Quantification of local water and biomass in wild type PA01 biofilms by Confocal Raman Microspectroscopy

Confocal Raman Microspectroscopy (CRM) can be used as a tool for the in situ evaluation of the chemical composition of living, fully submerged, unstained biofilms. In this study the estimation of the local water content in Pseudomonas aeruginosa PA01 biofilms is given as an example. The ratio of the area of the O-H stretching vibration band at 3450 cm(-1), (water), to that of the C-H stretching bands at 2950 cm(-1) (biomass), was used to estimate the relative biofilm water content. The quantification of biofilm water and biomass was based on calibration curves generated from protein solutions. Water/biomass ratios (W:BR) equivalent to that of a 30% (w/v) protein solution were observed within some biofilm colonies.     

Effects of water gradients and use of urea on skin ultrastructure evaluated by confocal Raman microspectroscopy

The rather thin outermost layer of the mammalian skin, stratum corneum (SC), is a complex biomembrane which separates the water rich inside of the body from the dry outside. The skin surface can be exposed to rather extreme variations in ambient conditions (e.g. water activity, temperature and pH), with potential effects on the barrier function. Increased understanding of how the barrier is affected by such changes is highly relevant for regulation of transdermal uptake of exogenous chemicals. In the present study we investigate the effect of hydration and the use of a well-known humectant, urea, on skin barrier ultrastructure by means of confocal Raman microspectroscopy. We also perform dynamic vapor sorption (DVS) microbalance measurements to examine the water uptake capacity of SC pretreated with urea. Based on novel Raman images, constructed from 2D spectral maps, we can distinguish large water inclusions within the skin membrane exceeding the size of fully hydrated corneocytes. We show that these inclusions contain water with spectral properties similar to that of bulk water. The results furthermore show that the ambient water activity has an important impact on the formation of these water inclusions as well as on the hydration profile across the membrane. Urea significantly increases the water uptake when present in skin, as compared to skin without urea, and it promotes formation of larger water inclusions in the tissue. The results confirm that urea can be used as a humectant to increase skin hydration.     

Follow-up of drug permeation through excised human skin with confocal Raman microspectroscopy

Skin is a multilayered organ which covers and protects the surface of human body by providing a barrier function against exogenous agents. Meanwhile, the efficacy of several topically applicated drugs is directly related to their penetration through the skin barrier. Several techniques are commonly used to evaluate the rate, the speed and the depth of penetration of these drugs, but few of them can provide real-time results. Therefore, the use of nondestructive and structurally informative techniques permits a real breakthrough in the investigations on skin penetration at a microscopic scale. Confocal Raman microspectroscopy is a nondestructive and rapid technique which allows information to be obtained from deep layers under the skin surface, giving the possibility of a real-time tracking of the drug in the skin layers. The specific Raman signature of the drug enables its identification in the skin. In this study, we try to follow the penetration of Metronidazole, a drug produced by Galderma as a therapeutic agent for Rosacea treatment, through the skin. The first step was the spectral characterization of Metronidazole in the skin. Then micro-axial profiles were conducted to follow the penetration of the drug in the superficial layers, on excised human skin specimens. For more accurate information, transverse sections were cut from the skin and spectral images were conducted, giving information down to several millimeters deep. Moreover, the collected spectra permit us to follow the structural modifications, induced by the Metronidazole on the skin, by studying the changes in the spectral signature of the skin constituents. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Quantification of extracellular polymeric substances in biofilms by confocal laser scanning microscopy

Extracellular polymeric substances (EPS) in a biofilm were quantified by measuring the total cell volume from a 3-D image of the biofilm using confocal laser scanning microscope after staining cells with a fluorescent dye specific for nucleic acids. The EPS content was the difference between the volatile solids in the biofilm and the total cell mass, which could be quantified from the measured cell volume.     

Hydrohalite spatial distribution in frozen cell cultures measured using confocal Raman microscopy

Hydrohalite, a crystalline rock salt hydrate, (NaCl·2H₂O), can form in cryopreservation samples under certain circumstances changing the local chemical environment of the preserved cells. Evidence of this crystalline phase was recently found by microspectroscopy measurements, and believed to form exclusively extracellular. We have studied the spatial distribution of hydrohalite in frozen mouse fibroblast cell samples by means of confocal Raman scanning microscopy (CRM). Hydrohalite has a unique Raman spectrum with several bands in the high frequency tail of the OH-stretching band which can be used for unambiguous identification. Hydrohalite can only form through eutectic crystallization in saline solutions without any cryoprotective agents and the spatial distribution thus gives a more detailed view on this crystallization process. This is important since eutectic crystallization has been empirically correlated to cell death, but the exact injury mechanism is unclear. By the means of colocalization of Raman bands we show that hydrohalite can indeed form intracellularly and is not a strictly extracellular phenomenon. We furthermore found that intracellular ice and intracellular hydrohalite very often coincide. Finally we show that the addition of 0.5 wt.% dimethyl sulfoxide (Me₂SO) inhibits formation of hydrohalite. This study shows how Raman microscopy and successive analysis can be employed non-invasively within cryobiology to give additional chemical and structural information compared to conventional imaging techniques.     

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.     

Direct detection of analyte binding to single molecularly imprinted polymer particles by confocal Raman spectroscopy

We describe the use of Raman spectroscopy to detect and quantify, for the first time, the presence of the imprinting template in single molecularly imprinted polymer microspheres. The polymers were imprinted with the β-blocking drugs propranolol and atenolol, and precipitation polymerization was used to obtain spherical particles of diameters of 200 nm and 1.5 μm. The size of the Raman laser spot being between 1 μm and a few μm, the nanoparticles were used for bulk detection whereas with micrometer-sized particles, quantitative measurements on single particles were possible. The laser power, and consequently the acquisition times, needed to be adapted as a function of the polymer and template used in order to avoid burning. Analyte quantification from Raman spectra is straightforward by determining the peak height of a typical Raman band of the analyte, and by using a typical polymer peak for normalization. Relatively low detection limits down to 1 μM have been reached for the detection of S-propranolol through bulk measurements on MIP nanoparticles.     

Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ

AIMS: To investigate the use of confocal Raman microspectroscopy (CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. METHODS AND RESULTS: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium (artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances (EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in-plane and -depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology - characteristic of the seeding dispersal process - were also observed. CONCLUSIONS: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.     

Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺, that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal “in Escherichia coli (E. coli) colony” screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca²⁺ indicator after Ca²⁺ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca²⁺ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP₃. Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.     

Intracellular dynamics of topoisomerase I inhibitor,CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The use of laser scanning confocal microscopy to characterize mycorrhizas of Pinus strobus L. and to localize associated bacteria

 Laser scanning confocal microscopy (LSCM), light microscopy (LM) and epifluorescence microscopy (FM) were used to observe the extramatrical hyphae, mantle patterns and associated bacteria on mycorrhizal tips of Pinus strobus L. seedlings grown in pot cultures. Laccaria sp. and Tuber sp. formed ectomycorrhizas with Pinus strobus, while Phialophora finlandia Wang & Wilcox and E-strain (sensu Danielson 1982) formed ectendomycorrhizas. Distinct mantle patterns and cystidia were observed with greater resolution using LSCM, and intracellular hyphae were visualized in three dimensions. Trypan blue penetrated fresh whole mounts to 20 μm and was an excellent stain for visualizing fungal hyphae and bacteria with LSCM. Fluorescein isothiocyanate and acridine orange were used in conjunction with LSCM and FM to localize bacteria on ectomycorrhizal tips. With LSCM, bacteria were visible in the surface mucigel, and optical sectioning through the root tip showed that bacteria were also present within the mantle. LSCM is a non-intrusive and fast method for visualizing mycorrhizal structures and their associated bacteria on fresh, whole root tips. Accepted: 17 July 1996     

Homogenization of Pseudomonas aeruginosa PAO1 biofilms visualized by freeze‐substitution electron microscopy

A knowledge of the mechanical properties of bacterial biofilms is required to more fully understand the processes of biofilm formation such as initial adhesion or detachment. The main contribution of this article is to demonstrate the use of homogenization techniques to compute mechanical parameters of Pseudomonas aeruginosa PAO1 biofilms. For this purpose, homogenization techniques are used to analyze freeze substitution electron micrographs of the biofilm cross‐sections. The concept of a representative volume element and the study about his representativeness allows us to determine the optimal size in order to analyze these biofilm images. Results demonstrate significant heterogeneities with respect to stiffness and these can be explained by varying cell density distribution throughout the bacterial biofilms. These stiffness variations lead to different mechanical properties along the height of the biofilm. Moreover, a numerical shear stress test shows the impact of these heterogeneities on the detachment process. Several modes of detachment are highlighted according to the local strain energy in the different parts of the biofilm. Knowing where, and how, a biofilm may detach will allow better prediction of accumulation and biomass detachment. Biotechnol. Bioeng. 2013; 110: 1405–1418. © 2012 Wiley Periodicals, Inc.     

Non‐invasive identification of proteoglycans and chondrocyte differentiation state by Raman microspectroscopy

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG‐associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser‐based, marker‐free and non‐destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non‐invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non‐invasive cell and tissue state monitoring of cartilage in biomedical research. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The molecular structure and physical properties of elastin fibers as revealed by Raman microspectroscopy

Raman microspectroscopy has been used to investigate the structure of alpha-elastin and fibrous elastin from ligament and aorta, and to explore changes associated with mechanical strain and temperature. Although no vibrational modes associated with cross-linking of the fibers could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from alpha-elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% beta-structures, 46% unordered, and 18% alpha-helix. alpha-Elastin contained 29% beta-structures, 53% unordered, and 18% alpha-helix. In nuchal fibers the amide I mode was polarized, consistent with the peptide bond. Strains of up to 60% in ligament fiber bundles resulted in no significant shifts in peak position or in secondary structure. Polarization measurements revealed that the peptide bonds and several side chains re-orientated closer to the fiber axis. Heating nuchal fibers to 60 degrees C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of beta-structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 931-940, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Characterization and application of Raman labels for confocal Raman microspectroscopic detection of cellular proteins in single cells

A method using confocal Raman microspectroscopy for the detection of cellular proteins in single intact cells was developed. Two approaches were used to improve the detection of these cellular components. First, compounds with high Raman scattering were investigated for potential use as Raman labels. Raman labels were conjugated to either biomolecules or biotin and used as markers in the detection of cellular enzymes and receptors. Second, silver colloids were used to increase the surface-enhanced Raman scatter (SERS) of these Raman labels. Cresyl violet and dimethylaminoazobenzene are Raman labels that provide very sensitive SERS detection by a confocal Raman microscope with a HeNe laser at wavelength of 632.8 nm. The detection of 12-lipoxygenase and cyclooxygenase-1 in single bovine coronary artery endothelial cells and the binding of angiotensin II to its receptors in zona glomerulosa cells was demonstrated.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Insights into the chemical composition of <Emphasis Type="Italic">Equisetum hyemale</Emphasis> by high resolution Raman imaging

Equisetaceae has been of research interest for decades, as it is one of the oldest living plant families, and also due to its high accumulation of silica up to 25% dry wt. Aspects of silica deposition, its association with other biomolecules, as well as the chemical composition of the outer strengthening tissue still remain unclear. These questions were addressed by using high resolution (<1 μm) Confocal Raman microscopy. Two-dimensional spectral maps were acquired on cross sections of Equisetum hyemale and Raman images calculated by integrating over the intensity of characteristic spectral regions. This enabled direct visualization of differences in chemical composition and extraction of average spectra from defined regions for detailed analyses, including principal component analysis (PCA) and basis analysis (partial least square fit based on model spectra). Accumulation of silica was imaged in the knobs and in a thin layer below the cuticula. In the spectrum extracted from the knob region as main contributions, a broad band below 500 cm⁻¹ attributed to amorphous silica, and a band at 976 cm⁻¹ assigned to silanol groups, were found. From this, we concluded that these protrusions were almost pure amorphous, hydrated silica. No silanol group vibration was detected in the silicified epidermal layer below and association with pectin and hemicelluloses indicated. Pectin and hemicelluloses (glucomannan) were found in high levels in the epidermal layer and in a clearly distinguished outer part of the hypodermal sterome fibers. The inner part of the two-layered cells revealed as almost pure cellulose, oriented parallel along the fiber.     

Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy

Raman spectroscopy has strong potential for providing noninvasive dermatological diagnosis of skin cancer. In this study, confocal Raman microscopy was applied to the dermatological diagnosis for one of the most common skin cancers, basal cell carcinoma (BCC). BCC tissues were obtained from 10 BCC patients using a routine biopsy and used for confocal Raman measurements. Autofluorescence signals from tissues, which interfere with the Raman signals, were greatly reduced using a confocal slit adjustment. Distinct Raman band differences between normal and BCC tissues for the amide I mode and the PO2- symmetric stretching mode showed that this technique has strong potential for use as a dermatological diagnostic tool without the need for statistical treatment of spectral data. It was also possible to precisely differentiate BCC tissue from surrounding noncancerous tissue using the confocal Raman depth profiling technique. We propose that confocal Raman microscopy provides a novel method for dermatological diagnosis since direct observations of spectral differences between normal and BCC tissues are possible.