Silvie Bernatová

Raman microspectroscopy of algal lipid bodies: β-carotene quantification

Advanced optical instruments can serve for analysis and manipulation of individual living cells and their internal structures. We have used Raman microspectroscopic analysis for assessment of β-carotene concentration in algal lipid bodies (LBs) in vivo. Some algae contain β-carotene in high amounts in their LBs, including strains which are considered useful in biotechnology for lipid and pigment production. We have devised a simple method to measure the concentration of β-carotene in a mixture of algal storage lipids from the ratio of their Raman vibrations. This finding may allow fast acquisition of β-carotene concentration valuable, e.g., for Raman microspectroscopy assisted cell sorting for selection of the overproducing strains. Furthermore, we demonstrate that β-carotene concentration can be proportional to LB volume and light intensity during the cultivation. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on the spectroscopic features of the individual living cells. The resultant apparatus could find its use in demanding biotechnological applications such as selection of rare natural mutants or artificially modified cells resulting from genetic manipulations.

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.

Candida parapsilosis Biofilm Identification by Raman Spectroscopy

Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made.

Identification of individual biofilm-forming bacterial cells using Raman tweezers.

Abstract. A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

Raman microspectroscopy of algal lipid bodies: β-carotene as a volume sensor

Advanced optical instruments are useful for analysis and manipulation of individual living cells and their internal structures. We have employed Raman microspectroscopic analysis for assessment of algal lipid body (LB) volume in vivo. Some algae contain β-carotene in high amounts in their LBs, including strains which are considered useful in biotechnology for lipid and pigment production. We have detected proportionality between the Raman vibrations of β-carotene and the LB volume. This finding may allow fast acquisition of LB volume approximation valuable e.g. for Raman microspectroscopy assisted cell sorting. We combine optical manipulation and analysis on a microfluidic platform in order to achieve fast, effective, and non-invasive sorting based on spectroscopic features of the individual living cells. The resultant apparatus could find its use in demanding biotechnological applications such as selection of rare natural mutants or artificially modified cells resulting from genetic manipulations.

Influence of Culture Media on Microbial Fingerprints Using Raman Spectroscopy.

Raman spectroscopy has a broad range of applications across numerous scientific fields, including microbiology. Our work here monitors the influence of culture media on the Raman spectra of clinically important microorganisms (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans). Choosing an adequate medium may enhance the reproducibility of the method as well as simplifying the data processing and the evaluation. We tested four different media per organism depending on the nutritional requirements and clinical usage directly on a Petri dish. Some of the media have a significant influence on the microbial fingerprint (Roosvelt-Park Institute Medium, CHROMagar) and should not be used for the acquisition of Raman spectra. It was found that the most suitable medium for microbiological experiments regarding these organisms was Mueller-Hinton agar.

Rapid identification of staphylococci by Raman spectroscopy

Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

Natural user interface as a supplement of the holographic Raman tweezers

Holographic Raman tweezers (HRT) manipulates with microobjects by controlling the positions of multiple optical traps via the mouse or joystick. Several attempts have appeared recently to exploit touch tablets, 2D cameras or Kinect game console instead. We proposed a multimodal “Natural User Interface” (NUI) approach integrating hands tracking, gestures recognition, eye tracking and speech recognition. For this purpose we exploited “Leap Motion” and “MyGaze” low-cost sensors and a simple speech recognition program “Tazti”. We developed own NUI software which processes signals from the sensors and sends the control commands to HRT which subsequently controls the positions of trapping beams, micropositioning stage and the acquisition system of Raman spectra. System allows various modes of operation proper for specific tasks. Virtual tools (called “pin” and “tweezers”) serving for the manipulation with particles are displayed on the transparent “overlay” window above the live camera image. Eye tracker identifies the position of the observed particle and uses it for the autofocus. Laser trap manipulation navigated by the dominant hand can be combined with the gestures recognition of the secondary hand. Speech commands recognition is useful if both hands are busy. Proposed methods make manual control of HRT more efficient and they are also a good platform for its future semi-automated and fully automated work.

Characterization of microorganisms using Raman tweezers

The ability to identify and characterize microorganisms (algae, bacteria, eukaryotic cells) from minute sample volumes in a rapid and reliable way is the crucial first step in their classification and characterization. In the light of this challenge related to microorganisms exploitation Raman spectroscopy can be used as a powerful tool for chemical analysis. Raman spectroscopy can elucidate fundamental questions about the metabolic processes and intercellular variability on a single cell level. Moreover, Raman spectroscopy can be combined with optical tweezers and with microfluidic chips to measure nutrient dynamics and metabolism in vivo, in real-time, and label free. We demonstrate the feasibility to employ Raman spectroscopy-based sensor to sort microorganisms (bacteria, algae) according to the Raman spectra. It is now quite feasible to sort algal cells according to the degree of unsaturation (iodine value) in lipid storage bodies.

Thermal tuning of spectral emission from optically trapped liquid-crystal droplet resonators

Surfactant-stabilized emulsion droplets of liquid crystals (LCs) suspended in water and labeled with a fluorescent dye form active, anisotropic optofluidic microresonators. These microresonators can host whispering gallery modes (WGMs), high-quality morphology-dependent optical resonances that are supported due to the contrast of refractive index between the LC droplets and the surrounding aqueous medium. In addition, owing to the refractive index contrast, such LC emulsion droplets can be stably trapped in three dimensions using optical tweezers, enabling long-term investigation of their spectral characteristics. We explore various combinations of fluorescently dyed LC droplets and host liquid-surfactant systems and show that the WGM emission spectra of optical resonators based on optically trapped LC emulsion droplets can be largely and (almost) reversibly tuned by controlled changes of the ambient temperature. Depending on the actual range of temperature modulation and LC phase of the studied droplet, thermally induced effects can either lead to phase transitions in the LC droplets or cause modifications of their refractive index profile without changing their LC phase. Our results indicate feasibility of this approach for creating miniature thermally tunable sources of coherent light that can be manipulated and stabilized by optical forces.