Henk-Jan van Manen

Noninvasive Imaging of Protein Metabolic Labeling in Single Human Cells Using Stable Isotopes and Raman Microscopy

We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 cm(-1) spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.

Intracellular degradation of microspheres based on cross-linked dextran hydrogels or amphiphilic block copolymers: A comparative Raman microscopy study

AIMS Clusters of nanoparticles may significantly improve the sensitivity of diagnostics and the safety and efficacy of therapeutic nanotechnologies in medicine. We report methods for the formation of nanoparticle clusters and for monitoring their accumulation in cancer cells. METHODS The accumulation of gold nanoparticles in tumor cells was studied using flow cytometry, optical scattering and fluorescent, atomic force, photothermal and scanning electron microscopy. RESULTS Incubation of cells at 37 degrees C for 30 min or more with 10-30-nm nanoparticles resulted in the formation of clusters of nanoparticles as large as 20 nanoparticles or more. CONCLUSIONS Specific targeting using a monoclonal antibody as a vector increases the concentration of nanoparticles on the surface of target cells compared with nonspecific nanoparticle accumulation. In turn, an increased concentration of nanoparticles on the target surface yields larger nanoparticle clusters inside the cells due to endocytosis. Photothermal and scattering microscopy were found to be the most sensitive methods for imaging nanoparticle clusters in living cells.Micro- and nanospheres composed of biodegradable polymers show promise as versatile devices for the controlled delivery of biopharmaceuticals. Whereas important properties such as drug release profiles, biocompatibility, and (bio)degradability have been determined for many types of biodegradable particles, information about particle degradation inside phagocytic cells is usually lacking. Here, we report the use of confocal Raman microscopy to obtain chemical information about cross-linked dextran hydrogel microspheres and amphiphilic poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) microspheres inside RAW 264.7 macrophage phagosomes. Using quantitative Raman microspectroscopy, we show that the dextran concentration inside phagocytosed dextran microspheres decreases with cell incubation time. In contrast to dextran microspheres, we did not observe PEGT/PBT microsphere degradation after 1 week of internalization by macrophages, confirming previous studies showing that dextran microsphere degradation proceeds faster than PEGT/PBT degradation. Raman microscopy further showed the conversion of macrophages to lipid-laden foam cells upon prolonged incubation with both types of microspheres, suggesting that a cellular inflammatory response is induced by these biomaterials in cell culture. Our results exemplify the power of Raman microscopy to characterize microsphere degradation in cells and offer exciting prospects for this technique as a noninvasive, label-free optical tool in biomaterials histology and tissue engineering.

Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation.

Surface‐Confined Metallodendrimers: Isolated Nanosize Molecules

The incorporation of dialkyl sulfide side chains in metallodendrimers is a simple method for their insertion into a monolayer of decanethiol formed by self-assembly on a gold surface. The dendrimer binds through the sulfide group to a defect in the monolayer on the gold surface (see picture). The surface concentration of the isolated dendrimer adsorbate can be regulated by the adsorption time (for example, 55 adsorbates on a surface of 200x200 nm(2) after 20 h).

Coordination Chemistry of SCS PdII Pincer Systems

We have studied the coordination of substituted pyridines, and phosphorus- and sulfur-containing ligands to an SCS PdII pincer system. These ligands coordinate to PdII (trans to the cyclopalladated aryl group) by quantitative substitution of the labile acetonitrile ligand in complex 1. Competition experiments showed that both electronic and steric effects influence the strength of coordination to the PdII pincer of the substituted pyridines. A quantitative analysis of the substituent effect was achieved by a Hammett correlation. Phosphorus-containing ligands also coordinate to this SCS PdII motif, as evidenced by NMR spectroscopy and single-crystal X-ray diffraction studies. They are much stronger ligands than the pyridines. The coordination strength of the thioureas falls in between those of the pyridines and phosphanes/phosphites. Our results lead therefore to the following order of ligand strength towards PdII in SCS PdII pincers: PR3 > P(OR)3 > N,N-disubstituted thiourea > (substituted) pyridines > MeCN.

Microbioreactors for Raman Microscopy of Stromal Cell Differentiation

We present the development of microbioreactors with a sensitive and accurate optical coupling to a confocal Raman microspectrometer. We show that such devices enable in situ and in vitro investigation of cell cultures for tissue engineering by chemically sensitive Raman spectroscopic imaging techniques. The optical resolution of the Raman microspectrometer allows recognition and chemical analysis of subcellular features. Human bone marrow stromal cells (hBMSCs) have been followed after seeding through a phase of early proliferation until typically 21 days later, well after the cells have differentiated to osteoblasts. Long-term perfusion of cells in the dynamic culture conditions was shown to be compatible with experimental optical demands and off-line optical analysis. We show that Raman optical analysis of cells and cellular differentiation in microbioreactors is feasible down to the level of subcellular organelles during development. We conclude that microbioreactors combined with Raman microspectroscopy are a valuable tool to study hBMSC proliferation, differentiation, and development into tissues under in situ and in vitro conditions.

Noncovalent synthesis of water-soluble SCS PdII pincer assemblies

This article describes the noncovalent synthesis of water-soluble coordination assemblies based on SCS PdII pincer moieties. Two neutral solubilizing moieties, one based on a linear carbohydrate chain and the other on tetraethylene glycol residues, have been functionalized with pyridine and phosphane ligands. The coordination of the resulting molecules to various hydrophobic, cationic mono- and multimeric SCS PdII pincer systems has been investigated by 1H and 31P NMR spectroscopy, and by MALDI-TOF mass spectrometry. In general, the assemblies with tetraethylene glycol chains display a higher solubility in water than the ones containing linear sugar moieties. A hexapincer core decorated with six linear carbohydrates forms a hydrogel. Finally, a noncovalent water-soluble metallodendrimer having 18 peripheral tetraethylene glycol groups was constructed in a convergent manner.

Data size reduction strategy for the classification of breath and air samples using multicapillary column-ion mobility spectrometry.

Ion mobility spectrometry combined with multicapillary column separation (MCC-IMS) is a well-known technology for detecting volatile organic compounds (VOCs) in gaseous samples. Due to their large data size, processing of MCC-IMS spectra is still the main bottleneck of data analysis, and there is an increasing need for data analysis strategies in which the size of MCC-IMS data is reduced to enable further analysis. In our study, the first untargeted chemometric strategy is developed and employed in the analysis of MCC-IMS spectra from 264 breath and ambient air samples. This strategy does not comprise identification of compounds as a primary step but includes several preprocessing steps and a discriminant analysis. Data size is significantly reduced in three steps. Wavelet transform, mask construction, and sparse-partial least squares-discriminant analysis (s-PLS-DA) allow data size reduction with down to 50 variables relevant to the goal of analysis. The influence and compatibility of the data reduction tools are studied by applying different settings of the developed strategy. Loss of information after preprocessing is evaluated, e.g., by comparing the performance of classification models for different classes of samples. Finally, the interpretability of the classification models is evaluated, and regions of spectra that are related to the identification of potential analytical biomarkers are successfully determined. This work will greatly enable the standardization of analytical procedures across different instrumentation types promoting the adoption of MCC-IMS technology in a wide range of diverse application fields.

Trägerfixierte Metallodendrimere: isolierte Moleküle im Nanomaßstab

Die Einfuhrung von Dialkylsulfidseitenketten in Metallodendrimere ist eine einfache Strategie fur deren Einbau in eine Decanthiolmonoschicht, die zuvor durch Selbstorganisation auf einer Goldoberflache gebildet wurde. Uber den Sulfidanker kann das Dendrimer in Defektstellen der Monoschicht an das Goldsubstrat binden (siehe Bild). Die Oberflachenkonzentration der isolierten Dendrimeradsorbate kann uber die Adsorptionszeit gesteuert werden (z.B. 55 Adsorbate auf einer Flache von 200×200 nm2 nach 20 h.

Raman microscopy of phagocytosis: shedding light on macrophage foam cell formation

The phagocyte NADPH oxidase is a crucial enzyme in the innate immune response of leukocytes against invading microorganisms. The superoxide (O2-) that is generated by this enzyme upon infection is directly and indirectly used in bacterial killing. The catalytic subunit of NADPH oxidase, the membrane-bound protein heterodimer flavocytochrome b558, contains two heme moieties. Here, we first briefly discuss our recent confocal resonant Raman (RR) spectroscopy and microscopy experiments on flavocytochrome b558 in both resting and phagocytosing neutrophilic granulocytes. Such experiments allow the determination of the redox state of flavocytochrome b558 inside the cell, which directly reflects the electron transporting activity of NADPH oxidase. Subsequently, we report that incubation of murine RAW 264.7 macrophages with PolyActive microspheres for 1 week in culture medium leads to morphological and biochemical changes in the macrophages that are characteristic for the generation of macrophage-derived foam cells. Lipid-laden foam cells are the hallmark of early atherosclerotic lesions. Using nonresonant Raman spectroscopy and microscopy, we demonstrate that the numerous intracellular droplets in macrophages exposed to microspheres are rich in cholesteryl esters. The finding that phagocytic processes may trigger foam cell formation reinforces the current belief that (chronic) infection and inflammation are linked to the initiation and progression of atherosclerotic lesions. The study of such a connection may reveal new therapeutic targets for atherosclerosis treatment or prevention.