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Summary Designing improved vaccines against mutable viruses such as dengue and influenza would be helped by a better understanding of how the B‐cell memory compartment responds to variant antigens. Towards this we have recently shown, after secondary immunization of mice with a widely variant dengue virus envelope protein with only 63% amino acid identity, that IgM+ memory B cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, further investigation of memory responses to variant proteins, using more closely related influenza virus haemagglutinins (HA) that were 82% identical, produced a variant‐induced boost response in the GC dominated by highly mutated B cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over a certain level of antigenic differences, cross‐reactive memory B‐cell populations have reduced competency for affinity maturation. Combined with our previous observations, these findings also provide new parameters of success and failure in antibody memory responses.

A powerful vaccine against mutable viruses might induce memory antibodies that either strongly bound antigenic variants or that could rapidly undergo secondary affinity maturation to achieve this. We have recently shown after secondary immunization of mice with a widely variant protein (Burton et al. 2018) that IgM+ memory B-cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, boosting with more closely related proteins produced a GC response dominated by highly mutated B-cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over certain antigenic differences, a cross reactive, mutated, memory B-cell compartment can be an impediment to affinity maturation.

Twentieth and twenty-first centuries saw number of research on teaching second language to different learners. It tried to answer different issues, which existed in the classroom and persons who handle it with different theories with its specialisation. Since the specialisation of one theory neglected the other theories in textbook preparation and in the teaching process, it paved way for new theories. Hence, the method proposed in the textbook was different from the needs of the learners. In that case, teachers coerced to fulfil the procedures of the method, but not the needs of the students. Such demands forced the teachers and theorists to look for a theory as alternative to all the language issues. One such theory, which gained the attention of the teachers, to answer the multiple issues was eclecticism, which was the combination of different theories in a single domain. This research carried out in a heterogeneous learning environment in a micro level to observe and analyse the relevance and irrelevance of eclectic method in the complex situation.

Yeast propagation using 50% diluted hydrolysate in water was utilized for the fermentation of hydrolysate derived from pre-treated ensiled sweet sorghum. The purpose was to condition the yeast to the inhibitors generated during the ensiling of sweet sorghum. The conditioned seed cultures exhibited similar fermentation performance and superior kinetics than the inoculum prepared in YPD medium. Furthermore, the conditioned yeast showed increased tolerance to the increased levels of these inhibitors, including ethanol, acetic and lactic acids, demonstrating an effective way to increase the robustness of yeast fermentation for ethanol production.

Vaccines induce memory B-cells that provide high affinity secondary antibody responses to identical antigens. Memory B-cells can also re-instigate affinity maturation, but how this happens against antigenic variants is poorly understood despite its potential impact on driving broadly protective immunity against pathogens such as Influenza and Dengue. We immunised mice sequentially with identical or variant Dengue-virus envelope proteins and analysed antibody and germinal-centre (GC) responses. Variant protein boosts induced GCs with a higher proportion of IgM+ B cells. The most variant protein re-stimulated GCs with the highest proportion of IgM+ cells with the most diverse, least mutated V-genes and with a slower but efficient serum antibody response. Recombinant antibodies from GC B-cells showed a higher affinity for the variant antigen than antibodies from a primary response, confirming a memory origin. This reveals a new process of antibody memory, that IgM memory cells with fewer mutations participate in secondary responses to variant antigens, demonstrating how the hierarchical structure of B-cell memory is used and indicating the potential and limits of cross-reactive antibody based immunity., eLife digest Many devastating infectious diseases are caused by viruses that change over time. When a vaccine exists, it usually protects against a particular strain of virus, but often fails to defend against new versions of the microbe. This is why the flu vaccine has to be ‘updated’ every year, for example. Vaccines rely on the memory of our immune system. When a virus enters the body, a group of immune cells known as B cells gets activated. Certain B cells can recognise the invader and produce specific proteins, the antibodies, which can target and kill the invader. During the infection some of these B cells become ‘memory B cells’, having gone through a maturation process that hones their ability to specifically recognize this particular microbe. If the same virus enters the organism again, the memory B cells rapidly identify it and produce a quicker and more efficient immune response than during the first attack. This is how vaccines work. However memory B cells may not be able to recognize a previous intruder if it has changed too much. The memory B cell population is diverse. Some cells are fully mature and can quickly recognize the original virus. But others have not finished their maturation process: these cells are less focussed, and cannot target the original microbe with the same exact precision shown by mature memory cells. For almost forty years it was thought that this reduced focus might make the immature cells better at identifying new versions of the original attacker, but up until now, it was not clear what these memory cells could do. Here Burton, Tennant et al. injected a group of mice with proteins from the Dengue virus, which prompted an immune reaction. After several weeks, the animals received either the same proteins again, or proteins that were different. Compared to the fully mature cells, the immature memory B cells were much better at recognizing the variants of the proteins, and these cells then multiplied and mounted an immune response. Without the original protein injection, the response without the immature memory B cells was not as efficient. The body therefore has a pool of memory B cells that can recognise a wider range of virus protein variants than the ones that caused the first immune reaction. Understanding the role of immature memory B cells in immunity could help design vaccines that protect against several strains or fast-evolving viruses. This could have the potential to reduce the severity of diseases that affect hundreds of millions of people every year.

We acknowledge the support of NERC (Grant No NE/L014076/1) in the research presented here (AJM and RLO) to develop ZnS-based quantum dots technology via resource recovery from waste. The underpinning evaluation of commercial quantum dots in the three test photobiological systems was supported by the Discipline Hopping Award scheme co-funded by EPSRC, BBSRC, and MRC. The support of BBSRC is acknowledged for MRes studentships (AG and FW).

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

There is significant interest in optical fibers whose cross sections contain more than one single-mode core. Optical fibers are normally spliced together by removing the coating, cleaving the fiber ends so that their end faces are flat and perpendicular to the fiber axis and butting them together in a V-groove. The near field refers to the field of a fiber or waveguide on the far end face in air. The far field of a fiber or waveguide is the field emerging from its far end into air as measured at a sufficiently large distance from the end face. For single-mode fibers and waveguides with relatively small core sizes of the order of a few wavelengths of the source light, the far field is diffracted at the end face. Waveguides and fibers are illuminated by a variety of sources, including lasers, LEDs, or light propagating out of another fiber or waveguide.

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Microalgae are widely viewed as a promising and sustainable source of renewable chemicals and biofuels. Botryococcus braunii synthesizes and secretes significant amounts of long‐chain (C30‐C40) hydrocarbons that can be subsequently converted into gasoline, diesel, and aviation fuel. B. braunii cultures are not axenic and the effects of co‐cultured microorganisms on B. braunii growth and hydrocarbon yield are important, but sometimes contradictory. To understand the composition of the B. braunii microbial consortium, we used high throughput Illumina sequencing of metagenomic DNA to profile the microbiota within a well established, stable B. braunii culture and characterized the demographic changes in the microcosm following modification to the culture conditions. DNA sequences attributed to B. braunii were present in equal quantities in all treatments, whereas sequences assigned to the associated microbial community were dramatically altered. Bacterial species least affected by treatments, and more robustly associated with the algal cells, included members of Rhizobiales, comprising Bradyrhizobium and Methylobacterium, and representatives of Dyadobacter, Achromobacter and Asticcacaulis. The presence of bacterial species identified by metagenomics was confirmed by additional 16S rDNA analysis of bacterial isolates. Our study demonstrates the advantages of high throughput sequencing and robust metagenomic analyses to define microcosms and further our understanding of microbial ecology.

Background: In Scotland, a national HPV immunisation programme began in 2008 for 12- to 13-year olds, with a catch-up campaign from 2008 to 2011 for those under the age of 18. To monitor the impact of HPV immunisation on cervical disease at the population level, a programme of national surveillance was established. Methods: We analysed colposcopy data from a cohort of women born between 1988 and 1992 who entered the Scottish Cervical Screening Programme (SCSP) and were aged 20–21 in 2008–2012. Results: By linking datasets from the SCSP and colposcopy services, we observed a significant reduction in diagnoses of cervical intraepithelial neoplasia 1 (CIN 1; RR 0.71, 95% CI 0.58 to 0.87; P=0.0008), CIN 2 (RR 0.5, 95% CI 0.4 to 0.63; P

The use of citizen science to obtain annotations from multiple annotators has been shown to be an effective method for annotating datasets in which computational methods alone are not feasible. The way in which the annotations are obtained is an important consideration which affects the quality of the resulting consensus annotation. In this paper, we examine three separate approaches to obtaining consensus scores for instances rather than merely binary classifications. To obtain a consensus score, annotators were asked to make annotations in one of three paradigms: classification, scoring and ranking. A web-based citizen science experiment is described which implements the three approaches as crowdsourced annotation tasks. The tasks are evaluated in relation to the accuracy and agreement among the participants using both simulated and real-world data from the experiment. The results show a clear difference in performance between the three tasks, with the ranking task obtaining the highest accuracy and agreement among the participants. We show how a simple evolutionary optimiser may be used to improve the performance by reweighting the importance of annotators.

1532-1827 Kavanagh, K Pollock, K G J Potts, A Love, J Cuschieri, K Cubie, H Robertson, C Donaghy, M Journal article Br J Cancer. 2014 Apr 15. doi: 10.1038/bjc.2014.198.; International audience; Background:In 2008, a national human papillomavirus (HPV) immunisation programme began in Scotland for 12-13 year old females with a three-year catch-up campaign for those under the age of 18. Since 2008, three-dose uptake of bivalent vaccine in the routine cohort aged 12-13 has exceeded 90% annually, while in the catch-up cohort overall uptake is 66%.Methods:To monitor the impact of HPV immunisation, a programme of national surveillance was established (pre and post introduction) which included yearly sampling and HPV genotyping of women attending for cervical screening at age 20. By linking individual vaccination, screening and HPV testing records, we aim to determine the impact of the immunisation programme on circulating type-specific HPV infection particularly for four outcomes: (i) the vaccine types HPV 16 or 18 (ii) types considered to be associated with cross-protection: HPV 31, 33 or 45; (iii) all other high-risk types and (iv) any HPV.Results:From a total of 4679 samples tested, we demonstrate that three doses (n=1100) of bivalent vaccine are associated with a significant reduction in prevalence of HPV 16 and 18 from 29.8% (95% confidence interval 28.3, 31.3%) to 13.6% (95% confidence interval 11.7, 15.8%). The data also suggest cross-protection against HPV 31, 33 and 45. HPV 51 and 56 emerged as the most prevalent (10.5% and 9.6%, respectively) non-vaccine high-risk types in those vaccinated, but at lower rates than HPV 16 (25.9%) in those unvaccinated.Conclusions:This data demonstrate the positive impact of bivalent vaccination on the prevalence of HPV 16, 18, 31, 33 and 45 in the target population and is encouraging for countries which have achieved high-vaccine uptake.British Journal of Cancer advance online publication, 15 April 2014; doi:10.1038/bjc.2014.198 www.bjcancer.com.

Aims: The IATPAD (Improvement in Access to Treatment for People with Alcohol and Drug Related Problems) study explored barriers and facilitators to accessing alcohol and drug treatment services in eight European countries.Methods: Results from individual interviews and focus group discussions with 246 service users who had experienced alcohol and drug treatment in Bulgaria, Greece, Italy, Poland, Slovakia, Slovenia, Spain and Scotland are described. Thematic analysis was used to interpret the data.Results: The study identified a number of consistent facilitating factors and barriers across all eight European countries, despite different health care systems. The results suggest that five main factors were implicated in the help-seeking behaviour of the service users interviewed. These were: information, service organisation, staff attitudes, significant others and personal disposition. All these factors could be viewed as both facilitators and barriers.Conclusions: To enhance access to alcohol and drug tre...

Radiocarbon (14C) accelerator mass spectrometry (AMS) dating has played a significant role towards improving our understanding of the timing of events and rates of change in archaeological and environmental records over the last 50 000 years. Although it is not always possible to find suitable macrofossils for 14C dating, microfossils, notably plant pollen, are a viable alternative. Obtaining preserved pollen samples of known provenance and of sufficient quantity for dating by 14C AMS is, however, challenging because of time-consuming methods of extraction and purification and possible contamination from other organic material. Here we report a new, rapid and straightforward method using flow cytometry (FCM) to distinguish, sort and collect sufficient quantities of fossil pollen with minimal contamination from lake sediments for radiocarbon dating. Using this approach, we demonstrate 14C AMS ages that date back to at least 40 ka BP. While future work may be required to refine purification methodologies for different sample types and to precisely quantify the dating limit of this approach, FCM dating of microfossils shows considerable promise in generating robust geochronological frameworks for sequences that have previously proved problematic. Copyright © 2013 John Wiley & Sons, Ltd.

A variety of bacteria associate with the hydrocarbon-producing microalga Botryococcus braunii , some of which may influence its growth. We report here the genome sequences for Achromobacter piechaudii GCS2, Agrobacterium sp. strain SUL3, Microbacterium sp. strain GCS4, and Shinella sp. strains GWS1 and SUS2, isolated from a laboratory culture of B. braunii , race B, strain Guadeloupe.

A variety of bacteria associate with the hydrocarbon-producing microalga Botryococcus braunii, some of which may influence its growth. We report here the genome sequences for Achromobacter piechaudii GCS2, Agrobacterium sp. strain SUL3, Microbacterium sp. strain GCS4, and Shinella sp. strains GWS1 and SUS2, isolated from a laboratory culture of B. braunii, race B, strain Guadeloupe.

Sub-fossil diatoms represent an important archive of past environmental change in both terrestrial and marine settings. In recent years the isotopic analysis of diatom frustules has developed into an important area of paleo-environmental research. The extraction and concentration of diatoms from marine and lake sediments has proven difficult, particularly in silica rich samples. Here we present a new method for the rapid extraction of diatom frustules from lacustrine sediment using flow cytometry. This new technique produces samples suitable for geochemical and isotope-based research with a high degree of purity over a much shorter time frame than existing techniques, hours rather than days.

Purpose: This study aims to find out the dual-perspectives on authenticity in English language teaching.Problem: The concept of authenticity has generated quite a ripple in ESL/EFL classrooms. Contrasting perspectives have emerged over the years in reaction to this concept. Some regard authenticity as an inevitable part of teaching as it provides students with opportunities to interact with reality in the classroom itself, whereas, some others consider it an ambitious construct and suggest approaching it with caution.Method: In order to verify these perspectives, a survey of literature has been done to trace the genesis of authenticity and its various facets.Conclusion: As a result, we have found that a balanced outlook on authenticity is required by the teachers to promote better teaching-learning experiences.

Objectives: Existing fluoride concentration and consumption data were used to estimate fluoride intakes from the diet and toothpaste use, for New Zealand sub-populations, to identify any population groups at risk of high-fluoride intake. Methods: For each sub-population, two separate dietary intake estimates were made – one based on a non-fluoridated water supply (fluoride concentration of 0.1 mg/L), and the other based on a water supply fluoridated to a concentration of 1.0 mg/L. Fluoride concentration data were taken from historical surveys, while food consumption data were taken from national 24-hour dietary recall surveys or from simulated diets. Results: Mean and 95th percentile estimations of dietary fluoride intake were well below the upper level of intake (UL), whether intakes were calculated on the basis of a non-fluoridated or fluoridated water supply. The use of fluoride-containing toothpastes provides additional fluoride intake. For many of the population groups considered, mean fluoride intakes were below the adequate intake (AI) level for caries protection, even after inclusion of the fluoride contribution from toothpaste. Intake of fluoride was driven by consumption of dietary staples (bread, potatoes), beverages (particularly tea, soft drinks, and beer), and the fluoride status of drinking water. Conclusion: Estimates of fluoride intake from the diet and toothpaste did not identify any groups at risk of exceeding the UL, with the exception of infants (6-12 months) living in areas with fluoridated water supplies and using high-fluoride toothpaste. In contrast, much of the adult population may be receiving insufficient fluoride for optimum caries protection from these sources, as represented by the AI.

Two diastereoselective, scaleable routes towards trans -3,4-disubstituted piperidines with a 4-hydroxymethyl-3-hydroxy or 4-aminomethyl-3-hydroxy substitution pattern are being described. In the first route, the 3,4-trans configuration was introduced regio- and diastereoselectively via a hydroboration/oxidation sequence starting from 4-hydroxymethylpyridine. In the second route, regioselective epoxide ring opening of N -benzyl-3,4-epoxy-piperidine was achieved with LiCN, in situ generated from acetocyanohydrin and LiNH 2 . The regioselectivity of both the hydroboration and the epoxide ring opening was positively influenced by the presence of the basic piperidine nitrogen. Both routes have been optimized to be performed at large scale.

Transcriptional feedback loops are a feature of circadian clocks in both animals and plants. We show that the plant circadian clock also incorporates the cytosolic signaling molecule cyclic adenosine diphosphate ribose (cADPR). cADPR modulates the circadian oscillator's transcriptional feedback loops and drives circadian oscillations of Ca 2+ release. The effects of antagonists of cADPR signaling, manipulation of cADPR synthesis, and mathematical simulation of the interaction of cADPR with the circadian clock indicate that cADPR forms a feedback loop within the plant circadian clock.

In addition to the large number of patients with isolated limb injuries, many patients with major blunt or penetrating trauma harbor extremity injuries as a component of their overall clinical picture. Extremity injuries range from gross deformities and amputations to more subtle injuries, potentially difficult to diagnose, or may escape detection in unconscious or intoxicated patients. However, many soft tissue and vascular injuries require time sensitive interventions to ensure salvage of the limb and the best outcome for the patient. This article reviews the acute management of vascular and soft tissue injuries in the emergency department.

Signal transduction events that lead to circadian control of physiology are poorly understood. Signalling elements that could transmit time information include transcription factors, reversible phosphorylation, and changes in the concentration of cytosolic free calcium ([Ca 2+ ] cyt ). [Ca 2+ ] cyt oscillates with a circadian rhythm in Arabidopsis and Nicotiana , but does not have a defined role in circadian signalling. [Ca 2+ ] cyt oscillations with shorter periods encode specific signals in several cell types, therefore circadian [Ca 2+ ] cyt oscillations provide a potential mechanism for signalling time information. Cell types such as stomatal guard cells and legume pulvini represent attractive model systems for dissecting circadian Ca 2+ signalling.

Calreticulin (CRT) is a eukaryotic, highly conserved, Ca2+-binding protein predominantly located in the endoplasmic reticulum (ER) lumen. In addition to being involved in the regulation of cellular Ca2+, calreticulin is a key quality control element during protein folding in the ER lumen. Tobacco (Nicotiana tabacum L.) suspension cells overexpressing a maize CRT (CRT1a) were used here to examine the properties of CRT in growing plant cells with respect to stress exposure. The endogenous CRT gene was induced rapidly after subculturing of the cells to new medium. In accordance, the CRT protein levels increased, peaking at days 3-4. At day 5, when the CRT transcript levels had levelled off, a further increase in endogenous CRT expression was obtained when the cells were treated with excess Ca2+ or the N-linked glycosylation inhibitor tunicamycin. Whereas the response to Ca2+ occurred within 30 min, the induction by tunicamycin took several hours to be established. Transforming tobacco cells with maize CRT1a, under a constitutive mannopine synthase promoter, resulted in a stable level of expressed CRT1a during the growth cycle compared with endogenous CRT. The CRTs showed differences in attached glycans, but both contained the high mannose-rich-type glycans characteristic of ER proteins. In agreement with an ER location, both tobacco CRT and the transgene product CRT1a codistributed with the ER marker NADH cytochrome c reductase after density gradient centrifugation of microsomal fractions from tobacco cells. Increased production of CRT, as was obtained in the transgenic tobacco cell lines, made cells more tolerant than wild-type cells to high Ca2+ during growth. These data suggest that overexpression of CRT1a in plant cells results in a more efficient calcium buffering capacity in the ER. (Less)

The stability of the self-contained structure of green fluorescent protein (GFP) has made it the most widely utilized fluorescent marker for gene expression and subcellular localization studies ([Chalfie et al., 1994][1]; [Tsien, 1998][2]; [De Giorgi et al., 1999][3]; [Haseloff et al., 1999][4]).

We recently reported the genome sequence of a free-living strain of Vibrio furnissii (NCTC 11218) harvested from an estuarine environment. V. furnissii is a widespread, free-living proteobacterium and emerging pathogen that can cause acute gastroenteritis in humans and lethal zoonoses in aquatic invertebrates, including farmed crustaceans and molluscs. Here we present the fully annotated genome of Vibrio furnissii NCTC11218 and analyses to further assess the potential pathogenic impact of V. furnissii. We compared the complete genome of V. furnissii with 8 other emerging and pathogenic Vibrio species. We selected and analysed more deeply 10 genomic regions based upon unique or common features, and used 3 of these regions to construct a phylogenetic tree. Thus, we positioned V. furnissii more accurately than before and revealed a closer relationship between V. furnissii and V. cholerae than previously thought. However, V. furnissii lacks several important features normally associated with virulence in the human pathogens V. cholera and V. vulnificus. We systematically built phylogenetic trees of all the predicted proteins and grouped them according GO categories. A striking feature of the V. furnissii genome is the hugely increased Super Integron, compared to the other Vibrio. Analyses of predicted genomic islands resulted in the discovery of a protein sequence that is present only in Vibrio associated with diseases in aquatic animals. We also discovered evidence of high levels horizontal gene transfer in V. furnissii. V. furnissii seems therefore to have a dynamic and fluid genome that could quickly adapt to environmental perturbation or increase its pathogenicity. Taken together, these analyses confirm the potential of V. furnissii as an emerging marine and possible human pathogen, especially in the developing, tropical, coastal regions that are most at risk from climate change.

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximise the information gained from advances in fluorescent protein labelling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the perfluorocarbons PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using 4 advanced modes of microscopy: laser scanning confocal microscopy (LSCM), Two-photon fluorescence (TPF) microscopy, second harmonic generation (SHG) microscopy and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image perfluorocarbons directly in the mesophyll and thereby easily delimit the negative space within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.