A new approach to perform patch-clamp experiments on living cells under controlled anoxic and normoxic conditions was developed and tested. To provide an optimal control over the oxygen content and the biochemical environment a patch-clamp recording micropipette was integrated within an oxygen tight poly-methyl methacrylate (PMMA) based microchip. The oxygen content within the microfluidic chamber surrounding patch-clamp micropipette was maintained at 0.5-1.5 % by a continuous flow of artificial extracellular solution purged with nitrogen. The nerve and glial cells acutely obtained from the male rat brain were trapped by the optical tweezers and steered towards the patch-clamp micropipette through the channels of the microchip in order to achieve a close contact between the pipette and the cellular membrane. The patch-clamp recordings revealed that optical tweezers did not affect the electrophysiological properties of the tested cells suggesting that optical trapping is a safe and non-traumatizing method to manipulate living cells in the microfluidic system. Thus, our approach of combining optical tweezers and a gas-tight microfluidic chamber may be applied in various electrophysiological investigations of single cells were optimal control of the experimental conditions and the sample in a closed environment are necessary.

We present a new approach of combining Lab-on-a-chip technologies with optical manipulation technique for accurate investigations in the field of cell biology. A general concept was to develop and combine different methods to perform advanced electrophysiological investigations of an individual living cell under optimal control of the surrounding environment. The conventional patch clamp technique was customized by modifying the open system with a gas-tight multifunctional microfluidics system and optical trapping technique (optical tweezers).The system offers possibilities to measure the electrical signaling and activity of the neuron under optimum conditions of hypoxia and anoxia while the oxygenation state is controlled optically by means of a spectroscopic technique. A cellbased microfluidics system with an integrated patch clamp pipette was developed successfully. Selectively, an individual neuron is manipulated within the microchannels of the microfluidic system under a sufficient control of the environment. Experiments were performed to manipulate single yeast cell and red blood cell (RBC) optically through the microfluidics system toward an integrated patch clamp pipette. An absorption spectrum of a single RCB was recorded which showed that laser light did not impinge on the spectroscopic spectrum of light. This is promising for further development of a complete lab-on-a-chip system for patch clamp measurements.

Introduktion: Syftet med studien är att studera enstaka nervcellers respons vid syrebrist i ett mikroflödessystem för att förstå nervcellens respons vid stroke. Målet med studien var att utveckla ett slutet mikroflödessystem som ger optimal kontroll av den omgivande miljön och samtidigt möjliggöra elektrofysiologiska undersökningar under kontrollerade syreförhållande. Material och metoder: Mikroflödescellen utvecklades för ett inverterat mikroskop, utrustad med en optisk pincett och optisk spektroskopi samt patch-clamp för elektrofysiologiska studier på en enstaka nervcell. Istället för att föra en pipett mot en cell i ett öppet system fångades en enskild cell optiskt i ett slutet mikroflödessystem och fördes mot en fixerad patch-clamp mikropipett. Cellen utsattes för olika syrehalter och övervakades av ett UV-Vis spektroskop medan cellens elektrofysiologiska aktivitet registreras med patch-clamp. Det slutna mikroflödessystemet med integrerad mikropipett, kopplades till ett pumpsystem för införandet av celler och buffert med olika kemiska egenskaper och syrehalter. I ett inverterat mikroskop integrerades optisk pincett, UV-Vis spektrometer och patch-clamp. Resultat och diskussion: För att pröva konceptet fångades och fördes en röd blodcell optiskt mot mikropipetten som befann sig på en fast position i mikroflödescellen. Cellens syrebindningstillstånd varierades genom att tillsätta syrefri eller syresatt buffert och registrerades med UV-Vis spektrometern. I ett vidare experiment manipulerades en nervcell optiskt i ett öppet system mot patch-clamp pipetten och elektrofysiologiska mätningar utfördes. Vi kunde verifiera att den optiska pincetten inte påverkade den elektrofysiologiska mätningen. För närvarandet utförs elektrofysiologiska mätningar i det slutna mikroflödessystemet för att se hur nervcellerna reagerar under varierande syrehalt. Genom mätningarna hoppas vi att få mer kunskap om försvarsmekanismerna som igångsätts av neuroner under syrefattiga förhållanden.

The response and the reaction of the brain system to hypoxia is a vital research subject that requires special instrumentation. With this research subject in focus, a new multifunctional lab-on-a-chip (LOC) system with control over the oxygen content for studies on biological cells was developed. The chip was designed to incorporate the patch clamp technique, optical tweezers and absorption spectroscopy. The performance of the LOC was tested by a series of experiments. The oxygen content within the channels of the LOC was monitored by an oxygen sensor and verified by simultaneously studying the oxygenation state of chicken red blood cells (RBCs) with absorption spectra. The chicken RBCs were manipulated optically and steered in three dimensions towards a patch-clamp micropipette in a closed microfluidic channel. The oxygen level within the channels could be changed from a normoxic value of 18% O 2 to an anoxic value of 0.0-0.5% O 2. A time series of 3 experiments were performed, showing that the spectral transfer from the oxygenated to the deoxygenated state occurred after about 227 ± 1 s and a fully developed deoxygenated spectrum was observed after 298 ± 1 s, a mean value of 3 experiments. The tightness of the chamber to oxygen diffusion was verified by stopping the flow into the channel system while continuously recording absorption spectra showing an unchanged deoxygenated state during 5400 ± 2 s. A transfer of the oxygenated absorption spectra was achieved after 426 ± 1 s when exposing the cell to normoxic buffer. This showed the long time viability of the investigated cells. Successful patching and sealing were established on a trapped RBC and the whole-cell access (Ra) and membrane (Rm) resistances were measured to be 5.033 ± 0.412 M Ω and 889.7 ± 1.74 M Ω respectively.

This paper reviews microfluidic technologies with emphasis on applications in the fields of pharmacy, biology, and tissue engineering. Design and fabrication of microfluidic systems are discussed with respect to specific biological concerns, such as biocompatibility and cell viability. Recent applications and developments on genetic analysis, cell culture, cell manipulation, biosensors, pathogen detection systems, diagnostic devices, high-throughput screening and biomaterial synthesis for tissue engineering are presented. The pros and cons of materials like polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), glass, and silicon are discussed in terms of biocompatibility and fabrication aspects. Microfluidic devices are widely used in life sciences. Here, commercialization and research trends of microfluidics as new, easy to use, and cost-effective measurement tools at the cell/tissue level are critically reviewed.

We present a new concept of integrating a micropipette within a closed microfluidic system equipped with optical tweezers and a UV-Vis spectrometer. A single red blood cell (RBC) was optically trapped and steered in three dimensions towards a micropipette that was integrated in the microfluidic system. Different oxygenation states of the RBC, triggered by altering the oxygen content in the microchannels through a pump system, were optically monitored by a UV-Vis spectrometer. The built setup is aimed to act as a multifunctional system where the biochemical content and the electrophysiological reaction of a single cell can be monitored simultaneously. The system can be used for other applications like single cell sorting, in vitro fertilization or electrophysiological experiments with precise environmental control of the gas-, and chemical content.

Spectroscopic holography refers to techniques in which the detected hologram contains information about specific species in the medium under study. In general, at least two lasers are required with wavelengths chosen carefully to fit the interaction process utilized. In this process, energy from the shorter wavelength laser beam is transferred to the longer wavelength coherently through the process of stimulated emission. Two interaction mechanisms are considered; Stimulated Laser Induced Fluorescence (LIF) and Stimulated Raman Scattering (SRS), which both are species specific with the ability of coherent interaction. In this paper, the fundamental properties of spectroscopic holography is presented and demonstrated with a few idealized experiments. These validation experiments are performed in a gas chamber in which different gases may be blended and the gas pressure changed between 1-12 bars. In addition, two examples of applications are presented. In the first set of experiments, LIF holography is used to image light absorption and laser heating in a dye simultaneously. The second set of experiments is performed in a ow of methane gas. It is demonstrated that the combination of holographic phase measurements and SRS gain images may be used for calibration. This calibration may further be used to measure absolute concentration in a burning flame.

Hemoproteins usually supply cells and tissue with oxygen. A new hemoprotein mainly present in nerve cells called Neuroglobin was recently discovered. Enhanced expression of the protein has been shown to reduce hypoxic neural injury but the mechanism behind this function remains unknown. Methods enabling investigation of the protein in single functional neurons need to be developed. Here, we have studied how the electrical signaling capacity of a neuron was affected by hypoxic environments. Preliminary results show a trend of higher noise-level when a neuron is exposed to hypoxic compared to normoxic surroundings, which implies increased ion-channel activity. The setup used today shows shortages such as reduced control over the oxygen content due to leakage. Therefore, a gas-tight, multifunctional microfluidic system is under development which enables us to study influences of Neuroglobin concentrations on neuronal activity during hypoxia and anoxia. For electrophysiological recordings a patch-clamp micro pipette will be molded into the walls of the microfluidic system. A single biological cell is steered towards the pipette and attached there by means of optical tweezers. The Neuroglobin oxygen binding state will be studied using optical spectroscopy and the neuron environment will be manipulated by applying flows of varying oxygen content through the microfluidic system. This system will constitute a powerful tool in the investigation of the Neuroglobin mechanism of action.

The tactile resonance method (TRM) and Raman spectroscopy (RS) are promising for tissue characterisation in vivo. Our goal is to combine these techniques into one instrument, to use TRM for swift scanning, and RS for increasing the diagnostic power. The aim of this study was to determine the classification accuracy, using support vector machines, for measurements on porcine tissue and also produce preliminary data on human prostate tissue. This was done by developing a new experimental setup combining micro-scale TRM — scanning haptic microscopy (SHM) — for assessing stiffness on a micro-scale, with fibre optic RS measurements for assessing biochemical content. We compared the accuracy for using SHM alone versus SHM combined with RS, for different degrees of tissue homogeneity. The cross-validation classification accuracy for healthy porcine tissue types using SHM alone was 65–81%, and when RS was added it was increased to 81–87%. The accuracy for healthy and cancerous human tissue was 67–70% when only SHM was used, and increased to 72–77% for the combined measurements. This shows that the potential for swift and accurate classification of healthy and cancerous prostate tissue is high. This is promising for developing a tool for probing the surgical margins during prostate cancer surgery.

Tissue characterization is fundamental for identification of pathological conditions. Raman spectroscopy (RS) and tactile resonance measurement (TRM) are two promising techniques that measure biochemical content and stiffness, respectively. They have potential to complement the golden standard-–histological analysis. By combining RS and TRM, complementary information about tissue content can be obtained and specific drawbacks can be avoided. The aim of this study was to develop a multivariate approach to compare RS and TRM information. The approach was evaluated on measurements at the same points on porcine abdominal tissue. The measurement points were divided into five groups by multivariate analysis of the RS data. A regression analysis was performed and receiver operating characteristic (ROC) curves were used to compare the RS and TRM data. TRM identified one group efficiently (area under ROC curve 0.99). The RS data showed that the proportion of saturated fat was high in this group. The regression analysis showed that stiffness was mainly determined by the amount of fat and its composition. We concluded that RS provided additional, important information for tissue identification that was not provided by TRM alone. The results are promising for development of a method combining RS and TRM for intraoperative tissue characterization.

Prostate cancer is the most common form of cancer and the third leading cause of cancer-related death in European men. There is a need for new methods that can accurately localize and diagnose prostate cancer. In this study a new approach is presented: a combination of resonance sensor technology and Raman spectroscopy. Both methods have shown promising results for prostate cancer detection in vitro. The aim of this study was to evaluate the combined information from measurements with a Raman fiberoptic probe and a resonance sensor system. Pork belly tissue was used as a model system. A three-dimensional translation table was equipped with an in-house developed software, allowing measurements to be performed at the same point using two separate instruments. The Raman data was analyzed using principal component analysis and hierarchical clustering analysis. The spectra were divided into 5 distinct groups. The mean stiffness of each group was calculated from the resonance sensor measurements. One of the groups differed significantly (p < 0.05) from the others. A regression analysis, with the stiffness parameter as response variable and the principal component scores of the Raman data as the predictor variables, explained 67% of the total variability. The use of a smaller resonance sensor tip would probably increase the degree of correlation. In conclusion, Raman spectroscopy provides additional discriminatory power to the resonance sensor

Most Raman spectroscopic studies on tissue are performed in vitro. To assure that the results are applicable to in vivo examinations, preparation protocols and measurement procedures of tissue for in vitro studies should preserve tissue characteristics close to the native state. This study had two aims. The first was to elucidate if photoinduced effects arise during 5 minutes' continuous illumination of tissue with an 830 nm laser at an irradiance of 3 × 1010 W/m2. The second was to investigate the effects of snap-freezing of porcine prostate tissue in liquid nitrogen and subsequent storage at -80 °C, by means of multivariate analysis. 830 nm laser illumination of the specified irradiance did not affect the Raman spectra. A decrease of the spectral background was observed, likely due to photobleaching of tissue fluorophores. Snap-freezing and subsequent storage at -80 °C gave rise to subtle but significant alterations in Raman spectra, most likely related to changes in the protein conformations

The gold standard for detecting prostate cancer (PCa), systematic biopsy, lacks sensitivity as well as grading accuracy. PSA screening leads to over-treatment of many men, and it is unclear whether screening reduces PCa mortality. This review provides an understanding of the difficulties of localizing and diagnosing PCa. It summarizes recent developments of ultrasound (including elastography) and MRI, and discusses some alternative experimental techniques, such as resonance sensor technology and vibrational spectroscopy. A comparison between the different methods is presented. It is concluded that new ultrasound techniques are promising for targeted biopsy procedures, in order to detect more clinically significant cancers while reducing the number of cores. MRI advances are very promising, but MRI remains expensive and MR-guided biopsy is complex. Resonance sensor technology and vibrational spectroscopy have shown promising results in vitro. There is a need for large prospective multicentre trials that unambiguously prove the clinical benefits of these new techniques.

The depth-gating capacity of a spatially quasi-incoherent imaging interferometer is investigated in relation to the 3D correlation properties of diffraction field laser speckles. The system exploits a phase-stepped imaging Michelson-type interferometer in which spatially quasi-incoherent illumination is generated by passing an unexpanded laser beam through a rotating diffuser. Numerical simulations and optical experiments both verify that the depth-gating capacity of the imaging interferometer scales as 𝜆/2NA2𝑝λ/2NAp2, where 𝜆λ is the wavelength of the laser and NA𝑝NAp is the numerical aperture of the illumination. For a set depth gate of 150 µm, the depth-gating capacity of the interferometer is demonstrated by scanning a standard USAF target through the measurement volume. The results obtained show that an imaging tool of this kind is expected to provide useful capabilities for imaging through disturbing media and where a single wavelength is required.

An interferometric technique that utilize a spatially quasi-incoherent light source to perform interferometric measurements involving diffusely scattering objects is presented. The proposed technique is demonstrated with settings that give a depth gate of 90 µm.

We described a new concept on a depth-resolved investigation based on imaging interferometry. It exploits a quasi-incoherent imaging interferometry scheme and speckle image correlation techniques. We can spatially resolve a depth-gating capacity of the imaging interferometer with a resolution of around 26 µm.

We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.

Knowledge of the solid state is of great importance in the development of a new active pharmaceutical ingredient, since the solid form often dictates the properties and performance of the drug. In the present study, solid state characteristics of the sodium salt of the candidate cholesterol reducing compound eritadenine, 2(R), 3(R))-dihydroxy-4-(9-adenyl)-butanoic acid, were investigated. The compound was crystallized by slow cooling from water and various aqueous ethanol solutions, at different temperatures. Further, the compound solution was subjected to lyophilization and to high vacuum drying. The resulting solids were screened for polymorphism by micro Raman spectroscopy (λex = 830 nm) and the crystallinity was investigated by X-ray powder diffraction. Further, thermal analysis was applied to study possible occurrence of solvates or hydrates. Solids obtained from slow cooling showed crystallinity, whereas rapid cooling gave rise to more amorphous solids. Analysis of difference spectra of the Raman data for solids obtained from slow cooling of solution revealed subtle differences in the structures between crystals derived from pure water and crystals derived from aqueous ethanol solutions. Finally, from the thermal analysis it was deduced that crystals obtained from pure water were stoichiometrically dihydrates whereas crystals obtained from aqueous ethanol solutions were 2.5 hydrates; this formation of different hydrates were supported by the Raman difference analysis.

Eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid, is a cholesterol-reducing compound naturally occurring in the shitake mushroom (Lentinus edodes). To identify the unknown Raman spectrum of this compound, pure synthetic eritadenine was examined and the vibrational modes were assigned by following the synthesis pathway. This was accomplished by comparing the known spectra of the starting compounds adenine and D-ribose with the spectra of a synthesis intermediate, methyl 5-(6-Aminopurin-9H-9-yl)-2,3-O-isopropylidene-5-deoxy-β-D-ribofuranoside (MAIR) and eritadenine. In the Raman spectrum of eritadenine, a distinctive vibrational mode at 773 cm-1 was detected and ascribed to vibrations in the carbon chain, ν(C--C). A Raman line that arose at 1212 cm-1, both in the Raman spectrum of MAIR and eritadenine, was also assigned to ν(C--C). Additional Raman lines detected at 1526 and at 1583 cm-1 in the Raman spectrum of MAIR and eritadenine were assigned to ν(N--C) and a deformation of the purine ring structure. In these cases the vibrational modes are due to the linkage between adenine and the ribofuranoside moiety for MAIR, and between adenine and the carbon chain for eritadenine. This link is also the cause for the disappearance of adenine specific Raman lines in the spectrum of both MAIR and eritadenine. Several vibrations observed in the spectrum of D-ribose were not observed in the Raman spectrum of eritadenine due to the absence of the ribose ring structure. In the Raman spectrum of MAIR some of the D-ribose specific Raman lines disappeared due to the introduction of methyl and isopropylidene moieties to the ribose unit. With the approach presented in this study the so far unknown Raman spectrum of eritadenine could be successfully identified and is presented here for the first time.

We report on the development of an experimental platform where epi-fluorescence microscopy and optical tweezers are combined with a microfluidic system to enable the analysis of rapid cytological responses in single cells. The microfluidic system allows two different media to be merged in a Y-shaped channel. Microscale channel dimensions ensure purely laminar flow and, as a result, an environmental gradient can be created between the two media. Optical tweezers are used to move a single trapped cell repeatedly between the different environments. The cell is monitored continuously by fluorescence microscopy during the experiment. In a first experiment on yeast (Saccharomyces cerevisiae) we observed changes in cell volume as the cell was moved between environments with different osmolarity. This demonstrated that the platform allowed analysis of cytological alterations on a time scale shorter than 0.2 s. In a second experiment we observed the spatial migration of the Yap1p transcription factor fused to GFP as a cell was moved from an environment of low to high oxidative capacity. The system is universal allowing the response to numerous environmental changes to be studied on the sub second time scale in a variety of model cells. We intend to use the platform to study how the age of cells, their progression through the cell cycle, or their genetic landscape, alter their capacity (kinetics and amplitude) to respond to environmental changes.

Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

Species specific 3D imaging requires control of where in the sample stimulated Raman gain is achieved. By using a phase spatial light modulator the signal position can be calculated, controlled and directly imaged in 3D.

Stimulated Raman scattering is a phenomenon with potential use in providing real-time molecular information in three-dimensions (3D) of a sample using imaging. For precise imaging, the knowledge about the spatial generation of stimulated Raman scattering is essential. To investigate the spatial behavior in an idealized case, computer simulations and experiments were performed. For the computer simulations, diffraction theory was used for the beam propagation complemented with nonlinear phase modulation describing the interaction between the light and matter. For the experiments, a volume of ethanol was illuminated by an expanded light beam and a plane inside the volume was imaged in transmission. For generating stimulated Raman scattering, a pump beam was focused into this volume and led to a beam dump after passing the volume. The pulse duration of the two beams were 6 ns and the pump beam energy ranged from 1 to 27 mJ. The effect of increasing pump power on the spatial distribution of the Raman gain and the spatial growth of the signal at different interaction lengths between the beam and the sample was investigated. The spatial width of the region where the stimulated Raman scattering signal was generated for experiments and simulation was 0.21 and 0.09 mm, respectively. The experimental and simulation results showed that most of the stimulated Raman scattering is generated close to the pump beam focus and the maximum peak of the Stokes intensity spatially comes shortly after the peak of the pump intensity.

Raman spectroscopy allows the molecular chemical analysis of whole living cells by comparing them to known Raman signatures of specific vibrational bonds. In this work we used Raman spectroscopy to differentiate between wild type yeast cells and mutants characterized by increased or reduced mitochondrial fragmentation. To associate mitochondrial fragmentation with biochemical markers, we performed Linear Discriminant Analysis (LDA) of whole cell Raman spectra (~50–100 cells/spectrum). We show that the long-lived, less fragmented mutants fall into a significantly distant cluster from the wild type and short-lived, more fragmented mutants. Clustering depends on respiratory growth and coincides with that of membrane phospholipids and some respiratory chain components. Spectral clustering is supported by enzymatic activity measurements of OXPHOS Complexes. In addition, we find that NAD(P)H autofluorescence also correlates with mitochondrial fragmentation, representing another likely aging biomarker, besides phospholipids and OXPHOS components. In summary, we demonstrate that Raman spectroscopy has the potential to become a powerful tool for differentiating healthy from unhealthy aged tissues, as well as for the prognostic evaluation of mitochondrial function and fitness.

Mitochondrial Cytochrome c Oxidase Subunit 4 Isoform 2 (Cox4i2) is essential for acute oxygen sensing and signaling in pulmonary arterial smooth muscle cells (PASMCs) by triggering the production of superoxide during acute hypoxia [1]. However, the molecular mechanism underlying Cox4i2-dependent oxygen sensing remains elusive. We analysed oxygen-dependent respiration by high resolution respirometry, redox changes of the electron transport chain (ETC) by RAMAN spectroscopy, and supercomplex formation by blue native gel analysis of PASMCs isolated from wild type (WT) and Cox4i2-/- mice. To understand the role of Cox4i2-specific cysteine residues we determined hypoxia-induced superoxide production and oxygen affinity in a mouse epithelial cell line (CMT167 cells) overexpressing either Cox4i1, or WT Cox4i2, or Cox4i2 mutants (C41S, C55A, C109S). Respiration and supercomplex formation were similar in WT and Cox4i2-/- PASMCs. Interestingly, hypoxia-induced reduction of ETC components (NADH, ubiquinol, and reduced cytochrome c) was prevented in Cox4i2-/- PASMCs. CMT167 cells expressing either Cox4i1, or Cox4i2 mutants lacked hypoxia-induced superoxide release, which was detected only in cells expressing WT Cox4i2. In contrast, overexpression of Cox4i1, or Cox4i2, or Cox4i2 mutants did not affect oxygen affinity. Our findings suggest that Cox4i2 does not alter superoxide production by rearrangement of supercomplexes, whereas its specific cysteines are needed for the superoxide release. In conclusion, Cox4i2 plays a major role in the hypoxia-induced reduction of ETC components, likely mediated through its redox-active cysteine residues.

In this paper we will describe a system designed to combine optical tweezers and laser scalpels with confocal as well as epi-fluorescence microscopy. A continuos wave Nd:YVO4 laser is used to produce a dual optical tweezers, where each trap can be individually controlled. A second optical tweezers setup is based on a tunable titanium sapphire laser, which allows us to adjust the wavelength to minimize the damage to the cell under investigation. A pulsed nitrogen laser working at 337 nm forms a laser scalpel. The tweezers and scalpels are both incorporated in an inverted microscope equipped with epi-fluorescence and confocal imaging capabilities. In order to further control the sample we have developed a technique to tailor make the environment closest to the studied objects. Micrometer-sized structures such as channels and reservoirs have been produced in rubber silicon using lithographic methods. In combination with a micro-manipulator, our system can be used to extract single cells from a population of billions for further studies or growth.

In recent years there has been a growing interest in the use of optical manipulation techniques, such as optical tweezers, in biological research as the full potential of such applications are being realized. Biological research is developing towards the study of single entities to reveal new behaviors that cannot be discovered with more traditional ensemble techniques. To be able to study single cells we have developed a new method where a combination of micro-fluidics and optical tweezers was used. Micro-fluidic channels were fabricated using soft lithography. The channels consisted of a Y-shaped junction were two channels merged into one. By flowing different media in the two channels in laminar flow we were able to create a sharp concentration gradient at the junction. Single cells were trapped by the tweezers and the micro-fluidic system allowed fast environmental changes to be made for the cell in a reversible manner. The time required to change the surroundings of the cell was limited to how sharp mixing region the system could create, thus how far the cells had to be moved using the optical tweezers. With this new technique cellular response in single cells upon fast environmental changes could be investigated in real time. The cellular response was detected by monitoring variations in the cell by following the localization of fluorescently tagged proteins within the cell.

Despite that sodium silicate solutions of high pH are commonly used in industrial applications, most investigations are focused on low to medium values of pH. Therefore we have investigated such solutions in a broad modulus range and up to high pH values (similar to 14) by use of infrared (IR) spectroscopy and silicon nuclear magnetic resonance (Si-29-NMR). The results show that the modulus dependent pH value leads to more or less charged species, which affects the configurations of the silicate units. This in turn, influences the alkali-activation process of low CO2 footprint cements, i.e. materials based on industrial waste or by-products

There are indications on that the initial setting time is dependent on the relative ratio of Na2O to SiO2 when sodium silicate solutions (Na2SiO3) are used for the alkali-activation of ground granulated blast-furnace slag (GGBS). One possible reason for this is that the ratio (called modulus) not only influences the pH but also the viscosity of the solution. The viscosity is, in turn dependent on the structures in the liquid. Therefore, we have investigated the structure of sodium silicate solutions of different moduli by infrared spectroscopy (IR) and silicon nuclear magnetic resonance (Si-29-NMR). The results, which show that the silica configuration is highly dependent on the modulus, will be discussed in relation to the initial setting time of corresponding measurements on GGBS hydration

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O₂-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H₂O₂. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO₂). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp electrophysiology, and iii) live-cell imaging under precisely controlled pO₂ have therefore been developed. Merging these modalities allows label-free and simultaneous observation of oxygen-dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in-depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions. 

Geobacter sulfurreducens is a good candidate as a chassis-organism due to its ability to form thick, conductive biofilms, enabling long distance extracellular electron transfer (EET). Due to the complexity of EET pathways in G. sulfurreducens, a dynamic approach is required to study genetically modified EET rates in the biofilm. By coupling on-line resonance Raman microscopy with chronoamperometry, we were able to observe the dynamic discharge response in the biofilm's cytochromes to an increase in anode voltage. Measuring the heme redox state alongside the current allows for the fitting of a dynamic model using the current response and a subsequent validation of the model via the value of a reduced cytochrome c Raman peak. The modelled reduced cytochromes closely fitted the Raman response data from the G. sulfurreducens wild-type strain, showing the oxidation of heme groups in cytochromes until achieving a new steady state. Furthermore, the use of a dynamic model also allows for the calculation of internal rates, such as acetate and NADH consumption rates. The Raman response of a mutant lacking OmcS showed a sharper initial rate than predicted, followed by an almost linear decrease of the reduced mediators. The increased initial rate could be attributed to an increase in biofilm conductivity, previously observed in biofilms lacking OmcS. One explanation for this is that OmcS acts as a conduit between cytochromes; therefore deleting the gene restricts the electron transfer rate to the extracellular matrix. This could, however, be modelled assuming a linear oxidation rate of intercellular mediators.

IMPORTANCE Bioelectrochemical systems can fill a vast array of application niches, due to the control of redox reactions that it offers. Although native microorganisms are preferred for applications such as bioremediation, more control is required for applications such as biosensors or biocomputing. The development of a chassis organism, in which the EET is well defined and readily controllable, is therefore essential. The combined approach in this work offers a unique way of monitoring and describing the reaction kinetics of a G. sulfurreducens biofilm, as well as offering a dynamic model that can be used in conjunction with applications such as biosensors.

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many “on-line” systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ∆pilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ∆S. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ. 

This chapter reviews the powerful combination of optical tweezers and Raman spectroscopy as applied to small particles in solution. It first reviews the significant amount of work that has been published on particles in the micron range, in particular on biological cells. Raman Spectroscopy and laser trapping performed on dielectric particles, aerosols, gas bubbles, polymorphs and biological cells are discussed in detail. Results from experiments exploring heating and photo-induced damage caused by laser light from the UV to the NIR wavelength region are summarized. Metal nanoparticles, typically gold and silver nanoparticles, play an important role in optical tweezers for Raman analysis in the context of surface-enhanced Raman scattering (SERS). In addition, combined with other techniques, such as microfluidics, the applicability of optical tweezers for SERS can be even more expanded. A large number of studies have demonstrated that optical tweezers integrated with Raman spectroscopy have a very high potential for real-time chemical characterization and diagnosis of micron-sized dielectric beads and biological objects.

Prostate cancer (PCa) is the most common cancer form for men in Europe. A sensor system combining Raman spectroscopy and stiffness sensing with a resonance sensor has recently been developed by us for prostate cancer detection. In this study the sensor system has been used for measurements on two slices of fresh human prostate tissue. The stiffness sensor could detect locations slices with significantly different stiffness contrasts (p < 0.05). Raman spectroscopic measurements could be performed with the dual-modality probe for tissue classification. The findings are important for the continued development of a combination probe for prostate cancer detection.

Prostate cancer is the most common cancer for men in the western world. The most prevalent curative treatment is radical prostatectomy. However, prostate surgery can give unwanted side effects and there is a need for an instrument that can provide decision support to the surgeon during surgery on the presence of cancer cells in the surgical margin. A dual-modality probe, combining Raman spectroscopy and tactile resonance technology, has been used for detecting cancer in fresh human prostate tissue. The tactile resonance modality measures the tissue stiffness and Raman spectroscopy depicts the molecular content in tissue, both related to cancer. After ethical approval, the study investigated the potential of the dual-modality probe by testing its ability to differentiate between normal and cancerous prostate tissue ex vivo. It also investigated the minimal amount of measurement points needed to securely detect cancer on the surface of prostate tissue. Measurements on three prostate tissue slices show that the tactile resonance modality measuring stiffness was able to detect differences between normal and cancerous tissue on a significant level of 90%, but the sample size was too low to draw any firm conclusions. It was also suggested from the study results that the high wavenumber region in the Raman spectrum can give valuable information about cancer in prostate tissue. A number of 24 measurement points were enough for detecting cancer in prostate slices in this study. It can be suggested from this study that combining these two sensor modalities is promising for accurate detection of prostate cancer that is needed during prostate surgery, but more measurements including more prostates must be performed before the full value of the study result can be established.