Development of new photon-counting detectors for single-molecule fluorescence microscopy.

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Parallel multispot smFRET analysis using an 8-pixel SPAD array.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Epidemiology of insect venom sensitivity in children and its correlation to clinical and atopic features.

The aim of this study was to obtain more accurate figures of the prevalence of cutaneous sensitivity to Hymenoptera venoms (HV) and its correlation with other parameters of atopy in a population of primary schoolchildren. Parents filled out a structured questionnaire and children were tested with a panel of inhalant and food allergens as well as standardized freeze-dried extracts of HV. Among the 1175 children who completed the study there was a personal history of rhinoconjunctivitis in 242 (20.8%) and a current wheezing in 114 (9.78%). Two-hundred twenty-eight (19.40%) children had a history of Hymenoptera sting (HS) reactions (224 or 19.06% of local reactions and 4 or 0.34% of local and systemic reactions). Positive skin-prick tests (SPT) to any given HV extract were present in 43 children (3.66%). Most subjects had positive SPT to honey bee venom (35/1175; 2.98%); 17/1175 (1.45%) had positive SPT to wasp and only 12 subjects (1.02%) had positive SPT to polistes venom. There was a correlation between a positive SPT to HV and the history of clinical reactions to HS (P=0.0026). Positive SPT to at least one of the inhalant and food allergens tested were found in 353 subjects (30.04%). Factors such as age, sex, reactions to HV, positive SPT to mite, cat dander, grass, Alternaria, Parietaria, cow's milk, egg white and wheat were significantly associated with a positive SPT to HV using a univariate regression analysis. Only age, reactions to HV, a positive SPT to grass, Parietaria, cow's milk, and egg white were significantly associated with a positive SPT to HV using a multiple regression analysis. In this study, the frequency of immunological sensitization to HV in a population of unselected children is not so high as in adults. There is an association between the presence of positive SPT to HV and an atopy linked humoral IgE response. The presence of a significant and independent association between positive SPT to food of animal origin and positive SPT to HV is surprising and needs further study.

Prevalence and risk factors of latex sensitization in an unselected pediatric population.

BACKGROUND: The prevalence of latex sensitization has been investigated in population groups considered at high risk, but it has not been systematically surveyed among the general population. OBJECTIVE: We sought to determine the prevalence of and the risk factors associated with latex sensitization in a general pediatric population. METHODS: We investigated 1175 children (mean age +/- SD, 105 +/- 17.5 months) in 11 elementary schools in Tuscany (Italy). All parents answered a questionnaire, and children underwent skin prick tests (SPTs) with latex, six aeroallergens (Dermatophagoides pteronyssinus, D. farinae, cat, grass pollen, Alternaria tenuis, and Parietaria judaica), three food allergens (milk, egg white, and wheat), and three insect venoms (honeybee, wasp, and Polistes). RESULTS: Eight subjects (0.7%; mean age +/- SD, 123 +/- 9.28 months) had positive SPT responses to latex. No children showed allergic reactions to latex. One or more positive SPT responses to aeroallergens were present in 340 children (28.9%); one or more positive SPT responses to food allergens were present in 26 (2.2%); one or more positive SPT responses to aeroallergens, food allergens, or both were present in 353 (30.0%); and one or more positive SPT responses to one or more insect venoms were present in 43 subjects (3.7%). Significant (p < 0.05) risk factors associated with latex sensitization included: positive SPT responses to aeroallergens, food allergens, or both; a positive response to one or more insect venoms; a positive response to mite, milk, egg white, wheat, honeybee venom, wasp venom, Polistes venom, or a combination thereof; and increased age. CONCLUSION: This report shows a very low prevalence of latex sensitization with an absence of clinical symptoms to latex. This study demonstrates a significant association between latex sensitization and the presence of one or more positive SPT responses to aeroallergens, food allergens, or both; one or more positive SPT responses to one or more insect venoms; and increased age.