Characterizing and identifying cells in multicellular 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.
View Article and Find Full Text PDFFor 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.
View Article and Find Full Text PDFCRISPR 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.
View Article and Find Full Text PDFGenome 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.
View Article and Find Full Text PDFWhen 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.
View Article and Find Full Text PDFBackground: 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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFThe 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).
View Article and Find Full Text PDFGene 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.
View Article and Find Full Text PDFFar-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.
View Article and Find Full Text PDFCyanobacterial 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.
View Article and Find Full Text PDFHerein, 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.
View Article and Find Full Text PDFPhotosystem 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 , sp PCC 7002, and sp PCC 6803.
View Article and Find Full Text PDFFungi 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.
View Article and Find Full Text PDFCosmic 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).
View Article and Find Full Text PDFTotal 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.
View Article and Find Full Text PDFThe 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).
View Article and Find Full Text PDFWe 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).
View Article and Find Full Text PDFBiofuels 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.
View Article and Find Full Text PDFCarbon 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.
View Article and Find Full Text PDFPhotosynthetic organisms rely on antenna systems to harvest and deliver energy from light to reaction centers. In fluctuating photic environments, regulation of light harvesting is critical for a photosynthetic organism's survival. Here, we describe the use of a suite of phycobilisome mutants to probe the consequences of antenna truncation in the cyanobacterium Synechocystis sp.
View Article and Find Full Text PDFIncreased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub-organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy.
View Article and Find Full Text PDFMany membrane receptors are recruited to specific cell surface domains to form nanoscale clusters upon ligand activation. This step appears to be necessary to initiate cell signaling, including pathways in innate immune system activation. However, virulent pathogens such as Yersinia pestis (the causative agent of plague) are known to evade innate immune detection, in contrast to similar microbes (such as Escherichia coli) that elicit a robust response.
View Article and Find Full Text PDFCyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large membrane-extrinsic phycobilisome antenna in addition to membrane-bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess thylakoid membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively.
View Article and Find Full Text PDFHaematococcus pluvialis is a freshwater unicellular green microalga belonging to the class Chlorophyceae and is of commercial interest for its ability to accumulate massive amounts of the red ketocarotenoid astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione). Using confocal Raman microscopy and multivariate analysis, we demonstrate the ability to spectrally resolve resonance-enhanced Raman signatures associated with astaxanthin and β-carotene along with chlorophyll fluorescence. By mathematically isolating these spectral signatures, in turn, it is possible to locate these species independent of each other in living cells of H.
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