Zdeněk Pilát

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 (CH2 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.

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.

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.

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.

Optical trapping of microalgae at 735-1064 nm: photodamage assessment.

Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in that they have strong light absorption in the visible range, and by the fact that their reaction centers are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux density caused photodamage that was strongly wavelength dependent. The photochemical activity before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30s exposure to incident radiation at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable photodamage.

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.

Active sorting switch for biological objects

Active contactless optical sorting of microobjects represents very useful technique in many areas of biology, chemistry, and medicine. We suggest here a configuration that combines optical sorting, trapping, excitation, and detection paths and provides efficient sorting of biological samples according to their various parameters (fluorescence, Raman spectrum, CCD image, motion etc.). This approach is based on the shape of the laser beam and we succeeded in sorting of several types of living microorganisms.

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.

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.