Optical Micromanipulation Techniques Combined with Microspectroscopic Methods

Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.The subject of the presented Ph.D. thesis is a combination of optical micromanipulation and microspectroscopic methods. We used laser tweezers to transport and sort various living microorganisms, such as microalgal or yeast cells. We employed Raman microspectroscopy to analyze chemical composition of individual cells and we used the information about chemical composition to automatically select the cells of interest. We combined pulsed amplitude modulation fluorescence microspectroscopy, optical micromanipulation and other techniques to map the stress response of cells to various laser wavelengths, intensities and durations of optical trapping. We fabricated microfluidic chips of various designs and we constructed Raman-tweezers sorter of micro-objects such as living cells on a microfluidic platform.

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

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

Raman Spectroscopy for the characterization of algal cells

ABSTRACT Raman spectroscopy can elucidate fundamental questions about intercellular variability and what governs it. Moreover, knowing the metabolic response on single cell level this can significantly contribute to the study and use of microalgae in systems biology and biofuel technology. Raman spectroscopy is capable to measure nutrient dynamics and metabolism in vivo, in real-time, label free making it possible to monitor/evaluate population variability. Also, degree of unsaturation of the algae oil (iodine value) can be measured using Raman spectra obtained from single microalgae. The iodine value is the determination of the amount of unsaturation contained in fatty acids (in the form of double bonds). 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. 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

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 tweezers in microfluidic systems for automatic analysis and sorting of living cells

We have devised an automatic analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, together with computerized real time image analysis, spectra processing and micromanipulation. This device serves to identify and sort biological objects, such as living cells of various prokaryotic and eukaryotic organisms based on their Raman spectral properties. This approach allowed us to collect information about the chemical composition of the objects, such as the presence and composition of lipids, proteins, or nucleic acids without using artificial chemical probes such as fluorescent markers. The non-destructive and non-contact nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. The special microfluidic chip uses gravity to move the cells across the sorting area. Our system uses dedicated software to achieve fully automated spectral analysis and sorting. The devised system is a robust and universal platform for non-contact sorting of microobjects based on their chemical properties. It could find its use in many medical, biotechnological, and biological applications

Optofluidic techniques for directed evolution of enzymes

Enzymes are highly versatile and ubiquitous biological catalysts. They can greatly accelerate\nlarge variety of reactions, while ensuring appropriate catalytic activity and high selectivity.\nThese properties make enzymes attractive biocatalysts for a wide range of industrial and\nbiomedical applications. Over the last two decades, directed evolution of enzymes has\ntransformed the field of protein engineering

Fluorescence and surface enhanced Raman spectroscopy in microfluidics for monitoring of enzymatic reactions

We have implemented two different systems for detecting the concentration of molecules in microfluidic systems. The first method uses optical fibers and detects the intensity of fluorescence, while the second method is using surface enhanced Raman spectroscopy (SERS)

Raman tweezers for sorting of living cells

We have developed an instrument for automatized analysis and sorting of living cells of unicellular algae and other micro-objects based on laser tweezers and Raman spectroscopy. The system comprises the Raman tweezers setup, special microfluidic chip, and a specialized software allowing image recognition, spectral analysis, and automated sorting functions. The resulting instrument allows non-destructive analysis of chemical properties of living cells and their automatic separation for further examination or cultivation