Absolute frequencies of H13C14N hydrogen cyanide transitions in the 1.5-µm region with the saturated spectroscopy and a sub-kHz scanning laser.

The wide span and high density of lines in its rovibrational spectrum render hydrogen cyanide a useful spectroscopic media for referencing absolute frequencies of lasers in optical communication and dimensional metrology. We determined, for the first time to the best of our knowledge, the molecular transitions' center frequencies of the H13C14N isotope in the range from 1526 nm to 1566 nm with 1.3 × 10-10 fractional uncertainty. We investigated the molecular transitions with a highly coherent and widely tunable scanning laser that was precisely referenced to a hydrogen maser through an optical frequency comb. We demonstrated an approach to stabilize the operational conditions needed to maintain the constantly low pressure of the hydrogen cyanide to carry out the saturated spectroscopy with the third-harmonic synchronous demodulation. We demonstrated approximately a forty-fold improvement in the line centers' resolution compared to the previous result.

Analysis of Bacteriophage-Host Interaction by Raman Tweezers.

Bacteriophages, or "phages" for short, are viruses that replicate in bacteria. The therapeutic and biotechnological potential of phages and their lytic enzymes is of interest for their ability to selectively destroy pathogenic bacteria, including antibiotic-resistant strains. Introduction of phage preparations into medicine, biotechnology, and food industry requires a thorough characterization of phage-host interaction on a molecular level. We employed Raman tweezers to analyze the phage-host interaction of Staphylococcus aureus strain FS159 with a virulent phage JK2 (=812K1/420) of the Myoviridae family and a temperate phage 80α of the Siphoviridae family. We analyzed the timeline of phage-induced molecular changes in infected host cells. We reliably detected the presence of replicating phages in bacterial cells within 5 min after infection. Our results lay the foundations for building a Raman-based diagnostic instrument capable of real-time, in vivo, in situ, nondestructive characterization of the phage-host relationship on the level of individual cells, which has the potential of importantly contributing to the development of phage therapy and enzybiotics.

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics.

Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 °C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy.

The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress.

Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.

Detection of Chloroalkanes by Surface-Enhanced Raman Spectroscopy in Microfluidic Chips.

Optofluidics, a research discipline combining optics with microfluidics, currently aspires to revolutionize the analysis of biological and chemical samples, e.g., for medicine, pharmacology, or molecular biology. In order to detect low concentrations of analytes in water, we have developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The geometry of the gold surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system, which allowed transport and precise mixing of the analyzed fluids, while preventing contamination or abrasion of the highly sensitive substrate. To illustrate its practical use, we employed the device for quantitative detection of persistent environmental pollutant 1,2,3-trichloropropane in water in submillimolar concentrations. The developed sensor allows fast and simple quantification of halogenated compounds and it will contribute towards the environmental monitoring and enzymology experiments with engineered haloalkane dehalogenase enzymes.

Effects of Infrared Optical Trapping on Saccharomyces cerevisiae in a Microfluidic System.

Baker's yeast (Saccharomyces cerevisiae) represents a very popular single-celled eukaryotic model organism which has been studied extensively by various methods and whose genome has been completely sequenced. It was also among the first living organisms that were manipulated by optical tweezers and it is currently a frequent subject of optical micromanipulation experiments. We built a microfluidic system for optical trapping experiments with individual cells and used it for the assessment of cell tolerance to phototoxic stress. Using optical tweezers with the wavelength of 1064 nm, we trapped individual Saccharomyces cerevisiae cells for 15 min and, subsequently, observed their stress response in specially designed microfluidic chambers over time periods of several hours by time-lapse video-microscopy. We determined the time between successive bud formations after the exposure to the trapping light, took account of damaged cells, and calculated the population doubling period and cell areas for increasing trapping power at a constant trapping time. Our approach represents an attractive, versatile microfluidic platform for quantitative optical trapping experiments with living cells. We demonstrate its application potential by assessing the limits for safe, non-invasive optical trapping of Saccharomyces cerevisiae with infrared laser light.

Direct measurement of the temperature profile close to an optically trapped absorbing particle.

The surface temperature of an absorbing particle trapped in optical tweezers (OTs) is measured using a mixture of two fluorescent dyes. We analyze the dependence of temperature on both laser power and the radial distance from its surface, and we verify the 1/r decrease of temperature with increasing distance from the particle surface. We detect the variations of spectral profiles as the medium temperature changes. The temperature dependent signal, i.e., the ratio of summed intensities from two distinct spectral regions, is affected by the convolution of temperature profile with transfer function of the spectroscopic system. We analyze this effect and determine the temperature increase on the surface of a core-shell particle trapped by OTs.

Algal biomass analysis by laser-based analytical techniques--a review.

Algal biomass that is represented mainly by commercially grown algal strains has recently found many potential applications in various fields of interest. Its utilization has been found advantageous in the fields of bioremediation, biofuel production and the food industry. This paper reviews recent developments in the analysis of algal biomass with the main focus on the Laser-Induced Breakdown Spectroscopy, Raman spectroscopy, and partly Laser-Ablation Inductively Coupled Plasma techniques. The advantages of the selected laser-based analytical techniques are revealed and their fields of use are discussed in detail.

Spectral tuning of lasing emission from optofluidic droplet microlasers using optical stretching.

We introduce tunable optofluidic microlasers based on active optical resonant cavities formed by optically stretched, dye-doped emulsion droplets confined in a dual-beam optical trap. To achieve tunable dye lasing, optically pumped droplets of oil dispersed in water are stretched by light in the dual-beam trap. Subsequently, resonant path lengths of whispering gallery modes (WGMs) propagating in the droplet are modified, leading to shifts in the microlaser emission wavelengths. Using this technique, we present all-optical, almost reversible spectral tuning of the lasing WGMs and show that the direction of tuning depends on the position of the pump beam focus on the droplet. In addition, we study the effects of temperature changes on the spectral position of lasing WGMs and demonstrate that droplet heating leads to red-tuning of the droplet lasing wavelength.

Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy.

Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations-MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms' "fingerprint" (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.

SERS-Tags: Selective Immobilization and Detection of Bacteria by Strain-Specific Antibodies and Surface-Enhanced Raman Scattering.

Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.

Physiological and transcriptome profiling of Chlorella sorokiniana: A study on azo dye wastewater decolorization.

Over decades, synthetic dyes have become increasingly dominated by azo dyes posing a significant environmental risk due to their toxicity. Microalgae-based systems may offer an alternative for treatment of azo dye effluents to conventional physical-chemical methods. Here, microalgae were tested to decolorize industrial azo dye wastewater (ADW). Chlorella sorokiniana showed the highest decolorization efficiency in a preliminary screening test. Subsequently, the optimization of the experimental design resulted in 70% decolorization in a photobioreactor. Tolerance of this strain was evidenced using multiple approaches (growth and chlorophyll content assays, scanning electron microscopy (SEM), and antioxidant level measurements). Raman microspectroscopy was employed for the quantification of ADW-specific compounds accumulated by the microalgal biomass. Finally, RNA-seq revealed the transcriptome profile of C. sorokiniana exposed to ADW for 72 h. Activated DNA repair and primary metabolism provided sufficient energy for microalgal growth to overcome the adverse toxic conditions. Furthermore, several transporter genes, oxidoreductases-, and glycosyltransferases-encoding genes were upregulated to effectively sequestrate and detoxify the ADW. This work demonstrates the potential utilization of C. sorokiniana as a tolerant strain for industrial wastewater treatment, emphasizing the regulation of its molecular mechanisms to cope with unfavorable growth conditions.

Optically Transportable Optofluidic Microlasers with Liquid Crystal Cavities Tuned by the Electric Field.

Liquid crystal microdroplets with readily adjustable optical properties have attracted considerable attention for building reconfigurable optofluidic microsystems for sensing, imaging, and light routing applications. In this quest, development of active optical microcavities serving as versatile integrated sources of coherent light and ultra-sensitive environmental sensors has played a prominent role. Here, we study transportable optofluidic microlasers reversibly tunable by an external electric field, which are based on fluorophore-doped emulsion droplets of radial nematic liquid crystals manipulated by optical tweezers in microfluidic chips with embedded liquid electrodes. Full transparency of the electrodes formed by a concentrated electrolyte solution allows for applying an electric field to the optically trapped droplets without undesired heating caused by light absorption. Taking advantage of independent, precise control over the electric and thermal stimulation of the lasing liquid crystal droplets, we characterize their spectral tuning response at various optical trapping powers and study their relaxation upon a sudden decrease in the trapping power. Finally, we demonstrate that sufficiently strong applied electric fields can induce fully reversible phase transitions in the trapped droplets even below the bulk melting temperature of the used liquid crystal. Our observations indicate viability of creating electrically tunable, optically transported microlasers that can be prepared on-demand and operated within microfluidic chips to implement integrated microphotonic or sensing systems.

Raman Microspectroscopic Analysis of Selenium Bioaccumulation by Green Alga Chlorella vulgaris.

Selenium (Se) is an element with many commercial applications as well as an essential micronutrient. Dietary Se has antioxidant properties and it is known to play a role in cancer prevention. However, the general population often suffers from Se deficiency. Green algae, such as Chlorella vulgaris, cultivated in Se-enriched environment may be used as a food supplement to provide adequate levels of Se. We used Raman microspectroscopy (RS) for fast, reliable, and non-destructive measurement of Se concentration in living algal cells. We employed inductively coupled plasma-mass spectrometry as a reference method to RS and we found a substantial correlation between the Raman signal intensity at 252 cm-1 and total Se concentration in the studied cells. We used RS to assess the uptake of Se by living and inactivated algae and demonstrated the necessity of active cellular transport for Se accumulation. Additionally, we observed the intracellular Se being transformed into an insoluble elemental form, which we further supported by the energy-dispersive X-ray spectroscopy imaging.

Raman microspectroscopy of individual algal cells: sensing unsaturation of storage lipids in vivo.

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm(-1) (cis C═C stretching mode) and 1,445 cm(-1) (CH(2) scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.