Label-Free, Noninvasive Bone Cell Classification by Hyperspectral Confocal Raman Microscopy.

Characterizing and identifying cells in multicellular in vitro models remain a substantial challenge. Here, we utilize hyperspectral confocal Raman microscopy and principal component analysis coupled with linear discriminant analysis to form a label-free, noninvasive approach for classifying bone cells and osteosarcoma cells. Through the development of a library of hyperspectral Raman images of the K7M2-wt osteosarcoma cell lines, 7F2 osteoblast cell lines, RAW 264.7 macrophage cell line, and osteoclasts induced from RAW 264.7 macrophages, we built a linear discriminant model capable of correctly identifying each of these cell types. The model was cross-validated using a k-fold cross validation scheme. The results show a minimum of 72% accuracy in predicting cell type. We also utilize the model to reconstruct the spectra of K7M2 and 7F2 to determine whether osteosarcoma cancer cells and normal osteoblasts have any prominent differences that can be captured by Raman. We find that the main differences between these two cell types are the prominence of the ß-sheet protein secondary structure in K7M2 versus the α-helix protein secondary structure in 7F2. Additionally, differences in the CH2 deformation Raman feature highlight that the membrane lipid structure is different between these cells, which may affect the overall signaling and functional contrasts. Overall, we show that hyperspectral confocal Raman microscopy can serve as an effective tool for label-free, nondestructive cellular classification and that the spectral reconstructions can be used to gain deeper insight into the differences that drive different functional outcomes of different cells.

Singleplex, multiplex and pooled sample real-time RT-PCR assays for detection of SARS-CoV-2 in an occupational medicine setting.

For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24-48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid 'multiplex-pooled' strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics.

Gene editing and CRISPR in the clinic: current and future perspectives.

Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data.

CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays-even isolated arrays-from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.

Imaging effectiveness calculator for non-design microscope samples.

When attempting to integrate single-molecule fluorescence microscopy with microfabricated devices such as microfluidic channels, fabrication constraints may prevent using traditional coverslips. Instead, the fabricated devices may require imaging through material with a different thickness or index of refraction. Altering either can easily reduce the quality of the image formation (measured by the Strehl ratio) by a factor of 2 or more, reducing the signal-to-noise ratio accordingly. In such cases, successful detection of single-molecule fluorescence may prove difficult or impossible. Here we provide software to calculate the effect of non-design materials upon the Strehl ratio or ensquared energy and explore the impact of common materials used in microfabrication.

Internalization and accumulation of model lignin breakdown products in bacteria and fungi.

BACKGROUND: Valorization of lignin has the potential to significantly improve the economics of lignocellulosic biorefineries. However, its complex structure makes conversion to useful products elusive. One promising approach is depolymerization of lignin and subsequent bioconversion of breakdown products into value-added compounds. Optimizing transport of these depolymerization products into one or more organism(s) for biological conversion is important to maximize carbon utilization and minimize toxicity. Current methods assess internalization of depolymerization products indirectly-for example, growth on, or toxicity of, a substrate. Furthermore, no method has been shown to provide visualization of depolymerization products in individual cells. RESULTS: We applied mass spectrometry to provide direct measurements of relative internalized concentrations of several lignin depolymerization compounds and single-cell microscopy methods to visualize cell-to-cell differences in internalized amounts of two lignin depolymerization compounds. We characterized internalization of 4-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, and the model dimer guaiacylglycerol-beta-guaiacyl ether (GGE) in the lignolytic organisms Phanerochaete chrysosporium and Enterobacter lignolyticus and in the non-lignolytic but genetically tractable organisms Saccharomyces cerevisiae and Escherichia coli. The results show varying degrees of internalization in all organisms for all the tested compounds, including the model dimer, GGE. Phanerochaete chrysosporium internalizes all compounds in non-lignolytic and lignolytic conditions at comparable levels, indicating that the transporters for these compounds are not specific to the lignolytic secondary metabolic system. Single-cell microscopy shows that internalization of vanillic acid and 4-hydroxybenzoic acid analogs varies greatly among individual fungal and bacterial cells in a given population. Glucose starvation and chemical inhibition of ATP hydrolysis during internalization significantly reduced the internalized amount of vanillic acid in bacteria. CONCLUSIONS: Mass spectrometry and single-cell microscopy methods were developed to establish a toolset for providing direct measurement and visualization of relative internal concentrations of mono- and di-aryl compounds in microbes. Utilizing these methods, we observed broad variation in intracellular concentration between organisms and within populations and this may have important consequences for the efficiency and productivity of an industrial process for bioconversion. Subsequent application of this toolset will be useful in identifying and characterizing specific transporters for lignin-derived mono- and di-aryl compounds.

Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

Cellular localization of tolyporphins, unusual tetrapyrroles, in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy.

The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole macrocycles known as tolyporphins. Almost all known tolyporphins (A-M except K) contain a dioxobacteriochlorin chromophore and exhibit an absorption spectrum somewhat similar to that of chlorophyll a. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under various growth conditions (media, illumination, culture age). Cultures grown in nitrate-depleted media (BG-110 vs. nitrate-rich, BG-11) are known to increase the production of tolyporphins by orders of magnitude (rivaling that of chlorophyll a) over a period of 30-45 days. Multivariate curve resolution (MCR) was applied to an image set containing images from each condition to obtain pure component spectra of the endogenous pigments. The relative abundances of these components were then calculated for individual pixels in each image in the entire set, and 3D-volume renderings were obtained. At 30 days in media with or without nitrate, the chlorophyll a and phycobilisomes (combined phycocyanin and phycobilin components) co-localize in the filament outer cytoplasmic region. Tolyporphins localize in a distinct peripheral pattern in cells grown in BG-110 versus a diffuse pattern (mimicking the chlorophyll a localization) upon growth in BG-11. In BG-110, distinct puncta of tolyporphins were commonly found at the septa between cells and at the end of filaments. This work quantifies the relative abundance and envelope localization of tolyporphins in single cells, and illustrates the ability to identify novel tetrapyrroles in the presence of chlorophyll a in a photosynthetic microorganism within a non-axenic culture.

Delivering CRISPR: a review of the challenges and approaches.

Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.

Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll f.

Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. In this study, cyanobacteria containing chlorophyll f, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris, were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl f was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels.

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response to nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. We observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.

Multifunctional, Tunable Metal-Organic Framework Materials Platform for Bioimaging Applications.

Herein, we describe a novel multifunctional metal-organic framework (MOF) materials platform that displays both porosity and tunable emission properties as a function of the metal identity (Eu, Nd, and tuned compositions of Nd/Yb). Their emission collectively spans the deep red to near-infrared (NIR) spectral region (∼614-1350 nm), which is highly relevant for in vivo bioimaging. These new materials meet important prerequisites as relevant to biological processes: they are minimally toxic to living cells and retain structural integrity in water and phosphate-buffered saline. To assess their viability as optical bioimaging agents, we successfully synthesized the nanoscale Eu analog as a proof-of-concept system in this series. In vitro studies show that it is cell-permeable in individual RAW 264.7 mouse macrophage and HeLa human cervical cancer tissue culture cells. The efficient discrimination between the Eu emission and cell autofluorescence was achieved with hyperspectral confocal fluorescence microscopy, used here for the first time to characterize MOF materials. Importantly, this is the first report that documents the long-term conservation of the intrinsic emission in live cells of a fluorophore-based MOF to date (up to 48 h). This finding, in conjunction with the materials' very low toxicity, validates the biocompatibility in these systems and qualifies them as promising for use in long-term tracking and biodistribution studies.

Lateral Segregation of Photosystem I in Cyanobacterial Thylakoids.

Photosystem I (PSI) is the dominant photosystem in cyanobacteria and it plays a pivotal role in cyanobacterial metabolism. Despite its biological importance, the native organization of PSI in cyanobacterial thylakoid membranes is poorly understood. Here, we use atomic force microscopy (AFM) to show that ordered, extensive macromolecular arrays of PSI complexes are present in thylakoids from Thermosynechococcus elongatus, Synechococcus sp PCC 7002, and Synechocystis sp PCC 6803. Hyperspectral confocal fluorescence microscopy and three-dimensional structured illumination microscopy of Synechocystis sp PCC 6803 cells visualize PSI domains within the context of the complete thylakoid system. Crystallographic and AFM data were used to build a structural model of a membrane landscape comprising 96 PSI trimers and 27,648 chlorophyll a molecules. Rather than facilitating intertrimer energy transfer, the close associations between PSI primarily maximize packing efficiency; short-range interactions with Complex I and cytochrome b6f are excluded from these regions of the membrane, so PSI turnover is sustained by long-distance diffusion of the electron donors at the membrane surface. Elsewhere, PSI-photosystem II contact zones provide sites for docking phycobilisomes and the formation of megacomplexes. PSI-enriched domains in cyanobacteria might foreshadow the partitioning of PSI into stromal lamellae in plants, similarly sustained by long-distance diffusion of electron carriers.

Hyperspectral fluorescence microscopy detects autofluorescent factors that can be exploited as a diagnostic method for Candida species differentiation.

Fungi in the Candida genus are the most common fungal pathogens. They not only cause high morbidity and mortality but can also cost billions of dollars in healthcare. To alleviate this burden, early and accurate identification of Candida species is necessary. However, standard identification procedures can take days and have a large false negative error. The method described in this study takes advantage of hyperspectral confocal fluorescence microscopy, which enables the capability to quickly and accurately identify and characterize the unique autofluorescence spectra from different Candida species with up to 84% accuracy when grown in conditions that closely mimic physiological conditions.

Removing Cosmic Spikes Using a Hyperspectral Upper-Bound Spectrum Method.

Cosmic ray spikes are especially problematic for hyperspectral imaging because of the large number of spikes often present and their negative effects upon subsequent chemometric analysis. Fortunately, while the large number of spectra acquired in a hyperspectral imaging data set increases the probability and number of cosmic spikes observed, the multitude of spectra can also aid in the effective recognition and removal of the cosmic spikes. Zhang and Ben-Amotz were perhaps the first to leverage the additional spatial dimension of hyperspectral data matrices (DM). They integrated principal component analysis (PCA) into the upper bound spectrum method (UBS), resulting in a hybrid method (UBS-DM) for hyperspectral images. Here, we expand upon their use of PCA, recognizing that principal components primarily present in only a few pixels most likely correspond to cosmic spikes. Eliminating the contribution of those principal components in those pixels improves the cosmic spike removal. Both simulated and experimental hyperspectral Raman image data sets are used to test the newly developed UBS-DM-hyperspectral (UBS-DM-HS) method which extends the UBS-DM method by leveraging characteristics of hyperspectral data sets. A comparison is provided between the performance of the UBS-DM-HS method and other methods suitable for despiking hyperspectral images, evaluating both their ability to remove cosmic ray spikes and the extent to which they introduce spectral bias.

Dynamics and Interactions of Individual Proteins in the Membrane of Single, Living Cells.

Total internal reflection fluorescence (TIRF) microscopy is a powerful technique for interrogating protein dynamics in the membranes of living single cells. Receptor-ligand interactions are of particular interest for improving our understanding of cell signaling networks in a variety of applications. Here, we describe methods for fluorescently labeling individual receptors and their ligands, conducting single-molecule TIRF microscopy of receptors and ligands in single, living cells, and importantly, performing image analysis on the resulting time sequence of images to extract quantitative dynamics. While we use Toll-like receptor 4 and its ligand lipopolysaccharide as a specific example, the methods are general and readily extendable to other receptor-ligand systems of importance in cellular biology.

A complex carotenoid palette tunes avian colour vision.

The brilliantly coloured cone oil droplets of the avian retina function as long-pass cut-off filters that tune the spectral sensitivity of the photoreceptors and are hypothesized to enhance colour discrimination and improve colour constancy. Although it has long been known that these droplets are pigmented with carotenoids, their precise composition has remained uncertain owing to the technical challenges of measuring these very small, dense and highly refractile optical organelles. In this study, we integrated results from high-performance liquid chromatography, hyperspectral microscopy and microspectrophotometry to obtain a comprehensive understanding of oil droplet carotenoid pigmentation in the chicken (Gallus gallus). We find that each of the four carotenoid-containing droplet types consists of a complex mixture of carotenoids, with a single predominant carotenoid determining the wavelength of the spectral filtering cut-off. Consistent with previous reports, we find that the predominant carotenoid type in the oil droplets of long-wavelength-sensitive, medium-wavelength-sensitive and short-wavelength-sensitive type 2 cones are astaxanthin, zeaxanthin and galloxanthin, respectively. In addition, the oil droplet of the principal member of the double cone contains a mixture of galloxanthin and two hydroxycarotenoids (lutein and zeaxanthin). Short-wavelength-absorbing apocarotenoids are present in all of the droplet types, providing filtering of light in a region of the spectrum where filtering by hydroxy- and ketocarotenoids may be incomplete. Thus, birds rely on a complex palette of carotenoid pigments within their cone oil droplets to achieve finely tuned spectral filtering.

Spectroradiometric monitoring for open outdoor culturing of algae and cyanobacteria.

We assess the measurement of hyperspectral reflectance for outdoor monitoring of green algae and cyanobacteria cultures with a multichannel, fiber-coupled spectroradiometer. Reflectance data acquired over a 4-week period are interpreted via numerical inversion of a reflectance model, in which the above-water reflectance is expressed as a quadratic function of the single backscattering albedo, which is dependent on the absorption and backscatter coefficients. The absorption coefficient is treated as the sum of component spectra consisting of the cultured species (green algae or cyanobacteria), dissolved organic matter, and water (including the temperature dependence of the water absorption spectrum). The backscatter coefficient is approximated as the scaled Hilbert transform of the culture absorption spectrum with a wavelength-independent vertical offset. Additional terms in the reflectance model account for the pigment fluorescence features and the water-surface reflection of sunlight and skylight. For the green algae and cyanobacteria, the wavelength-independent vertical offset of the backscatter coefficient is found to scale linearly with daily dry weight measurements, providing the capability for a nonsampling measurement of biomass in outdoor ponds. Other fitting parameters in the reflectance model are compared with auxiliary measurements and physics-based calculations. The model-derived magnitudes of sunlight and skylight water-surface reflections compare favorably with Fresnel reflectance calculations, while the model-derived quantum efficiency of Chl-a fluorescence is found to be in agreement with literature values. Finally, the water temperatures derived from the reflectance model exhibit excellent agreement with thermocouple measurements during the morning hours but correspond to significantly elevated temperatures in the afternoon hours.

Host cell pigmentation in Scenedesmus dimorphus as a beacon for nascent parasite infection.

Biofuels derived from the mass cultivation of algae represent an emerging industry that aims to partially displace petroleum based fuels. Outdoor, open-pond, and raceway production facilities are attractive options for the mass culture of algae however, this mode of cultivation leaves the algae susceptible to epidemics from a variety of environmental challenges. Infestations can result in complete collapse of the algal populations and destruction of their valuable products making it paramount to understand the host-pathogen relationships of known algal pests in order to develop mitigation strategies. In the present work, we characterize the spatial-temporal response of photosynthetic pigments in Scenedesmus dimorphus to infection from Amoeboaphelidium protococcarum, a destructive endoparasite, with the goal of understanding the potential for early detection of infection via host pigment changes. We employed a hyperspectral confocal fluorescence microscope to quantify these changes in pigmentation with high spatial and spectral resolution during early parasite infection. Carotenoid abundance and autofluorescence increased within the first 24 h of infection while chlorophyll emission remained constant. Changes in host cell photosynthesis and bulk chlorophyll content were found to lag behind parasite replication. The results herein raise the possibility of using host-cell pigment changes as indicators of nascent parasite infection.

On-line stable isotope gas exchange reveals an inducible but leaky carbon concentrating mechanism in Nannochloropsis salina.

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line (13)CO2 gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO2 provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible "pump-leak" style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO2 and had higher rates of net CO2 uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO2 supply was much higher for the high CO2 conditions, we calculated that growing cells bubbled with low CO2 is about 40 % more efficient for carbon capture than bubbling with high CO2. We attribute this higher efficiency to the activity of a CCM under low CO2 conditions.