Natural Autofluorescence Research Articles

The human fluorescence discrimination as precondition for the use of fluorescence-aided identification techniques (FIT)

ObjectivesThe fluorescence-aided identification technique (FIT) is based on the fluorescence properties of dental materials, specifically the intensity of their fluorescence compared to the autofluorescence of hard dental substances; this creates a perceived contrast between dental material and tooth. However, no studies to date have determined the extent to which the fluorescence intensity of tooth-colored dental materials must differ from that of natural autofluorescence to ensure reliable visual detection. The aim of this study was therefore to determine, for the first time, how pronounced the difference between fluorescence intensity and autofluorescence must be to reliably identify tooth-colored material. MethodsTen dentists assessed six different resin-based composite (RBC) samples of varying fluorescence intensity placed in the cavities of ten extracted teeth under standardized fluorescence-exciting illumination. The outcome variable was fluorescence perceptibility. Their assessments of the outcome variable were compared with measurements of the fluorescence intensities of the RBCs and the surrounding dental hard tissues, which were expressed as a fluorescence intensity ratio. Demographic data of the participants, including age, gender, and professional experience, were also recorded. ResultsNo significant differences were found for visual fluorescence perceptibility in relation to the explanatory variables of gender (p = 0.14), age (p = 0.13), and professional experience (p = 0.34). In contrast, the fluorescence intensity ratio was significantly different (p < 0.0001). SignificanceFor both clinicians and manufacturers, fluorescence intensity levels are important when selecting or developing FIT-compatible materials. Our results suggest that the fluorescence intensity levels of dental materials should be no more than 75% and no less than 200% of tooth natural autofluorescence to ensure reliable detection of tooth-colored materials.

New Technologies to Improve Embryo Assessment

Embryo quality is a key factor in determining whether an IVF cycle will result in a live birth. Current approaches for assessing embryo quality are subjective (morphology) or invasive (biopsy) and are not always predictive of live birth. Development of an accurate and non-invasive method to assess embryo quality would likely improve IVF success. We have taken a different approach towards developing a non-invasive diagnostic for embryo quality: optical imaging. We use low levels of light to illuminate the embryo and obtain spatial information on cell metabolism as well as biophysical properties of the embryo. Here I will present our work showing that hyperspectral imaging – captures natural autofluorescence emitted by the embryo – can be used to accurately discriminate between euploid and aneuploid mouse blastocysts (inner cell mass). Importantly, we show that this form of imaging is safe: compared to embryos that were not imaged, hyperspectral imaged embryos had similar, and not significantly different, pregnancy and implantation rates, with offspring weight at weaning also comparable. Another potential optical approach to assess embryo quality is digital holographic imaging. To date, little attention has been paid to the physical parameters of an embryo and whether these could be markers of embryo quality. One such physical parameter is refractive index. We have shown that digital holographic microscopy is able to measure very small changes in refractive index between good and poor-quality mouse embryos. Importantly, these optical approaches occur in the absence of stains or exogenous tags. Used alone, or in combination, such optical approaches may prove beneficial in developing to an accurate, non-invasive diagnostic for embryo quality.

Sub-lethal toxicity of five disinfection by-products on microalgae determined by flow cytometry – Lines of evidence for adverse outcome pathways

Standardised tests are often used to determine the ecotoxicity of chemicals and focus mainly on one or a few generic endpoints (e.g. mortality, growth), but information on the sub-cellular processes leading to these effects remain usually partial or missing. Flow cytometry (FCM) can be a practical tool to study the physiological responses of individual cells (such as microalgae) exposed to a stress via the use of fluorochromes and their morphology and natural autofluorescence. This work aimed to assess the effects of five chlorine-based disinfection by-products (DBPs) taken individually on growth and sub-cellular endpoints of the green microalgae Raphidocelis subcapitata. These five DBPs, characteristic of a chlorinated effluent, are the following monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA) and 1,1-dichloropropan-2-one (1,1-DCP). Results showed that 1,1-DCP had the strongest effect on growth inhibition (EC50 = 1.8 mg.L-1), followed by MCAA, TCAA, BCAA and DCAA (EC50 of 10.1, 15.7, 27.3 and 64.5 mg.L-1 respectively). Neutral lipid content, reactive oxygen species (ROS) formation, red autofluorescence, green autofluorescence, size and intracellular complexity were significantly affected by the exposure to the five DBPs. Only mitochondrial membrane potential did not show any variation. Important cellular damages (>10%) were observed for only two of the chemicals (BCAA and 1,1-DCP) and were probably due to ROS formation. The most sensitive and informative sub-lethal parameter studied was metabolic activity (esterase activity), for which three types of response were observed. Combining all this information, an adverse outcome pathways framework was proposed to explain the effect of the targeted chemicals on R. subcapitata. Based on these results, both FCM sub-cellular analysis and conventional endpoint of algal toxicity were found to be complementary approaches.

Rapid detection of φX-174virus based on synchronous fluorescence of tryptophan.

The development of rapid methods for the detection of virus particles based on their intrinsic fluorescence (the native auto-fluorescence that originates from the non-labeled analyte) is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we developed a new method for the detection of the naked φX-174virus. We show that a separation of φX-174from its Escherichia coli host (grown on the standard cultivation medium nutrient agar) by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~ 70% of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with a very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we apply synchronous fluorescence to detect φX-174based on the spectral fingerprint of its native Trp content. Such a method is more rapid than usual traditional separation and detection methods which can take several hours and does not require any addition of labeling agents such as fluorescent dyes or antibodies for the detection. As other virus species contain Trp as one of the amino acids presents in their proteins, this method has the potential to apply to the detection of other viral species.

Label-Free Characterization and Quantification of Mucosal Inflammation in Common Murine Colitis Models With Multiphoton Imaging

Clinical challenges in inflammatory bowel diseases require microscopic in vivo evaluation of inflammation. Here, label-free imaging holds great potential, and recently, our group demonstrated the advantage of using in vivo multiphoton endomicroscopy for longitudinal animal studies. This article extends our previous work by in-depth analysis of label-free tissue features in common colitis models quantified by the multiphoton colitis score (MCS). Fresh mucosal tissues were evaluated from acute and chronic dextran sulfate sodium (DSS), TNBS, oxazolone, and transfer colitis. Label-free imaging was performed by using second harmonic generation and natural autofluorescence. Morphological changes in mucosal crypts, collagen fibers, and cellularity in the stroma were analyzed and graded. Our approach discriminated between healthy (mean MCS = 2.5) and inflamed tissue (mean MCS > 5) in all models, and the MCS was validated by hematoxylin and eosin scoring of the same samples (85.2% agreement). Moreover, specific characteristics of each phenotype were identified. While TNBS, oxazolone, and transfer colitis showed high cellularity in stroma, epithelial damage seemed specific for chronic, acute DSS and transfer colitis. Crypt deformations were mostly observed in acute DSS. Quantification of label-free imaging is promising for in vivo endoscopy. In the future, this could be valuable for monitoring of inflammatory pathways in murine models, which is highly relevant for the development of new inflammatory bowel disease therapeutics.

Autofluorescence Is a Common Trait in Different Oceanic Fungi.

Natural autofluorescence is a widespread phenomenon observed in different types of tissues and organisms. Depending on the origin of the autofluorescence, its intensity can provide insights on the physiological state of an organism. Fungal autofluorescence has been reported in terrestrial and human-derived fungal samples. Yet, despite the recently reported ubiquitous presence and importance of marine fungi in the ocean, the autofluorescence of pelagic fungi has never been examined. Here, we investigated the existence and intensity of autofluorescence in five different pelagic fungal isolates. Preliminary experiments of fungal autofluorescence at different growth stages and nutrient conditions were conducted, reflecting contrasting physiological states of the fungi. In addition, we analysed the effect of natural autofluorescence on co-staining with DAPI. We found that all the marine pelagic fungi that were studied exhibited autofluorescence. The intensity of fungal autofluorescence changed depending on the species and the excitation wavelength used. Furthermore, fungal autofluorescence varied depending on the growth stage and on the concentration of available nutrients. Collectively, our results indicate that marine fungi can be auto-fluorescent, although its intensity depends on the species and growth condition. Hence, oceanic fungal autofluorescence should be considered in future studies when fungal samples are stained with fluorescent probes (i.e., fluorescence in situ hybridization) since this could lead to misinterpretation of results.

Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis.

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.

How well can today's tooth-colored dental restorative materials reproduce the autofluorescence of human teeth? - Ambition and reality!

The autofluorescence of dental hard tissues has been known for over 100 years. Thus, manufacturers add fluorophores to dental restorative materials to improve the esthetic properties of these materials. So far, there has been no study evaluating the ability of these fluorophores to reproduce the autofluorescence of dental hard tissues. A total of 240 different color shades representing 17 different brands of fluorescent light-curing RBC and CAD/CAM restorative materials were analyzed with a monochromator-based microplate reader. Additionally, combined enamel-dentin specimens (n= 11) were analyzed as "gold standard". The total fluorescence (TF) and the physiologically relevant luminous efficiency function adjusted total fluorescence (TFa ) were determined. The differences between the brands and the enamel-dentin specimens were further evaluated and visualized as contour plots. Merely the TFa of the brands CERASMART™, Filtek Supreme XTE™, KZR-CAD HD 2, and LuxaCam composite were not significantly different to the enamel-dentin specimens. The analysis of the contour plots revealed that even these four materials showed a fluorescence excess for the excitation wavelengths below about 400 nm and a deficit above this wavelength. None of the materials analyzed in this study were able to reproduce the natural fluorescence spectrum of the enamel-dentin specimens. Unlike the statements and images of blue fluorescent materials in the manufacturers' brochures, none of the materials examined here is fully capable of reproducing the natural autofluorescence of teeth.

Three-Dimensional Imaging of Plant Cell Wall Deconstruction Using Fluorescence Confocal Microscopy

Lignocellulosic biomass (LB) is recalcitrant to enzymatic hydrolysis due to its compact and complex cell wall structure. To identify the parameters behind LB recalcitrance, experimental data over hydrolysis time must be collected. Here, we describe a novel method to collect time-lapse images during cell wall deconstruction by enzymatic hydrolysis. The protocol includes instructions for sample preparation, layout of a custom designed incubation chamber and instructions for confocal time lapse acquisition. The protocol sets out a detailed plan where cross-sections of untreated and pretreated poplar samples are mounted in a sealed frame containing a buffer and an enzymatic cocktail. The sealed frame is then placed into an incubator to maintain the sample at a constant temperature of 50 °C, which is optimal for enzymatic reaction while avoiding enzymatic cocktail evaporation. Using lignin natural autofluorescence, confocal z-stacks of untreated and pretreated samples were acquired at regular time intervals during enzymatic hydrolysis for 24 h. Acquisition parameters were optimized to compromise between image resolution and reduced photo-bleaching. The acquired image might then be processed by further development of algorithms to extract precise quantitative information on cell wall deconstruction. This protocol is an important first step towards elucidating the underlying parameters of LB recalcitrance by allowing the acquisition of high-quality images of LB hydrolysis for extracting quantitative data on LB deconstruction.

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.

Silk fibroin and silk sericin composite hydrogel encapsulated exosomes promote wound healing

Recent studies have shown that the hydrogel formed by composite biomaterials is more advantageous than that formed by homogenous material. Natural silk is mainly composed of two proteins: silk fibroin (SF) and silk sericin(SS). SF has favorable mechanical properties, while SS has high cell affinity and natural autofluorescence properties. In our study, we explored the beneficial effect of the composite hydrogel formed by a mixture of silk fibroin (SF) and silk sericin (SS) in tissue repair. The composite hydrogel was used to encapsulate and deliver the exosomes derived from human umbilical cord mesenchymal stem cells (UMSC-Exo).

Chemically labeled toxins or bioactive peptides show a heterogeneous intracellular distribution and low spatial overlap with autofluorescence in bloom-forming cyanobacteria

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N-propargyloxy-carbonyl-L-lysine or O-propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.

Functional analysis of vasa/PL10-like genes in the ovary of Schistosoma mansoni

The RNA helicase Vasa plays a pivotal role in the development of the germ line. To decipher the functional roles of vasa/PL10-like genes in the human blood fluke Schistosoma mansoni, we performed RNA interference followed by the analysis of the ovary in the adult female. Double-stranded RNA targeting the schistosome vasa-like gene Smvlg1 reduced the volume of the ovary. Changes in morphology of the ovary were analysed using carmine red-staining of the parasites followed by a novel confocal laser scanning microscopy (CLSM)-based approach to control for natural autofluorescence in female schistosome tissues. The reduction in the ovary volume may have been promoted by the loss of germ cells. By contrast, significant differences were not apparent in the number of eggs produced or hatching rate of eggs laid by the female schistosomes transfected with Smvlg1-specific dsRNA. The findings suggested a role for S. mansoni vasa/PL10-like gene -1 in germ cell development within the schistosome ovary that might impact in the pathogenesis and disease transmission by this neglected tropical disease pathogen.

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by Förster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

Autofluorescence excitation-emission matrices as a quantitative tool for the assessment of meat quality.

Commercially produced meat is currently graded by a complex and partly subjective multiparameter methodology; a quantitative method of grading, using small samples would be desirable. Here, we investigate the correlation between commercial grades of beef and spectral signatures of native fluorophores in such small samples. Beef samples of different commercial grades were characterized by fluorescence spectroscopy complemented by biochemical and histological assessment. The excitation-emission matrices of the specimens reveal five prominent native autofluorescence signatures in the excitation range from 250 to 350 nm, derived mainly from tryptophan and intramuscular fat. We found that these signatures reflect meat grade and can be used for its determination.

Lessons learned from quantitative fundus autofluorescence.

Quantitative fundus autofluorescence (qAF) is an approach that is built on a confocal scanning laser platform and used to measure the intensity of the inherent autofluorescence of retina elicited by short-wavelength (488 nm) excitation. Being non-invasive, qAF does not interrupt tissue architecture, thus allowing for structural correlations. The spectral features, cellular origin and topographic distribution of the natural autofluorescence of the fundus indicate that it is emitted from retinaldehyde-adducts that form in photoreceptor cells and accumulate, under most conditions, in retinal pigment epithelial cells. The distributions and intensities of fundus autofluorescence deviate from normal in many retinal disorders and it is widely recognized that these changing patterns can aid in the diagnosis and monitoring of retinal disease. The standardized protocol employed by qAF involves the normalization of fundus grey levels to a fluorescent reference installed in the imaging instrument. Together with corrections for magnification and anterior media absorption, this approach facilitates comparisons with serial images and images acquired within groups of patients. Here we provide a comprehensive summary of the principles and practice of qAF and we highlight recent efforts to elucidate retinal disease processes by combining qAF with multi-modal imaging.

A new fluorescent oligonucleotide probe for in-situ identification of Microcystis aeruginosa in freshwater

Cyanobacteria colonize different environments and blooms can occur in both contaminated and non-contaminated water bodies (freshwater, brackish and marine areas). Among 150 known cyanobacteria genera, >40 species are able to produce toxins, which are natural compounds that differ from both a chemical and toxicological point of view and are responsible for acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. Fast and accurate methods for unequivocally identifying microcystin-producing cyanobacteria, such as Microcystis aeruginosa in water bodies, are necessary to distinguish them from other non-toxic cyanobacteria and to manage and monitor algal blooms. For this purpose, we designed, developed and validated an oligonucleotide probe for FISH (Fluorescence In Situ Hybridization) analysis to detect Microcystis aeruginosa at the species level even at relatively low concentrations in freshwater. The FISH probe, MicAerD03, was designed using the ARB software with the Silva database within the framework of the MicroCoKit project, also with the intention of adding it to the microarray from the EU project, μAQUA, for freshwater pathogens, which had only genus level probes for Microcystis.We tested various fixative methods to minimize the natural autofluorescence from chlorophyll-a and certain accessory pigments (viz., phycobilins and carotenoids). The FISH probe was tested on pure cultures of Microcystis aeruginosa, and then successfully applied to water samples collected from different sampling points of the Tiber River (Italy), using a laser confocal microscope. Subsequently, the probe was also conjugated at the 5′ end with horse-radish peroxidase (HRP-MicAerD03) to apply the CAtalysed Reported Deposition-FISH (CARD-FISH) for increasing the fluorescence signal of the mono-fluorescently labelled probe and make it possible to detect M. aeruginosa using an epifluorescence microscope. Samples taken within the EU MicroCokit project indicated that microarray signals for Microcystis were coming from single cells and not colonial cells. We confirmed this with the CARD-FISH protocol used here to validate the microarray signals for Microcystis detected at the genus level in MicroCokit.This paper provides a new early warning tool for investigating M. aeruginosa at the species level even at low cell concentrations in surface water, which can be added to the μAqua microarray for all freshwater pathogens to complete the probe hierarchy for Microcystis aeruginosa.

FRET-SLiM on native autofluorescence: a fast and reliable method to study interactions between fluorescent probes and lignin in plant cell wall

BackgroundLignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that necessitates some pretreatments and several types of catalysts to be transformed efficiently. In particular, enzymes degrading lignocellulose can become inactivated due to their binding to lignin through non-specific interactions, leading to a loss in catalytic efficiency of industrial processes. Gaining more knowledge in the strength of interactions would allow optimizing enzymes and selecting appropriate pretreatments.ResultsMeasuring interactions directly in plant cell wall can theoretically be performed using confocal fluorescence techniques by evaluating fluorescence resonance energy transfer (FRET) between compatible fluorophores. In this study, autofluorescence of plant cell wall, mainly originating from lignin, was considered as a donor fluorophore while the acceptor was a common rhodamine-based fluorescent probe. To overcome complex plant cell wall fluorescence, which limits FRET analysis by standard techniques, we have developed an original approach, combining spectral and lifetime measurements. It consists in (1) dissecting autofluorescence signal in each spectral channel, (2) optimizing spectral channel choice for lifetime measurements and (3) achieving an unambiguous FRET signature with an autofluorescent donor fluorophore. Interactions between rhodamine-based probes of various sizes and untreated or pretreated wheat sample were evaluated, showing it was possible to discriminate interactions at the nano-scale, revealing some accessibility differences and the effect of pretreatment.ConclusionsSLiM measurement allows precise estimation of the optimal spectral range for FRET measurement. SLiM response allows for the first time doubtless FRET measurements between lignin as a donor, and an acceptor fluorophore with high accuracy and sensitivity related to lifetime decrease studies. As demonstrated, it thus becomes possible to measure interactions of fluorescent probes directly inside plant cell wall samples. This approach can thus be applied to various fields such as lignocellulose deconstruction to optimize the action of enzymes or plant cell wall development to assay in situ the biosynthesis of lignin.

Restricted Localization of Photosynthetic Intracytoplasmic Membranes (ICMs) in Multiple Genera of Purple Nonsulfur Bacteria.

ABSTRACTIn bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this organization entails the use of a light-harvesting, membrane-bound compartment known as the intracytoplasmic membrane (ICM). Here we show that ICMs are spatially and temporally localized in diverse patterns among PNSB. We visualized ICMs in live cells of 14 PNSB species across nine genera by exploiting the natural autofluorescence of the photosynthetic pigment bacteriochlorophyll (BChl). We then quantitatively characterized ICM localization using automated computational analysis of BChl fluorescence patterns within single cells across the population. We revealed that while many PNSB elaborate ICMs along the entirety of the cell, species across as least two genera restrict ICMs to discrete, nonrandom sites near cell poles in a manner coordinated with cell growth and division. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. The natural diversity of ICM localization revealed herein has implications for both the evolution of phototrophic organisms and their light-harvesting compartments and the mechanisms underpinning spatial organization of bacterial compartments.