly evolving. Reports of newly revealed pathomechanisms of the nature of these tumors are published daily. Nevertheless, many aspects of a pathologic evaluation are still uncertain. It is crucial to be able to pull out practical information that impacts the diagnostic process, called a pathologic evaluation. In this review, we comprehensively summarize some of the recent papers from the pathologists’ and clinicians’ points of view. We specifically focus on pathology assessment and reporting, to make them meaningful for clinical and research purposes. Lastly, we highlight novel diagnostic and research approaches, point out some missing pieces in the field, and suggest further study directions.
Abstract
Pancreatic cancer (PC) is one of the most aggressive and lethal malignant neoplasms, ranking in seventh place in the world in terms of the incidence of death, with overall 5-year survival rates still below 10%. The knowledge about PC pathomechanisms is rapidly expanding. Daily reports reveal new aspects of tumor biology, including its molecular and morphological heterogeneity, explain complicated “cross-talk” that happens between the cancer cells and tumor stroma, or the nature of the PC-associated neural remodeling (PANR). Staying up-to-date is hard and crucial at the same time. In this review, we are focusing on a comprehensive summary of PC aspects that are important in pathologic reporting, impact patients’ outcomes, and bring meaningful information for clinicians. Finally, we show promising new trends in diagnostic technologies that might bring a difference in PC early diagnosis.
... Additionally, morphology-dependent spectral distortions regularly encountered with human tissue samples continue to plague vibrational spectroscopists. The problems faced by spectral pathology have been documented [16,17] and new artefact removal algorithms are continually being developed [18]. Another obstacle to the wider application of vibrational spectroscopic pathology is the difficulty in obtaining sufficient reliable annotations. ...
Biomedical vibrational spectroscopy has come of age. The past twenty years have brought many advancements and new developments and now its practitioners face a new challenge: artificial intelligence. Artificial intelligence has the capability to detect meaningful relationships in data sets such as those found in an infrared or Raman spectrum. The present narrative assesses the degree to which biomedical vibrational spectroscopy has already embraced artificial intelligence and what can be expected going forward. This article belongs to the Special Issue Biomedical Applications of Infrared and Raman Spectroscopy.
... The custom-built sterile chamber (6) contains a CaF 2 IR transparent optical window (7) allowing the transmission of the beam to the sample (8). The reflected light then travels back to the NCI system via the external steering mirror (5) and is directed towards the detection optics with the two galvanometers (4) and (3) internally. A CaF 2 focusing lens (9) is then used before the reflectance signal is collected by a Zn doped MCT detector (10). ...
The objective differentiation of facets of cellular metabolism is important for several clinical applications, including accurate definition of tumour boundaries and targeted wound debridement. To this end, spectral biomarkers to differentiate live and necrotic/apoptotic cells have been defined using in vitro methods. The delineation of different cellular states using spectroscopic methods is difficult due to the complex nature of these biological processes. Sophisticated, objective classification methods will therefore be important for such differentiation. In this study, spectral data from healthy/traumatised cell samples using hyperspectral imaging between 2500–3500 nm were collected using a portable prototype device. Machine learning algorithms, in the form of clustering, have been performed on a variety of pre-processing data types including ‘raw’ unprocessed, smoothed resampling, background subtracted and spectral derivative. The resulting clusters were utilised as a diagnostic tool for the assessment of cellular health and quantified using both sensitivity and specificity to compare the different analysis methods. The raw data exhibited differences for one of the three different trauma types applied, although unable to accurately cluster all the traumatised samples due to signal contamination from the chemical insult. The background subtracted and smoothed data sets reduced the accuracy further, due to the apparent removal of key spectral features which exhibit cellular health. However, the spectral derivative data-types significantly improved the accuracy of clustering compared to other data types, with both sensitivity and specificity for the background subtracted data set being >94% highlighting its utility to account for unknown signal contamination while maintaining important cellular spectral features.
... This led to the discovery of the use of IR spectroscopy to study diseases and early cancers. 4,5 'Bio-spectroscopy' is the general term used to describe the application of different forms of spectroscopy to biological and biomedical studies. 6 In recent years, the rapid development of FTIR spectroscopy has allowed it to be utilised as a robust tool in cancer screening and diagnosis. ...
The effects of hydration on the DNA conformation in the colon biopsy tissues at different disease stages – hyperplasia, dysplasia, and cancer, and their subsequent classifications were investigated in this study. FTIR spectroscopic imaging was used to study the tissues whilst controlling the humidity from 16 %RH to 88 %RH using saturated salt solutions. A non-uniform uptake of water into the tissue at its maximum hydrated state was observed in the chemical images showing the distribution of the absorbance of the νas OH spectral band. The regions of high absorbance of this band in the tissues overlap with the regions of high absorbance of predominantly the phosphate (1143 – 1100 cm-1) and lipid (2879 – 2844 cm-1) bands. Analysis of the second derivative spectra of the hydrated and de-hydrated colon tissues further revealed significant peak shifts and changes in absorbance of the spectral bands that correspond to the vibrations of the phosphate group of DNA. These findings showed some disparities when compared to the effect of hydration on the infrared spectra of live cells and pure isolated DNA, possibly due to the presence of DNA mostly in its A- form in the formalin fixed tissues. Coupled with principal component analysis, the spectral biomarkers that differentiate the healthy colon tissues from the diseased tissues were identified to be in the phosphodiester spectral region (1300 – 1000 cm-1). This differentiation varied under different humidity conditions, with the highest sensitivity of ~98% found at the de-hydrated state of the tissues with random forest supervised classification.
