Chemical Fixation Methods Research Articles

Streamlined chemical fixation method for morphological investigation of Candida albicans with scanning electron microscopy

This method describes the preparation of Candida albicans specimen for scanning electron microscopy (SEM), starting from specimen collection to obtaining SEM images. Our approach involves preserving cellular structure and preventing cell degradation through chemical fixation and post-fixation without Osmium tetroxide (OsO4), followed by air dried specimen placed onto Si substrate. Additionally, the method outlines the steps for mounting the prepared specimen onto SEM sample holder aluminum stubs, followed by coating with an ultrathin gold layer via thermal evaporation to enhance specimen/substrate/stub conductivity. The specimens are then mounted in the SEM sample holder. This step-by-step procedure demonstrates successful morphological analysis of C. albicans, providing a comprehensive and effective specimen preparation for SEM analysis.•Effective preparation of Candida albicans specimens for SEM analysis, preserving cellular structure.•Step-by-step specimen mounting and gold coating for enhanced detailed SEM imaging.•Chemical fixation of yeast cells for electron microscopy visualization.

Chemical Fixation of Carbon Dioxide into Cyclic Carbonates Catalyzed by Porous Materials

AbstractThe reaction of carbon dioxide (CO2) with epoxide to generate cyclic carbonates is one of the most effective chemical fixation methods. The catalytic system significantly affects the carbon fixation efficiency of the reaction. Among the reported catalytic systems, porous materials as heterogeneous catalysts have attracted significant attention. This study aimed to summarize porous materials that catalyzed the conversion of CO2 into cyclic carbonates, including metal organic frameworks, covalent organic frameworks, poly(ionic) liquids, hyper crosslinked polymers, conjugated porous polymers and others. The study analyzed the catalytic properties of these materials and outlined future research directions.

Glutaraldehyde and uranyl acetate dual fixation combined sputtering/unroofing enables intracellular fatty acids TOF-SIMS imaging with organelle-corresponding subcellular distribution.

Fatty acids (FAs) have diverse functions in cellular activities. The intracellular distribution of FAs is critical for their functions. Imaging of FAs by time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been achieved. However, TOF-SIMS images of FAs so far do not have subcellular distribution due to inadequate sample preparation methods. In this study, we developed a chemical fixation method using glutaraldehyde (GA) with uranyl acetate (UA), which preserved cellular structure and intracellular FA distribution well. Combining GA+UA fixation with sputtering-based methods and unroofing-based methods, respectively, we successfully imaged intracellular lipids with the subcellular distribution.

Tree species-based differences vs. decay performance and mechanical properties following chemical and thermal treatments

Many thermal and chemical treatments are known to inhibit wood decay despite the wood grade processed, but their impact, e.g., chemicals’ leaching and decay resistance, may not be similar. The aim of this study was to test whether some model treatments retain their performance in different wood species. Additionally, the effects of thermal modification and linseed oil-based varnish treatments as means to mitigate water-soluble chemicals leaching were assessed. The mass loss caused by Trametes versicolor was measured after a 12-week exposure to analyze whether the different treatment approaches prevented the fungal decay after a standard leaching test. The mechanical properties before and after exposure were tested independently to determine whether the mechanical properties of different wood species were affected by the tested treatments and wood decay. The responses of the tested wood species were found to vary by treatments, but thermal and chemical fixation methods for water-soluble tannins were beneficial in all cases considering the mass loss and the degradation of modulus of rupture and modulus of elasticity of treated wood. Varnish was overall the most effective treatment against decay, but the results emphasize the need for testing potential preservation methods and chemicals on several species.

Intercellular nuclear migration in cryofixed tobacco male meiocytes.

In this study, intercellular nuclear migration (INM), also known as cytomixis, was documented in cryofixed plant meiocytes for the first time. Intact tobacco inflorescences and flower buds as well as dissected individual anthers were cryofixed in liquid nitrogen by plunge freezing. Cryosubstituted and cryosectioned male meiocytes were analyzed by light microscopy. For cryosubstitution, the frozen material was kept in acetic alcohol at - 70°C for 1week. For cryosectioning, the frozen material was sectioned at - 20°C, and fixed with precooled acetic alcohol. Fixation of the intact tobacco inflorescences in Carnoy's solution was used as a control. Microscopy revealed good preservation of cell structure in the cryofixed anthers, flower buds, and inflorescences. INM was detectable in all the studied cryofixed and chemically fixed samples. The cytological picture of INM observed in the cryofixed meiocytes did not noticeably differ from the picture obtained with the chemically fixed cells. These results indicate that INM is observable irrespective of whether a physical or chemical fixation method is employed, with minimal damage from handling. Our results contradict the notion that INM is a phenomenon caused by mechanical, osmotic, or chemical artifacts during sample preparation.

Natural rubber‐carbon black coagulation: Following the nanostructure evolution from a colloidal suspension to a composite

AbstractMaking elastomeric composite materials via heteroaggregation of a binary colloidal suspension of Natural Rubber (NR) latex and Carbon Black (CB) filler is an interesting production method to obtain an efficient dispersion in the polymer matrix. This study successfully employs an original approach of field emission scanning electron microscopy (FESEM) to investigate for the first time the nanostructure evolution of a coagulum originated from the aggregation of NR globules with CB filler in suspension. More specifically, we exploited a chemical fixation method allowing simultaneous acquisition of backscattered electron (BSE) and secondary electron (SE) imaging modes. Additionally, the role of external physical stresses, like mechanical shear and sonication was also investigated in terms of structural effect induced on the formed coagulum at the nanoscopic scale. Our results highlight destabilization of NR globules, either induced by direct interaction with small CB aggregates or governed by solvent evaporation. Reduction in the size of CB agglomerates, obtained using sonication, highly improved filler distribution and confirmed that the size of CB aggregates is an important parameter responsible for the destabilization of NR globules.

Assessment of the Effect of Secondary Fixation on the Structure of Meat Products Prepared for Scanning Electron Microscopy.

The aim of the research was to verify the necessity of secondary fixation with osmium tetroxide in various types of meat products and evaluation of structural changes of products using different fixation procedures. The material for the study consisted of 11 types of meat products that were analyzed using a scanning electron microscope (SEM) with two different methods of chemical fixation. The first method included the usual processing of biological samples: glutaraldehyde primary fixation, the use of a buffer, secondary fixation by osmium tetroxide (OsO4), buffer, and dehydration using ethanol of increasing concentrations. The second method comprised the glutaraldehyde primary fixation and dehydration using the ethanol of increasing concentrations only. The results unambiguously suggest that the main difference between these methods is in fixation and visibility of fat. Our analysis principally suggests that fixation of the product with OsO4 allows the tracking of all components (fat droplets, muscle fibers, connective tissue) in meat products. At the same time, our results also support the possibility that the secondary fixation can be skipped during the analysis, where the main objection is an observation of lipid-free structures of the meat products (e.g., connection between muscle and starches or spices) or meat products with an insignificant amount of fat.

The subcellular distribution of the human Fic protein, HYPE/FicD, using a high resolution, tomography compatible electron microscopic method

Localization of membrane proteins via electron microscopy (EM) at high resolution is dependent on a robust detection technology and a sample preparation method that confers superior ultrastructural preservation of membranes. Current methods for precise localization rely on chemical fixation of the sample followed by alcohol dehydration at room temperature. The combination of these traditional methods exhibit poor preservation of membrane, induces membrane artifacts and affects resolution. High pressure freezing (HPF) of cells followed by heavy metal staining via freeze substitution (FS) in acetone at sub‐zero temperatures are techniques that exhibit the best possible membrane preservation. Therefore, we have developed a hybrid method that combines high pressure freezing with an existing peroxide based detection technique typically used with chemical fixation methods. As proof of concept, we assessed the localization of a fusion construct, consisting of the N‐terminus of HMG CO‐A protein fused with a peroxidase tag (that faces the cytosol). We exploited the induction of OSERs (organized smooth ER) in cells by the over‐expression of this N‐terminal fragment, as a metric for specific staining and membrane preservation. We next extended our studies to assess the localization of the sole human Fic (filament induced by cyclic AMP) protein, HYPE. We previously showed that HYPE resides in the ER lumen, but various global transcriptomic and proteomic studies suggest the existence of cytosolic and extracellular substrates for HYPE. Using our hybrid methodology, here we provide unequivocal evidence that the HYPE is compartmentalized within the various subdomains of the ER. We further demonstrate via serial sectioning, that our technique enables the localization of HYPE through a large volume of the cell. Our work thus aligns well with the three‐dimensional mapping of membrane proteins in large organelles using EM‐tomography in a near‐native cellular context.Support or Funding InformationThis work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Project Development Team Award and 2) the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effects of chemical fixation on the cellular nanostructure

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.

Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development.

The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes. The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as 'amorphous,' lacking in ultrastructural features, and is often observed as a thin (~80-100nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.

Apoplasmic loading in the rice phloem supported by the presence of sucrose synthase and plasma membrane-localized proton pyrophosphatase.

Although Oryza sativa (rice) is one of the most important cereal crops, the mechanism by which sucrose, the major photosynthate, is loaded into its phloem is still a matter of debate. Current opinion holds that the phloem loading pathway in rice could involve either a symplasmic or an apoplasmic route. It was hypothesized, on the basis of a complementary body of evidence from arabidopsis, which is an apoplasmic loader, that the membrane specificity of proton pyrophosphatases (H(+)-PPases; OVPs) in the sieve element-companion cell (SE-CC) complexes of rice source leaves would support the existence of either of the aforementioned phloem loading mechanisms. Additionally, it was contended that the presence of sucrose synthase in the SE-CC complexes would be consistent with an apoplasmic sucrose loading route in rice. Conventional chemical fixation methods were used for immunohistochemical localization of H(+)-PPases and sucrose synthase in rice and arabidopsis at the light microscopy level, while ultrastructural immunogold labelling of H(+)-PPases and sucrose synthase was performed on high-pressure frozen source leaves of rice. Using immunogold labelling, it was found that OVPs predominantly localize at the plasma membrane (PM) of the SE-CC complexes in rice source leaf minor veins, while in the root meristematic cells, OVPs preferentially localize at the vacuoles. The PM specificity of OPVs in the SE-CC complexes was deemed to support apoplasmic loading in the rice phloem. Further backing for this interpretation came from the sucrose synthase-specific immunogold labelling at the SE-CC complexes of rice source leaves. These findings are consistent with the idea that, in the same way as in arabidopsis and a majority of grasses, sucrose is actively loaded into the SE-CC complexes of rice leaves using an apoplasmic step.

<italic>In situ</italic> catalytic hydrogenation of carbon dioxide

Carbon capture and sequestration (CCS) is being explored as potential methods for mitigating CO2 accumulation. However, the high energy input in desorption/compression processes is a barrier. As a nontoxic, abundant, renewable C1 building block, CO2 can be used as an alternative carbonylating reagent to replace phosgene and carbon monoxide in organic synthesis and the chemical industry. The strategy of turning waste gas CO2 into useful compounds and the use of CO2 in the production of high-value-added compounds are important topics, especially the reduction of CO2 to energy products. We describe a strategy involving a combination of CO2 capture and utilization, in which the captured CO2 is considered as the substrate for further in situ catalytic transformations. This process circumvents the desorption and compression steps, and solves the energy penalty problem in CCS. It also avoids the use of high-pressure CO2, which poses safety concerns and is not economically viable. CO2 molecules can also be activated upon adsorption, which is favorable for subsequent in situ conversion under mild conditions. Of the many possible pathways for CO2 use, in situ catalytic hydrogenation is important, and has a wide range of potential application. The CO2 captured as carbamate or carbonic salts can be in situ hydrogenated to formic acid, methanol, and other important energy products. In this review, CO2 adsorption and chemical fixation methods are briefly introduced, and the catalytic systems for CO2 hydrogenation are systematically summarized. In particular, the latest progress on in situ catalytic hydrogenation and mechanistic understanding at the molecular level is discussed in detail.

High-Pressure Freeze and Freeze Substitution Electron Microscopy in C. elegans.

While traditional chemical fixation methods for C. elegans electron microscopy (EM) have provided invaluable anatomical and structural information, the development of high-pressure freeze (HPF) and freeze substitution (FS) protocols offers advantages for high-resolution imaging. Specimens prepared using HPF methodology exhibit fewer distortion artifacts due to fixation and dehydration, have improved antigenicity, and result in a more physiologically accurate structural representation of the worm. In the HPF technique, freely moving worms are frozen at high-pressure (2100 bar) and low temperature (-180 °C) within milliseconds. These conditions prevent the formation of ice crystals that can damage cellular structures. Samples then undergo FS, during which worms are slowly brought to room temperature while substituting amorphous ice with organic solvents to preserve tissue in its near native state and provide contrast for imaging. FS can be performed in an automatic freeze substitution (AFS) machine or in makeshift, temperature controlled chambers. Fixed worms can be embedded in plastic resin and further processed for a variety of imaging techniques. Samples then viewed using scanning (SEM) or transmission electron microscopy (TEM) will show enhanced preservation of organelles, cell morphology, and antigenicity for immunocytochemistry.

Chemical fixation methods for Raman spectroscopy-based analysis of bacteria

Preservation of biological samples for downstream analysis is important for analytical methods that measure the biochemical composition of a sample. One such method, Raman microspectroscopy, is commonly used as a rapid phenotypic technique to measure biomolecular composition for the purposes of identification and discrimination of species and strains of bacteria, as well as investigating physiological responses to external stressors and the uptake of stable isotope-labelled substrates in single cells. This study examines the influence of a number of common chemical fixation and inactivation methods on the Raman spectrum of six species of bacteria. Modifications to the Raman-phenotype caused by fixation were compared to unfixed control samples using difference spectra and Principal Components Analysis (PCA). Additionally, the effect of fixation on the ability to accurately classify bacterial species using their Raman phenotype was determined. The results showed that common fixatives such as glutaraldehyde and ethanol cause significant changes to the Raman spectra of bacteria, whereas formaldehyde and sodium azide were better at preserving spectral features.

Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: A multidimensional view by means of correlative light and 3-D transmission electron microscopy

Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines.

Cellular organization in germ tube tips of Gigaspora and its phylogenetic implications

Comparative morphology of the fine structure of fungal hyphal tips often is phylogenetically informative. In particular, morphology of the Spitzenkörper varies among higher taxa. To date no one has thoroughly characterized the hyphal tips of members of the phylum Glomeromycota to compare them with other fungi. This is partly due to difficulty growing and manipulating living hyphae of these obligate symbionts. We observed growing germ tubes of Gigaspora gigantea, G. margarita and G. rosea with a combination of light microscopy (LM) and transmission electron microscopy (TEM). For TEM, we used both traditional chemical fixation and cryo-fixation methods. Germ tubes of all species were extremely sensitive to manipulation. Healthy germ tubes often showed rapid bidirectional cytoplasmic streaming, whereas germ tubes that had been disturbed showed reduced or no cytoplasmic movement. Actively growing germ tubes contain a cluster of 10–20 spherical bodies approximately 3–8 μm behind the apex. The bodies, which we hypothesize are lipid bodies, move rapidly in healthy germ tubes. These bodies disappear immediately after any cellular perturbation. Cells prepared with cryo-techniques had superior preservation compared to those that had been processed with traditional chemical protocols. For example, cryo-prepared samples displayed two cell-wall layers, at least three vesicle types near the tip and three distinct cytoplasmic zones were noted. We did not detect a Spitzenkörper with either LM or TEM techniques and the tip organization of Gigaspora germ tubes appeared to be similar to hyphae in zygomycetous fungi. This observation was supported by a phylogenetic analysis of microscopic characters of hyphal tips from members of five fungal phyla. Our work emphasizes the sensitive nature of cellular organization, and the need for as little manipulation as possible to observe germ tube structure accurately.

A quantitative, biomechanical analysis of radioulnar deviation between fresh frozen and soft embalmed human forearms

Fresh frozen human cadaveric specimens are considered the gold standard for biomechanical research; however, the testing period of these specimens is limited because they decay rapidly, can be difficult to obtain and can carry infectious diseases. Therefore, various chemical fixation methods to deal with these issues have been developed. Traditional formalin fixation has been shown to significantly affect the biomechanical properties of the tissues; however, a relatively new method called soft embalming could provide an alternative to fresh frozen specimens in the field of biomechanical research. The purpose of this pilot study was to determine the effects soft embalming has on gross anatomical movements. The parameters for testing were radioulnar deviation range of motion with in plane and out of plane angulation measured using an incremented tendon loading protocol. Five fresh frozen human cadaveric forearms were obtained and injected with tantalum tracer beads and tested pre and post embalming using 3D X‐rays. The results showed that the soft embalmed specimens retained their flexibility and was slightly greater than the fresh frozen state. Although, at lighter loading trials, the range of motion in soft embalmed specimens was larger than the fresh frozen state. The results here may provide an argument for the inclusion of soft embalmed specimens in biomechanical testing.

Optimal preservation of the shark retina for ultrastructural analysis: An assessment of chemical, microwave, and high‐pressure freezing fixation techniques

Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum, obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent.

Deformation behavior of the iliotibial tract under different states of fixation

Background and objectiveThe iliotibial tract (tract) is an important structure for the biomechanics of both the hip and knee joint. While a detailed characterization of its mechanical properties might help to better understand its specific role in the load transfer from the pelvis to femur and tibia, determination of those properties is complicated by its particular structure of thin fibers in the fresh state. Moreover, although the tracts mechanical properties are often derived from cadaveric material chemically fixed with either ethanol or formaldehyde, the influence of such fixation methods remains to be elucidated. Aim of this study was to determine Young's modulus (tensile modulus, YM) of the tract. We hypothesized that either ethanol or formaldehyde fixation would significantly increase the YM compared to the tracts condition in a fresh state. Material and methods13 specimens of tract were gained from donators. The ends of the probes were plastinated with resin creating a sharp interface between the clamp and the probe to prevent material slippage. The specimens were measured in their fresh state, under ethanol- and formaldehyde-fixed conditions and re-measured after rinsing with tap water. ResultsThe YM of the fresh probes averaged 397.3N/mm2 with a standard deviation (SD) of 151.5N/mm2. The YM of the ethanol-fixed specimens was significantly higher (673.2N/mm2, SD 328.5N/mm2, p<0.05). After rinsing with tap water, the YM decreased to 95% of the fresh condition value (377.4N/mm2, SD 144.5N/mm2, non-significant change from fresh). After formaldehyde fixation, the YM reached 490.3N/mm2 (SD 143.0N/mm2, p<0.05). When the formaldehyde-fixed specimens were rinsed, the YM was 114% of the value of the fresh condition (452.6N/mm2, SD 115.1N/mm2, non-significant change from fresh). ConclusionsThis study found a significant influence of the chemical fixation method on the YM of the IT tract. If such fixation is required, our results suggest using a treatment with ethanol and subsequent rinsing that results in minimal changes to the tracts YM. Furthermore, plastination of the ends of the specimens could be crucial to allow in vitro determination of valid YM of ligaments data that can then be integrated with confidence in further finite element analyses.

Ultrastructural observation of natural field phytoplankton cells by using rapid freezing and freeze substitution

Transmission electron microscopy (TEM) is a basic technique used to study the ultrastructures of phytoplankton. However, the use of chemical fixation to prepare phytoplankton samples for TEM is laborious and time-consuming. Furthermore, chemical fixation often causes artificial structural changes (artifacts) in phytoplankton cells. In this study, we induced and optimized a rapid freezing and freeze substitution (RFS) method, accompanied by a filter dehydration system, to prepare TEM samples of marine microalgae, including Heterosigma akashiwo, Chattonella antiqua (Raphidophyceae), Heterocapsa circularisquama (Dinophyceae), Chaetoceros tenuissimus (Bacillariophyceae), and Teleaulax amphioxeia (Cryptophyceae). The whole procedure was much more efficient than the traditional method. In addition, the fine structure of each microalgal cell was well preserved. For example, the fine structures of the nuclear membrane, nuclear pores, and several vesicles were clearly observed, which were comparatively difficult to observe using the chemical fixation method. In addition, this new technique was applicable to the natural blooming cells of H. akashiwo, and high-resolution ultrastructural images were successfully observed. This is the first report on the application of the modified-RFS method for natural phytoplankton assemblages. This method can be used for diverse phytoplankters in natural waters, and the high-quality observations are expected to provide useful information toward understanding phytoplankter physiology and ecology.