Development of lipid targeting Raman probes for in vivo imaging of Caenorhabditis elegans

A simple, sensitive, and highly specific lipid targeting Raman probe (Nile red coated silver nanoparticles) has been developed to image living nematode Caenorhabditis elegans (C. elegans). Our idea of imaging lipids in C. elegans is to combine the specificity of the fluorescent dye, Nile red, and the highly enhanced Raman scattering on the silver nanoparticles. Our strategy involves the fabrication of a lipid targeting probe, which is incorporated into the intracellular intestinal granules of C. elegans by incubating these worms in the solution containing Raman probes, resulting in an uptake and subsequent incorporation of these Raman probes into the intestinal granule, thus allowing fast visualization of lipid droplets through a conventional confocal imaging technique.     

Development of bioorthogonal SERS imaging probe in biological and biomedical applications

Living-cell imaging demands high specificity,sensitivity,and minimal background interference to the targets of interest.However,developing a desirable imaging probe that can possess all the above features is still challenging.The bioorthogonal surface-enhanced Raman scattering(SERS) imaging has been recently emerged through utilizing Raman reporters with characteristic peaks in Raman-silent region of cells(1800-2800 cm~(-1)),which opens a revolutionary avenue for living-cell imaging with multiplexing capability.In this review,we focus on the recent advances in the technology development and the biological and biomedical applications of the living-cell bioorthogonal SERS imaging technique.After introduction of fundamental principles for bioorthogonal tag or label,we present applications for visualization of various intracellular components and environment including proteins,nucleic acids,lipids,pH and hypoxia,even for cancer diagnosis in tissue samples.Then,various bioorthogonal SERS imaging-guided thera py strategies have been discussed such as photothera py and surge ry.In conclusion,this strategy has great potential to be a flexible and robust tool for visualization detection and diseases diagnosis.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

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.     

Raman spectroscopic imaging for in vivo detection of cerebral brain metastases

We report for the first time a proof-of-concept experiment employing Raman spectroscopy to detect intracerebral tumors in vivo by brain surface mapping. Raman spectroscopy is a non-destructive biophotonic method which probes molecular vibrations. It provides a specific fingerprint of the biochemical composition and structure of tissue without using any labels. Here, the Raman system was coupled to a fiber-optic probe. Metastatic brain tumors were induced by injection of murine melanoma cells into the carotid artery of mice, which led to subcortical and cortical tumor growth within 14 days. Before data acquisition, the cortex was exposed by creating a bony window covered by a calcium fluoride window. Spectral contributions were assigned to proteins, lipids, blood, water, bone, and melanin. Based on the spectral information, Raman images enabled the localization of cortical and subcortical tumor cell aggregates with accuracy of roughly 250 μm. This study demonstrates the prospects of Raman spectroscopy as an intravital tool to detect cerebral pathologies and opens the field for biophotonic imaging of the living brain. Future investigations aim to reduce the exposure time from minutes to seconds and improve the lateral resolution.     

Spectroscopic characterization of bionanoparticles originating from newly developed self-forming synthetic PEGylated lipids (QuSomes)

In this work, we have aimed to merge the advantages of nanotechnology and biophotonics in conjunction with vibrational spectroscopic techniques in order to understand the various aspects of new kinds of synthetic bionanoparticles originating from self-forming synthetic biopolymers known as polyethylene glycol (PEG)ylated lipids. In particular, two complementary molecular spectroscopic techniques based on thin-layered Fourier transform infrared and confocal laser tweezers. Raman spectroscopy has been employed for the investigations of newly developed artificial PEGylated lipids trademarked as QuSomes. These novel types of synthetic lipids are composed of 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12), 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol (GDO-12), and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23). The lipid labeled GDM-12 has saturated 14 acyl chains whereas GDO-12 is characterized by monounsaturated 18 acyl chains, and GDS-23 is composed of saturated 18 acyl chains in their hydrophobic chain. Similarly, GDM-12 and GDO-12 contain 12 units, and GDS-23 contains 23 units of hydrophilic PEG head groups. In contrast to conventional phospholipids, this novel kind of lipid can form liposomes spontaneously upon hydration, without the input of external activation energy. In addition, fluorescence correlation spectroscopy has been utilized to measure the size distribution of such nanoparticles in suspension as well as scanning electron microscopy has been applied for the imaging purposes. Although such PEGylated lipids show a common spectral pattern, important differences in the spectra have been observed, enabling us to distinguish these different lipids on the basis of characteristic features calculated from the spectroscopic band component analysis. Finally, in this study, detailed spectroscopic results due to the vibrational band assignments and band component analysis corresponding to various functional groups for individual nanoparticles have been analyzed and discussed.     

Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging.     

Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.     

Spectroscopic studies of polymerized surfactants: 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine

Spectroscopic studies have been performed on aqueous dispersions of the surfactant 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine before and after polymerization with ul-traviolet light. Monomers of this lipid can, under certain conditions, convert from the expected spherical liposomal form to a unique phase consisting of hollow tubules. To determine the molecular conformation of these structures we have used Raman and infrared spectroscopies to probe the structure of the hydrocarbon chains and head groups of the lipids, and used absorption spectroscopy and resonance enhanced Raman scattering of the colored polymer to monitor the length and structure of the diacetylenic polymer backbone. Unusual C? H stretch-ing Raman bands imply that the hydrocarbon chain packing in the monomeric bilayers is different from that observed in other phosphatidylcholines, and that a distrubance in alkyl chain packing occurs on polymerization. Depending on irradiation conditions and the dispersal state of the lipid the polymer chains may be of at least three different colors, from which distinct resonance Raman spectra are obtained. The effective bond conjugation lengths range from quite short in the yellow polymer produced in sonicated vesicles to extremely long in a blue component seen in polymerized tubules.     

New look inside human breast ducts with Raman imaging. Raman candidates as diagnostic markers for breast cancer prognosis: Mammaglobin,palmitic acid and sphingomyelin

Looking inside the human body fascinated mankind for thousands of years. Current diagnostic and therapy methods are often limited by inadequate sensitivity, specificity and spatial resolution. Raman imaging may bring revolution in monitoring of disease and treatment. The main advantage of Raman imaging is that it gives spatial information about various chemical constituents in defined cellular organelles in contrast to conventional methods (liquid chromatography/mass spectrometry, NMR, HPLC) that rely on bulk or fractionated analyses of extracted components. We demonstrated how Raman imaging can drive the progress on breast cancer just unimaginable a few years ago. We looked inside human breast ducts answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the stroma around the duct during cancer development. We have identified Raman candidates as diagnostic markers for breast cancer prognosis: carotenoids, mammaglobin, palmitic acid and sphingomyelin as key molecular targets in ductal breast cancer in situ, and propose the molecular mechanisms linking oncogenes with lipid programming.     

Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three‐Channel Imaging Correlation

Enzyme activity in live cells is dynamically regulated by small‐molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long‐standing challenge. Herein, we report a three‐channel imaging correlation by a single dual‐reactive fluorescent probe to measure the dependence of phosphatase activity on the H₂S level in cells. The two sites of the probe reactive to H₂S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three‐channel imaging correlation shows that cells have an ideal level of H₂S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H₂S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H₂S in cellular signaling and in various human diseases.     

Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy

Click-free imaging of the nuclear localization of an alkyne-tagged cell proliferation probe, EdU, in living cells was achieved for the first time by means of Raman microscopy. The alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous molecules. This approach may eliminate the need for click reactions in the detection of alkyne-labeled molecules.     

Near infrared Raman spectra of human brain lipids

Human brain tissue, in particular white matter, contains high lipid content. These brain lipids can be divided into three principal classes: neutral lipids including the steroid cholesterol, phospholipids and sphingolipids. Major lipids in normal human brain tissue are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, sphingomyelin, galactocerebrosides, gangliosides, sulfatides and cholesterol. Minor lipids are cholesterolester and triacylglycerides. During transformation from normal brain tissue to tumors, composition and concentration of lipids change in a specific way. Therefore, analysis of lipids might be used as a diagnostic parameter to distinguish normal tissue from tumors and to determine the tumor type and tumor grade. Raman spectroscopy has been suggested as an analytical tool to detect these changes even under intra-operative conditions. We recorded Raman spectra of the 12 major and minor brain lipids with 785 nm excitation in order to identify their spectral fingerprints for qualitative and quantitative analyses.     

Analysis of human tear fluid by Raman spectroscopy

Tear fluid is a complex aqueous solution containing proteins, metabolites, electrolytes and lipids. This study uses Raman spectroscopy to analyse the composition of human tear fluid from three healthy volunteers. Two different methods are used to obtain Raman spectra from the 3 μL tear samples: (i) solution-phase Raman spectroscopy, and (ii) drop coating deposition Raman spectroscopy (DCDRS). Tear samples were either basal fluid, or yawn reflex secreted fluid. Calibration of the solution technique with standard protein solutions (5-15 mg mL⁻¹) showed that this method could predict the protein concentration (cross-validation) with an error of less than 1 mg mL⁻¹. The Raman signals from the tear fluid were very weak but signals due to protein and urea were clearly observable in all samples. The drop coating deposition technique was shown to produce very high signal-to-noise spectra for relatively short acquisition times, and small sample volumes. Raman point mapping combined with principal components analysis showed that the protein, urea, bicarbonate and lipid could all be detected in the tear samples and that the distribution of these components was inhomogeneous. Their position within the drying pattern was shown to depend on their relative solubilities. The results of this study suggest that solution Raman measurements may be calibrated to give the total tear protein concentration and DCDRS could be used to give a fingerprint of the tear protein (and lipid) composition.     

Characterization of lipid extracts from brain tissue and tumors using Raman spectroscopy and mass spectrometry

Brain tissue is characterized by high lipid content. The amount of lipids decreases, and its composition changes in the most frequent primary brain tumor, the glioma. Scope of the current paper was to extract quantitatively lipids from porcine and human brain tissue as well as from five human gliomas using a modified protocol according to Folch. The lipid extracts were studied by Raman spectroscopy with 785 nm excitation and by mass spectrometry with electron impact ionization. Porcine and human brain tissues have similar water and lipid content and show similar Raman and mass spectra. In contrast, gliomas are characterized by increased water content and decreased lipid content. Elevated phosphatidylcholine to cholesterol ratios in lipid extracts of gliomas were indicated by Raman bands of the choline group and cholesterol. Due to its higher sensitivity, mass spectrometry detected increased levels of cholesterol ester relative to cholesterol in lipid extracts of gliomas. For comparison, thin tissue sections were prepared from the glioma specimens before lipid extraction; infrared spectroscopic images were recorded and analyzed by a supervised classification model. This study demonstrates how to improve the analysis of brain tumors and to complement the diagnosis of brain pathologies using a multimodal approach.     

Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy

Macrophages are phagocytic cells which are involved in the non-specific immune defense. Lipid uptake and storage behavior of macrophages also play a key role in the development of atherosclerotic lesions within walls of blood vessels. The allocation of exogenous lipids such as fatty acids in the blood stream dictates the accumulation and quantity of lipids within macrophages. In case of an overexposure, macrophages transform into foam cells because of the large amount of lipid droplets in the cytoplasm. Raman micro-spectroscopy is a powerful tool for studying single cells due to the combination of microscopic imaging with spectral information. With a spatial resolution restricted by the diffraction limit, it is possible to visualize lipid droplets within macrophages. With stable isotopic labeling of fatty acids with deuterium, the uptake and storage of exogenously provided fatty acids can be investigated. In this study, we present the results of time-dependent Raman spectroscopic imaging of single THP-1 macrophages incubated with deuterated arachidonic acid. The polyunsaturated fatty acid plays an important role in the cellular signaling pathway as being the precursor of icosanoids. We show that arachidonic acid is stored in lipid droplets but foam cell formation is less pronounced as with other fatty acids. The storage efficiency in lipid droplets is lower than in cells incubated with deuterated palmitic acid. We validate our results with gas chromatography and gain information on the relative content of arachidonic acid and its metabolites in treated macrophages. These analyses also provide evidence that significant amounts of the intracellular arachidonic acid is elongated to adrenic acid but is not metabolized any further. The co-supplementation of deuterated arachidonic acid and deuterated palmitic acid leads to a non-homogenous storage pattern in lipid droplets within single cells. Figure a

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Raman microspectroscopy of live cells under autophagy-inducing conditions

The role of autophagy in numerous physiological responses triggered by a variety of mechanisms both in states of health and disease has raised considerable interest in this cellular process. However, the current analytical tools to study autophagy are either invasive or require genetic manipulation. Raman microspectroscopy is a potentially quantitative analytical method that has been shown to be useful for the label-free, non-destructive analysis of living biological cells and tissues. We present in this study initial efforts to study autophagy using Raman spectroscopy. The response of adherent mouse and human cancer cells to starvation conditions (glutamine deprivation and amino acid deprivation) was probed by Raman spectroscopy and compared to fluorescence microscopy results using autophagy-specific markers. We also demonstrate the capability of Raman spectroscopy to monitor the recovery dynamics of starved cells and to probe the heterogeneity in the response to starvation that can arise in cell populations. Finally, this work suggests that the 718 cm(-1) Raman line associated with phospholipids may be a useful spectral marker indicative of an autophagic response to starvation stimuli. Overall, this study establishes the utility of Raman spectroscopy to non-invasively detect biologically relevant changes in live cells exposed to conditions known to trigger autophagy.     

Enhanced transfection efficiency of multicomponent lipoplexes in the regime of optimal membrane charge density

Recently, membrane charge density of lipid membranes, sigma M, has been recognized as a universal parameter that controls the transfection efficiency of complexes made of binary cationic liposomes and DNA (binary lipoplexes). Three distinct regimes, most likely related to interactions between complexes and cells, have also been identified. The purpose of this work was to investigate the transfection efficiency behavior of multicomponent lipoplexes in the regime of optimal membrane charge density (1< sigma M < 2 x 10 (-2) e/A (2)) and compare their performance with that of binary lipoplexes usually employed for gene delivery purposes. We found remarkable differences in transfection efficiency due to lipid composition, with maximum in efficiency being obtained when multicomponent lipoplexes were used to transfect NIH 3T3 cells, while binary lipoplexes were definitely less efficient. These findings suggested that multicomponent systems are especially promising lipoplex candidates. With the aim of providing new insights into the mechanism of transfection, we investigated the structural evolution of lipoplexes when interacting with anionic (cellular) lipids by means of synchrotron small-angle X-ray diffraction (SAXD), while the extent of DNA release upon interaction with anionic lipids was measured by electrophoresis on agarose gels. Interestingly, a clear trend was found that the transfection activity increased with the number of lipid components. These results highlight the compositional properties of carrier lipid/cellular lipid mixtures as decisive factors for transfection and suggest a strategy for the rational design of superior cationic lipid carriers.     

Effect of coenzyme Q(10) incorporation on the characteristics of nanoliposomes

Coenzyme Q(10) (CoQ(10)) is incorporated in nanoliposomes composed of egg yolk phospholipid, cholesterol, and Tween 80. Atomic force microscopy, performed to characterize vesicle surface topology, shows some visible influence of CoQ(10) on the nanoliposomal structure. CoQ(10) incorporation can suppress the increase of the z-average diameter of nanoliposomes during storage for 8 months at 4 degrees C. The liposomal lipid peroxidation caused by Fe(III)/ascorbate is also significantly inhibited. Perturbation of acyl chain motion of lipids due to the presence of CoQ(10) in the bilayer is examined by fluorescence probe diphenyl-hexatriene and Raman spectroscopy. Fluorescence probe studies indicate that CoQ(10) incorporation results in the microviscosity increase of nanoliposomes. The steric structure of nanoliposomes reflected by Raman spectroscopy changes obviously and shows CoQ(10) content dependency. The order parameters for the lateral interaction between chains increase. The trans conformation decrease and the gauche conformation increase as the weight contents of CoQ(10) incorporation are at 1%, 5%, 10%, and 32.5%. However, the order parameters for the longitudinal interaction in chains was higher than that of pure nanoliposomes as the weight content of CoQ(10) is at 25%. Results suggest that CoQ(10)might intercalate between lipid molecules and perturb the bilayer structure.