We report on the development of a set of Raman based techniques to monitor a large variety of biological and chemical analytes, such as various microorganisms, gels of hyaluronic acid and selected halogenated hydrocarbons using Raman spectroscopy, Raman tweezers and surface-enhanced Raman spectroscopy (SERS). We analyzed individual microbial cells with Raman tweezers to provide solutions for fast and label-free identification of specific bacterial or yeast species. We designed an optofluidic SERS platform for quantification of sub-millimolar concentrations of halogenated environmental pollutants such as 1,2,3-trichloropropane and chloroform. We also examined the gel structure of hyaluronic acid by Raman spectroscopy.
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Because of 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 <i>Escherichia coli.</i> We used laser tweezers Raman spectroscopy (LTRS) in a microchamber chip to manipulate and analyze individual <i>E. coli cells</i>. We subjected the cells to antibiotic cefotaxime, and we observed the changes by the time-lapse microscopy and Raman spectroscopy. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments .
A biomass of yeast strains has been studied using Raman spectroscopy due to their potential applications in the field of biofuel generation, food industry and biotechnological applications. In order to utilize biomass for efficient industrial/biotechnological production, the optimal cultivation parameters have to be determined which in turn lead to high production of desired substances such as oil, carotenoids, and pigments in the selected cell line of yeast. Therefore, we focused on different cultivation conditions (the effects of temperature regime and medium composition) and their influence on microorganisms growth and metabolic changes.
Emulsion droplets of liquid crystals (LC) suspended in water and labeled with a suitable fluorescent dye can serve as active optofluidic microcavities, since the contrast of refractive index between the LC droplets and the surrounding aqueous medium allows excitation of whispering gallery modes (WGMs) in the droplets. In addition, such emulsion droplets can be also stably trapped in three-dimensions using optical tweezers which stabilizes the droplets while investigating their spectral characteristics. We explore various combinations of fluorescently dyed LC droplets and host liquid - surfactant systems and show that the WGM emission spectrum of an optically trapped LC droplet-based cavity can be largely and (almost) reversibly tuned by controlled changes of the ambient temperature that induce phase transitions in the LC droplets. Our results indicate feasibility of this approach for creating miniature tunable sources of coherent light.
The combination of optical tweezers and Raman micro-spectroscopy is frequently referred as Raman tweezers. A single focused laser beam is utilized here both as a source of Raman scattering and a source forming an optical trap. Raman tweezers have been recently used in variety of applications in cell biology as a useful tool for non-contact and non-destructive determination of living cells properties. Here we use Raman tweezers to follow response of cells on the length of their cultivation in mineral oil. Analyses of obtained Raman spectra are based on 2D correlation analysis and allow us to determine the chemical background of the cell response in a gentle way.
The main goal of our investigations is to focus on the basic physiological mechanisms of microorganisms (yeast and bacteria), exposed to different conditions, by time-resolved Raman spectroscopy. This study provides an insight into the mechanism of targeted stress factors or the influence of different cultivation times on species metabolism <i>in vivo</i>, in realtime and label free. We also focused on time-course study of physico-chemical properties of bacterial cells and cell cytoplasm with respect to the intracellular content of polyhydroxyalkanoates and to the production of yeast lipids or carotenoids.
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.
We report on Raman spectroscopy measurements - separated by a given time intervals - for the selected yeast strains (biofilm positive and biofilm negative) on colonies grown directly on the Petri dishes or on the well-plate. Chemometric principal component analysis of these spectra sets generated clusters of data points, from which the reproducibility of the measurement could be analysed. Consequently, these resulted in clusters coinciding well with the biofilm positive and biofilm negative strains measurement of a particular sample dish, suggesting good reproducibility of our measurement procedure, even when the samples were prepared and measured days up to months apart. This suggests the potential of Raman spectroscopy in routine clinical diagnostic.
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.
Raman tweezers represents a unique method for identification of different microorganisms on the basis of Raman scattering. Raman tweezers allows us to fix and sterile manipulate with the trapped object and in the same time check the growth, viability, response to the external environment etc. by Raman signal evaluating. The investigations presented here include distinction of bacteria in general (staphylococcal cells), identification of bacteria strains (biofilm-positive and biofilm-negative) by using principal component analysis (PCA) and monitoring the influence of antibiotics.
Here we report on combination of the data obtained from MICs (minimum inhibitory concentrations) with infor- mation of microoragnisms fingerprint provided by Raman spectroscopy. In our feasibility study we could follow mechanisms of the bacteriostatic versus bactericidal action on biofilm-positive <i>Staphylococcus epidermidis</i> simply by monitoring Raman bands corresponding to DNA translating the changes introduced by selected antibiotics. The Raman spectra of <i>Staphylococcus epidermidis</i> treated with a bacteriostatic agent show little effect on DNA which is in contrast with the action of a bactericidal agent where decreased in dedicated Raman spectra signal strength suggests DNA fragmentation. Moreover, we demonstrate that Raman tweezers are indeed able to distinguish strains of biofilm-forming (biofilm-positive) and biofilm-negative <i>Staphylococcus epidermidis</i> strains using principal component analysis (PCA).
The main goal of our investigation is to use Raman tweezers technique so that the responce of Raman scattering on
microorganisms suspended in liquid media (bacteria, algae and yeast cells in microfluidic chips) can be used to identify
different species. The investigations presented here include identification of different bacteria strains (biofilm-positive
and biofilm-negative) and yeast cells by using principal component analysis (PCA). The main driving force behind our
investigation was a common problem in the clinical microbiology laboratory - how to distinguish between contaminant
and invasive isolates. Invasive bacterial/yeast isolates can be assumed to form a biofilm, while isolates which do not
form a biofilm can be treated as contaminant. Thus, the latter do not represent an important virulence factor.
We have constructed a device for active optical manipulation and Raman spectral analysis in a microfluidic channel for efficient, nondestructive and contactless sorting of biological samples based on the Raman spectroscopic characteristics of living cells. In our previous work, we have linked such Raman spectral characteristics of microalgal lipid bodies with the unsaturation or carotene concentration via a calibration curve. As the sorting platform we have used a combination of fast galvano-optic laser steering system and specially designed microfluidic chips. We used X shaped channels with two input and output ports, and also several differently shaped variants. The steerable trapping laser beam was designed to move the cell to the specified locations and confine the cell for the time period needed for the analysis.
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.
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
Raman spectroscopy is a powerful tool for chemical analysis. This technique can elucidate fundamental questions about
the metabolic processes and intercellular variability on a single cell level. Therefore, Raman spectroscopy can
significantly contribute to the study and use of microalgae in systems biology and biofuel technology. Raman
spectroscopy can be combined with optical tweezers. We have employed microfluidic system to deliver the sampled
microalgae to the Raman-tweezers. This instrument is able to measure chemical composition of cells and to track
metabolic processes in vivo, in real-time and label-free making it possible to detect population variability in a wide array
of traits. Moreover, employing an active sorting switch, cells can be separated depending on input parameters obtained
from Raman spectra. We focus on algal lipids which are promising potential products for biofuel as well as for nutrition.
Important parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids. We
demonstrate the capacity of our Raman tweezers based sensor to sort cells according to the degree of unsaturation in
lipid storage bodies of individual living algal cells.