Automated Microscopy: Macro Language Controlling a Confocal Microscope and its External Illumination: Adaptation for Photosynthetic Organisms

Photosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (through Time Controller offered by Olympus or Experiment Designer offered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with the Cell plus sign in circleFinder software was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) the Cell plus sign in circleFinder software with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser. Cell plus sign in circleFinder can be downloaded from http://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity in Synechocystis sp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light

Effects of bark beetle disturbance on soil nutrient retention and lake chemistry in glacial catchment

Forest ecosystems worldwide are subjected to human-induced stressors, including eutrophication and acidification, and to natural disturbances (for example, insect infestation, windstorms, fires). The occurrence of the later is expected to increase due to the ongoing climate change. These multi-stressor forcings modify ecosystem biogeochemistry, including the retention of limiting nutrients, with implications for terrestrial and aquatic biodiversity. Here we present whole ecosystem nutrient (N, Ca, Mg, K) mass balances in the forested catchment of Plešné Lake, CZ, which has undergone transient changes linked to the recovery from anthropogenic acidification and to the forest disturbances caused by severe infestations by the bark beetle (Ips typographus). Measured fluxes and storage of nutrients in the lake-catchment ecosystem were used to constrain the processoriented biogeochemical model MAGIC (Model of Acidification of Groundwater In Catchments). Simulated lake water chemistry and changes in soil nutrient pools fitted observed data and revealed that (1) the ecosystem N retention declined, thus nitrate leaching increased for 10 years following the bark beetle disturbance, with transient adverse effects on the acid–base status of lake water, (2) the kinetics of nutrient mineralisation from decaying biomass coupled with nutrient immobilisation in regrowing vegetation constrained the magnitude and duration of ecosystem losses of N, Ca and Mg, (3) the excess of mineralised base cations from decomposing biomass replenished the soil cation exchange matrix, which led to increased soil base saturation, and (4) the improvement of the catchment soil acid–base status led to an increase of lake water pH and acid neutralising capacity. Forested ecosystems underlain by nutrient-poor soils and bedrock are prone to human-induced damages caused by acidification and eutrophication, and any natural disturbance may further lead to nutrient imbalances. We demonstrated that in this natural forest ecosystem protected from human intervention, disturbances together with natural post-disturbance vegetation recovery have temporally positive effects on the nutrient stores in the soil

Photosynthesis in Chromera velia Represents a Simple System with High Efficiency

Chromera velia (Alveolata) is a close relative to apicomplexan parasites with a functional photosynthetic plastid. Even though C. velia has a primitive complement of pigments (lacks chlorophyll c) and uses an ancient type II form of RuBISCO, we found that its photosynthesis is very efficient with the ability to acclimate to a wide range of irradiances. C. velia maintain similar maximal photosynthetic rates when grown under continual light-limited (low light) or light-saturated (high light) conditions. This flexible acclimation to continuous light is provided by an increase of the chlorophyll content and photosystem II connectivity under light limited conditions and by an increase in the content of protective carotenoids together with stimulation of effective non-photochemical quenching under high light. C. velia is able to significantly increase photosynthetic rates when grown under a light-dark cycle with sinusoidal changes in light intensity. Photosynthetic activities were nonlinearly related to light intensity, with maximum performance measured at mid-morning. C. velia efficiently acclimates to changing irradiance by stimulation of photorespiration and non-photochemical quenching, thus avoiding any measurable photoinhibition. We suggest that the very high CO(2) assimilation rates under sinusoidal light regime are allowed by activation of the oxygen consuming process (possibly chlororespiration) that maintains high efficiency of RuBISCO (type II). Despite the overall simplicity of the C. velia photosynthetic system, it operates with great efficiency

Non-Photochemical Quenching in Cryptophyte Alga Rhodomonas salina Is Located in Chlorophyll a/c Antennae

Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates – e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching

On estimation of diffusion coefficient based on spatio-temporal FRAP images: An inverse ill-posed problem

This contribution contains a description and comparison of two methods applied to exposure optimization applied to moulding process in the automotive industry

On estimation of diffusion coefficient based on spatio-temporal FRAP images: An inverse ill-posed problem

summary:We present the method for determination of phycobilisomes diffusivity (diffusion coefficient $D$) on thylakoid membrane from fluorescence recovery after photobleaching (FRAP) experiments. This was usually done by analytical models consisting mainly of a simple curve fitting procedure. However, analytical models need some unrealistic conditions to be supposed. Our method, based on finite difference approximation of the process governed by the Fickian diffusion equation and on the minimization of an objective function representing the disparity between the measured and simulated time-varying fluorescent particles concentration profiles, naturally accounts for experimentally measured time-varying fluorescent particles concentration profiles, naturally accounts for experimentally measured time-varying Dirichlet boundary conditions and can include a reaction term as well. The result we get is the overall (time averaged) diffusion coefficient $D$ and the sequence of diffusivities $D_j$ based on two successive fluorescence profiles in $j-th$ time interval. Due to the ill-posedness of our inverse problem, regularization algorithms are implemented. On the synthetic example, we illustrate the behaviour of solution depending on regularization parameter for different signal to noise ratio