Introduction to Infrared and Raman-Based Biomedical Molecular Imaging and Comparison with Other Modalities

Molecular imaging has rapidly developed to answer the need of image contrast in medical diagnostic imaging to go beyond morphological information to include functional differences in imaged tissues at the cellular and molecular levels. Vibrational (infrared (IR) and Raman) imaging has rapidly emerged among the molecular imaging modalities available, due to its label-free combination of high spatial resolution with chemical specificity. This article presents the physical basis of vibrational spectroscopy and imaging, followed by illustration of their preclinical in vitro applications in body fluids and cells, ex vivo tissues and in vivo small animals and ending with a brief discussion of their clinical translation. After comparing the advantages and disadvantages of IR/Raman imaging with the other main modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography/single-photon emission-computed tomography (PET/SPECT), ultrasound (US) and photoacoustic imaging (PAI), the design of multimodal probes combining vibrational imaging with other modalities is discussed, illustrated by some preclinical proof-of-concept examples.     

Density functional theory study of Fourier transform infrared and Raman spectra of 2-amino-5-chloro benzonitrile

The vibrational spectra of 2-amino-5-chloro benzonitrile (ACB) have been obtained by density functional theory (DFT) calculations. Normal coordinate analysis has been carried out to support the vibrational analysis. The results were compared with the experimental values. With the help of scaling procedures, the observed FTIR and FT Raman vibrational frequencies were analysed and compared with the theoretically predicted vibrational spectra. The assignments of bands to various normal modes of the molecules were also carried out.     

Label‐Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches

Raman spectroscopy is an emerging technique in bioanalysis and imaging of biomaterials owing to its unique capability of generating spectroscopic fingerprints. Imaging cells and tissues by Raman microspectroscopy represents a nondestructive and label‐free approach. All components of cells or tissues contribute to the Raman signals, giving rise to complex spectral signatures. Resonance Raman scattering and surface‐enhanced Raman scattering can be used to enhance the signals and reduce the spectral complexity. Raman‐active labels can be introduced to increase specificity and multimodality. In addition, nonlinear coherent Raman scattering methods offer higher sensitivities, which enable the rapid imaging of larger sampling areas. Finally, fiber‐based imaging techniques pave the way towards in vivo applications of Raman spectroscopy. This Review summarizes the basic principles behind medical Raman imaging and its progress since 2012.     

Scaled quantum chemical studies on the vibrational spectra of 4-bromo benzonitrile

The vibrational spectra of 4-bromo benzonitrile have been reported. The fundamental vibrational frequencies and intensity of vibrational bands were evaluated using density functional theory (DFT) with the standard B3LYP/6-311+G basis set combination and were scaled using various scale factors which yielded a good agreement between observed and calculated frequencies. The vibrational spectra were interpreted with the aid of normal coordinate analysis. The results of the calculations were applied to simulated infrared and Raman spectra of the title compound which showed excellent agreement with the observed spectra.     

Peroxisome proliferator-activated receptor alpha antagonism inhibits hepatitis C virus replication

Hepatitis C virus (HCV) is a global health problem and a leading cause of liver disease. Here, we demonstrate that the replication of HCV replicon RNA in Huh-7 cells is inhibited by a peroxisome proliferator-activated receptor (PPAR) antagonist, 2-chloro-5-nitro-N-(pyridyl)benzamide (BA). Downregulation of PPARgamma with RNA interference approaches had no effect on HCV replication in Huh-7 cells, whereas PPARalpha downregulation inhibited HCV replication. Fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy demonstrate a clear buildup of lipids upon treatment with BA. These observations are consistent with the misregulation of lipid metabolism, phospholipid secretion, cholesterol catabolism, and triglyceride clearance events associated with the inhibition of PPARalpha. The inhibition of HCV replication by BA may result from disrupting lipidation of host proteins associated with the HCV replication complex or, more generally, by disrupting the membranous web where HCV replicates.     

Vibrational fundamentals and natural bond orbitals analysis of some tri-fluorinated benzonitriles in ground state

The theoretical IR and Raman spectra of the 2,3,4-, 2,3,6-, 2,4,5- and 3,4,5-tri-fluorobenzonitrile molecules have been calculated by using the density functional method in the ground state. The rigorous normal coordinate analyses based upon both an empirical force field and quantum chemical calculations have been performed and the detailed vibrational assignment has been made on the basis of the calculated potential energy distributions (PEDs). A comparison of molecular geometries, atomic charges and vibrational fundamentals of these molecules has been reported. The effects of fluorination upon the geometries, atomic charges and vibrational frequencies of benzonitrile have been discussed. Several ambiguities and contradictions in the previously reported vibrational assignments have been clarified. In addition, the variation of Raman intensity with excitation frequency and with temperature has also been studied.     

Density functional theory study on anti-resonance in preresonance Raman scattering for naphthalene molecules

The anti-resonance phenomenon in preresonance Raman scattering is investigated on the basis of the direct Taylor expansion of the electric dipole transition moments in vibrational Raman tensors with respect to vibrational normal coordinates. A time-dependent density functional theory treatment is applied to compute the anti-resonance of a nontotally symmetric vibrational model for naphthalene molecules, and the model spectra agree favorably with experiment. This direct evaluation approach may provide a method of predicting anti-resonance and studying its origin.     

Infrared and Raman Spectroscopy of Methylcyanodiacetylene (CH3C5N)

A spectroscopic study combining IR absorption and Raman scattering is presented for methylcyanodiacetylene (CH₃C₅N). Gas‐phase, cryogenic matrix‐isolated, and pure solid‐phase substance was analyzed. Out of 16 normal vibrational modes, 14 were directly observed. The analysis of the spectra was assisted by quantum chemical calculations of vibrational frequencies, IR absorption intensities, and Raman scattering activities at density functional theory and ab initio levels. Previous assignments of gas‐phase IR absorption bands were revisited and extended.     

Crisp and soft multivariate methods visualize individual cell nuclei in Raman images of liver tissue sections

Raman and infrared spectroscopy have been recognized to be promising tools in clinical diagnostics because they provide molecular contrast without external stains. Here, vertex component analysis (VCA) was applied to Raman and Fourier transform infrared (FTIR) images of liver tissue sections and the results were compared with K-means cluster analysis, fuzzy C-means cluster analysis and principal component analysis. The main components of VCA from three Raman images were assigned to the central vein, periportal vein, cell nuclei, liver parenchyma and bile duct. After resonant Mie scattering correction, VCA of FTIR images identified veins, liver parenchyma, cracks, but no cell nuclei. The advantages of VCA in the context of tissue characterization by vibrational spectroscopic imaging are that the tissue architecture is visualized and the spectral information is reconstructed. Composite images were constructed that revealed a high molecular contrast and that can be interpreted in a similar way like hematoxylin and eosin stained tissue sections.     

Hepatic Vitamin A Content Investigation Using Coherent Anti‐Stokes Raman Scattering Microscopy

Standard techniques for examining the distribution of vitamin A in liver either require staining or lead to rapid photobleaching of the molecule. A potentially better alternative approach is to use coherent anti‐Stokes Raman scattering (CARS) microscopy; a fast, label‐free, non‐disruptive imaging method that provides contrast based on molecular vibrations. This contribution evaluates the viability of CARS microscopy for imaging vitamin A within thick hepatic tissue under physiological conditions by tuning into its characteristic vibrational band in the fingerprint region. Additional information about the morphology and architecture of the tissue was acquired using second harmonic generation (SHG) and multi‐photon excited fluorescence (MPEF) to help mapping the intra‐lobular positions of the vitamin A droplets. We demonstrate the capability of our multimodal imaging framework to selectively image lipid‐soluble vitamin A droplets deep in bulk liver tissue with a high contrast while co‐registering a complementary morphological background that clearly visualizes hepatic lobules. The results obtained envisage the good prospect of the technique for in vivo studies assessing vitamin A distribution heterogeneity and how it is affected by the progression of hepatic diseases.     

Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of PCBs and PBDEs in water and urine samples

We report on silver–gold core-shell nanostructures that contain Methylene Blue (MB) at the gold–silver interface. They can be used as reporter molecules in surface-enhanced Raman scattering (SERS) labels. The labels are stable and have strong SERS activity. TEM imaging revealed that these nanoparticles display bright and dark stripe structures. In addition, these labels can act as probes that can be detected and imaged through the specific Raman signatures of the reporters. We show that such SERS probes can identify cellular structures due to enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the core-shell nanostructures. They also provide structural information on the cellular environment as demonstrated for these nanoparticles as new SERS-active and biocompatible substrates for imaging of live cells.

The many facets of Raman spectroscopy for biomedical analysis

A critical review is presented on the use of linear and nonlinear Raman microspectroscopy in biomedical diagnostics of bacteria, cells, and tissues. This contribution is combined with an overview of the achievements of our research group. Linear Raman spectroscopy offers a wealth of chemical and molecular information. Its routine clinical application poses a challenge due to relatively weak signal intensities and confounding overlapping effects. Nonlinear variants of Raman spectroscopy such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have been recognized as tools for rapid image acquisition. Imaging applications benefit from the fact that contrast is based on the chemical composition and molecular structures in a label-free and nondestructive manner. Although not label-free, surface enhanced Raman scattering (SERS) has also been recognized as a complementary biomedical tool to increase sensitivity. The current state of the art is evaluated, illustrative examples are given, future developments are pointed out, and important reviews and references from the current literature are selected. The topics are identification of bacteria and single cells, imaging of single cells, Raman activated cell sorting, diagnosis of tissue sections, fiber optic Raman spectroscopy, and progress in coherent Raman scattering in tissue diagnosis. The roles of networks—such as Raman4clinics and CLIRSPEC on a European level—and early adopters in the translation, dissemination, and validation of new methods are discussed.     

丙氨酸晶体太赫兹振动光谱的实验和理论研究

利用太赫兹时域光谱和低频拉曼光谱仪研究了丙氨酸晶体在0.2-2.6 THz 范围内的太赫兹吸收和拉曼散射光谱. 研究表明: 在该低频范围有四个振动模式, 其中两个只具有拉曼活性, 其余两个同时具有红外和拉曼活性. 基于B3LYP杂化密度泛函的自洽场晶体轨道法对丙氨酸周期性结构进行了理论研究和光谱计算. 通过比较实验和理论结果, 指认了实验光谱特征峰所属的不可约表示. 通过理论计算得到的图形, 得出在此低频范围的振动模式主要包含分子间氢键的扭转和摇摆运动.     

Vibrational properties of the free and adsorbed acridone

Theoretical density functional theory (DFT) calculation, ab initio and experimental vibrational characterization of acridone were performed. The computed vibrational modes agree well with the experimental values of the related crystal structure. Surface enhanced Raman scattering (SERS) of acridone in silver colloids with different surface potential values was studied. FT-SERS spectrum of acridone revealed different adsorption behavior of the title compound on the silver particles.     

Raman, FTIR, photoacoustic-FTIR and inelastic neutron scattering spectra of alkaline earth and lanthanide salts of hexahydridoruthenate(II), A2RuH6, (A = Ca, Sr, Eu) and their deuterides

The vibrational spectra (Raman, photoacoustic and Fourier transform infrared and inelastic neutron scattering) of the calcium, strontium and europium salts of hexahydrido- and hexadeuteridoruthenate(II) have been analyzed. All observed fundamental modes and overtone and combination bands are assigned. Density functional theory calculations of the vibrational frequencies assist in the analysis.     

Quantitative detection of C-deuterated drugs by CARS microscopy and Raman microspectroscopy

The introduction of carbon-deuterium (C-D) bonds into drug compounds by organic synthesis is a non-invasive labelling approach, which does not alter the chemical and physiological properties of the drug itself. C-deuterated drugs exhibit characteristic vibrational signatures in the C-D stretching region around 2100-2300 cm(-1), which avoids spectral interference with contributions from a complex biological environment. In this paper, the quantitative detection of C-deuterated drugs by Raman microspectroscopy and single-band CARS microscopy is examined. Concentration-dependent studies on drugs with aliphatic and aromatic C-D moieties were performed in a two-channel microfluidic chip, using the corresponding non-deuterated (C-H) isotopologues as an internal reference.     

Raman spectroscopic studies of terthiophenes for molecular electronics

The effect of thiol and selenol functionalization on the vibrational spectra and photochemical stability of terthiophene based molecular wires was investigated using surface-enhanced Raman scattering (SERS). The molecules were found to exhibit markedly different properties at the silver surface of the SERS substrate, despite having almost identical Raman spectra in solution and in the solid state. In contrast to terthiophene (3T), the bisthiolterthiophene (T3) and biselenol-terthiophene (Se3) molecules were stable against photoinduced structural changes when adsorbed to the metal surface at low concentrations. This indicates that the strong bonds to the silver surface, via S or Se terminal atoms, leads to a rapid decay of photoexcited states. Comparison with ab initio calculations shows that both T3 and Se3 bind with only one of the functional groups to the Ag surface.     

Quantitative detection of dye labelled DNA using surface enhanced resonance Raman scattering (SERRS) from silver nanoparticles

The detection of dye labelled DNA by surface enhanced resonance Raman scattering (SERRS) is reported. The dye labels used are commercially available and have not previously been used as SERRS dyes. Detection limits using two excitation frequencies were determined for each label. This expands the range of labels which can be used for surface enhanced resonance Raman scattering with silver nanoparticles.     

A Theoretical Investigation of Surface‐enhanced Sum‐frequency Generation

Following the surface enhanced Raman scattering (SERS), we shall investigate the possibility of observing surface‐enhanced sum‐frequency generation (SESFG), which refers to the transformation of ordinary vibrational SFG (i.e. singly resonant) into SESFG. Two mechanisms of SESFG will be studied; one is due to the transformation of singly‐resonant vibrational SFG into doubly resonant vibrational SFG (that is, both vibrationally resonant and Raman‐scattering resonant) and the other is due to the enhancement of the polarizability in addition to the original vibrational resonance in vibrational SFG.     

Substituent Effects on the Vibrational Properties of the CN Stretch Mode of Aromatic Nitriles: IR Probes Useful for Time-resolved IR Spectroscopy

Developing ideal IR probes is essential to understand the structure and dynamics of biomolecules with time-resolved IR spectroscopies and imaging techniques. Especially, nitrile (CN) group has recently been proposed to serve as IR probes of the local environment of proteins. Herein, we investigated the effect of a substituent on the vibrational properties of the benzonitrile. The electron-donating and withdrawing character of p-substituent on benzonitrile are expected to modulate the vibrational frequency, molar extinction coefficient, and vibrational lifetime of CN probe. FT-IR revealed the positive correlation between electron-donating character and the molar extinction coefficient of CN stretch mode. Infrared pump-probe (IR-PP) measurements showed that the vibrational lifetime of CN stretch mode exhibits a relatively weak correlation with the electron-donating strength. Among the investigated samples, 4-dimethylamino benzonitrile with the strongest electron-donating strength shows enhanced absorption and extended vibrational lifetime. Utilizing substituent effects will be a practical strategy to improve the performance of the IR probe.