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.

Imaging-in-flow: digital holographic microscopy as a novel tool to detect and classify nanoplanktonic organisms

Traditional taxonomic identification of planktonic organisms is based on light microscopy, which is both time-consuming and tedious. In response, novel ways of automated (machine) identification, such as flow cytometry, have been investigated over the last two decades. To improve the taxonomic resolution of particle analysis, recent developments have focused on "imaging-in-flow," i.e., the ability to acquire microscopic images of planktonic cells in a flow-through mode. Imaging-in-flow systems are traditionally based on classical brightfield microscopy and are faced with a number of issues that decrease the classification performance and accuracy (e. g., projection variance of cells, migration of cells out of the focus plane). Here, we demonstrate that a combination of digital holographic microscopy (DHM) with imaging-in-flow can improve the detection and classification of planktonic organisms. In addition to light intensity information, DHM provides quantitative phase information, which generates an additional and independent set of features that can be used in classification algorithms. Moreover, the capability of digitally refocusing greatly increases the depth of field, enables a more accurate focusing of cells, and reduces the effects of position variance. Nanoplanktonic organisms similar in shape were successfully classified from images captured with an off-axis DHM with partial coherence. Textural features based on DHM phase information proved more efficient in separating the three tested phytoplankton species compared with shape-based features or textural features based on light intensity. An overall classification score of 92.4% demonstrates the potential of holographic-based imaging-in-flow for similar looking organisms in the nanoplankton range

3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology

A new photobioreactor for continuous microalgal production in hatcheries based on external-loop airlift and swirling flow

This study deals with the scale of a new photobioreactor for continuous microalgal production in hatcheries. The combination of the state-of-art with the constraints inherent to hatcheries has turned the design into a closed, artificially illuminated and external-loop airlift configuration based on a succession of elementary modules, each one being composed of two transparent vertical interconnected columns. The liquid circulation is ensured pneumatically (air injections) with respect to a swirling motion (tangential inlets). A single module of the whole photobioreactor was built-up to investigate how parameters, such as air sparger type, gas flow rate, tangential inlet, column radius and height can influence radiative transfer, hydrodynamics, mass transfer and biological performances. The volumetric productivities were predicted by modeling radiative transfer and growth of Isochrysis affinis galbana (clone Tahiti). The hydrodynamics of the liquid phase was modeled in terms of global flow behavior (circulation and mixing times, Péclet number) and of swirling motion decay along the column (Particle Image Velocimetry). The aeration performances were determined by overall volumetric mass transfer measurements. Continuous cultures of Isochrysis affinis galbana (clone Tahiti) were run in two geometrical configurations, generating either an axial or a swirling flow. Lastly, the definitive options of design are presented as well as a 120 Liter prototype, currently implemented in a French mollusk hatchery and commercialized

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 355)

This bibliography lists 147 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during October, 1991. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

Aerospace Medicine and Biology: A continuing bibliography with indexes (supplement 274)

This bibliography lists 128 reports, articles, and other documents introduced into the NASA scientific and technical information system in July 1985

In Situ Determination of the Effects of Lead and Copper on Cyanobacterial Populations in Microcosms

BACKGROUND: Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth. METHODOLOGY/PRINCIPAL FINDINGS: Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine in situ the effect of Pb and Cu stress in cyanobacterial populations. CONCLUSIONS/SIGNIFICANCE: The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and Microcoleus sp. (the dominant filamentous cyanobacterium in microbial mats) was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants

On-Line Monitoring of Biological Parameters in Microalgal Bioprocesses Using Optical Methods

Microalgae are promising sources of fuels and other chemicals. To operate microalgal cultivations efficiently, process control based on monitoring of process variables is needed. On-line sensing has important advantages over off-line and other analytical and sensing methods in minimizing the measurement delay. Consequently, on-line, in-situ sensors are preferred. In this respect, optical sensors occupy a central position since they are versatile and readily implemented in an on-line format. In biotechnological processes, measurements are performed in three phases (gaseous, liquid and solid (biomass)), and monitored process variables can be classified as physical, chemical and biological. On-line sensing technologies that rely on standard industrial sensors employed in chemical processes are already well-established for monitoring the physical and chemical environment of an algal cultivation. In contrast, on-line sensors for the process variables of the biological phase, whether biomass, intracellular or extracellular products, or the physiological state of living cells, are at an earlier developmental stage and are the focus of this review. On-line monitoring of biological process variables is much more difficult and sometimes impossible and must rely on indirect measurement and extensive data processing. In contrast to other recent reviews, this review concentrates on current methods and technologies for monitoring of biological parameters in microalgal cultivations that are suitable for the on-line and in-situ implementation. These parameters include cell concentration, chlorophyll content, irradiance, and lipid and pigment concentration and are measured using NMR, IR spectrophotometry, dielectric scattering, and multispectral methods. An important part of the review is the computer-aided monitoring of microalgal cultivations in the form of software sensors, the use of multi-parameter measurements in mathematical process models, fuzzy logic and artificial neural networks. In the future, software sensors will play an increasing role in the real-time estimation of biological variables because of their flexibility and extendibility

A quick and effective estimation of algal density by turbidimetry developed with Chlorella vulgaris cultures

El uso de Chlorella vulgaris Beijerinck como alimento para el zooplancton implica la necesidad de optimizar el cultivo de algas para mantener su crecimiento en el tiempo. En este trabajo se desarrolló un método que relaciona la densidad del cultivo con la turbidez para estimar la biomasa algal. Esta técnica se ha mejorado mediante la aplicación del análisis digital para el recuento de las algas que promueve la exactitud y reduce el disturbio en el cultivo, con obtención rápida y fácil de resultados repetibles. Se realizaron dos cultivos de C. vulgaris en recipientes de 3 L con aireación e iluminación continua (50 µmol fotones m–2 ·s–1 a 660 nm), alcanzando 214 y 280 NTU, respectivamente. El recuento de las muestras se realizó por medio de imágenes digitales tomadas con un microscopio invertido. Se aplicaron dos técnicas para el recuento de las alícuotas: la sedimentación directa y la sedimentación con homogeneización previa. Con el fin de comparar el ajuste de ambos métodos de sedimentación, las fotografías fueron tomadas en los sectores central, medio y periférico de la cámara de sedimentación. Para ambas técnicas se contaron un mínimo de 17 individuos imagen–1 y un máximo de 404 individuos imagen–1, con un tiempo promedio de un minuto por imagen. A niveles bajos de turbidez (< 40 NTU) la dispersión de los datos fue similar entre ambas técnicas (rango error: 16-60 %). Para niveles superiores de turbidez, en la técnica de sedimentación directa se observó un mayor rango de error (31-50 %) mientras que en la técnica con homogenización previa osciló entre 5 y 13 %. El análisis de regresión evidenció un bajo ajuste de los datos (67 %), que en la sedimentación sin homogeneización responde a un patrón reiterado de aumento de densidad algal desde la periferia hacia el centro de la cámara de sedimentación. La inclusión de una homogeneización previa promueve un mejor ajuste del modelo (99 %) y determina un incremento en la consistencia del método. Con los resultados obtenidos se demuestra que la técnica turbidimétrica desarrollada puede ser utilizada con éxito en cultivos de especies de algas cuyas formas geométricas sean reconocidas por el procesador de imágenes.The use of Chlorella vulgaris Beijerinck as a food source for zooplankton requires the optimization of algal-culture conditions for prolonged growth maintenance. In this study, we developed a method that relates algal density to culture turbidity to estimate culture biomass. This method was improved by applying digital analysis for algal counting, which promotes accuracy, low culture disturbance, easy repetition and the rapid acquisition of results. Two 3-L cultures of C. vulgaris, maintained for two weeks with continuous lighting (eight light-emitting diodes at 50 µmol photons m–2 ·s–1, at 660 nm) and aerators to prevent algal sedimentation, reached turbidities of 214 and 280 NTUs, respectively. Sample counting was performed using digital images obtained with an inverted microscope. Aliquot sedimentation was compared with or without previous homogenization through photographs taken in the central, middle, and peripheral sectors of the Utermöhl settling chambers. For each procedure, we counted between 17 and 404 individuals image–1, requiring, on average, one minute image–1. At low turbidity (< 40 NTU), the data dispersion was similar between the two protocols (error range, 16 to 60 %); at higher turbidity, the direct sedimentation alone gave a larger error (31-50 %) than with prior homogenization (5-13 %). Regression analysis at low data fit (67 %) suggested that the sedimentation heterogeneity of non-homogenized samples corresponded to a pattern of settled algae having increasing density from the periphery to the centre of the chamber, but with homogenization, a better model fitting (99 %) resulted, contributing to greater consistency with that procedure. We consider that this turbidometric protocol could be used successfully with cultures of algae that have geometrical shapes recognizable by the image software.Fil: Ferrando, Noelia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Benitez, Hernan Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Gabellone, Nestor Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Claps, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Altamirano, Pablo Rodrigo. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentin