Thus, precedents from other systems support our findings that spe

Thus, precedents from other systems support our findings that spectrin, adducin, and p4.1 can act independently during bacterial pathogenesis. Conclusions Invasion of intestinal epithelial cells and comet tail-based motility in host cells are key for S. flexneri to access replicative niches and disseminate throughout host tissues [2]. Here we have demonstrated that the actin-rich structures generated by these microbes also employ another cytoskeletal system, the spectrin cytoskeleton. Our identification of this structural network at these sites further highlights the importance of this system in bacterial pathogenesis and

indicates that these crucial segments in the pathogenesis of S. flexneri require a hybrid cytoskeletal meshwork, previously thought to be exclusive to actin. Methods Cells, bacteria and growth conditions HeLa cells (ATCC) were grown on #1 cover slips in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The bacterial strain utilized was S. flexneri (strain M90T). Bacteria were grown in standard trypticase soy. Infections HeLa cells were grown to approximately 70% confluency prior to infections. S. flexneri were grown overnight in standing culture, then diluted 80×, followed by growth in shaking

selleck culture at 37°C for 2.5 hours (OD600 nm = 0.6) after which 400 μl of the culture was added to the cells with 200 μl of growth media [31]. Infections were initiated by centrifugation for 10 mins at 700 g and 21°C. To quantify invasion events, investigate initial tail formation and study comet tails, total infection times consisted of 0.5, 2.5 and 4.5 hours respectively. For classical invasion assays, cells were washed 2× with PBS after 20 minutes of infections and incubated in 100 ug/mL of gentamycin in 10% DMEM for 1 hour. Cells were washed 3× with PBS, lysed using 1% triton and plated for CFU counts. Invasion assays examined by microscopy

To quantify S. flexneri invasion, mafosfamide similar infection parameters were followed as in the classical invasion assay, however after 1 hour of gentmycin treatment the cells were washed with PBS three times prior to fixation and quantification of bacterial invasion via microscopy. Immunofluorescence Immunofluorescence procedures were performed as described previously [20]. Briefly, samples were fixed using 3% paraformaldehyde for 15 minutes then permeabilized using 0.1% Triton X-100 in PBS (without calcium or magnesium) (Hyclone) for 5 minutes. Prior to primary antibody treatments, samples were blocked in 5% normal goat serum in TPBS/0.1% BSA (0.05% Tween-20 and 0.1% BSA in PBS) for 20 minutes. Antibodies were then incubated on the cover slips overnight at 4°C. The next day secondary antibodies were applied for 1.5 hrs at 37°C. The cover slips were then mounted on glass slides using Prolong Gold with DAPI (Invitrogen).

One of our sequences affiliated with Crenarchaea cluster 1 1b, wh

One of our sequences affiliated with Crenarchaea cluster 1.1b, which includes several putative AOA [54–56]. However, it has recently been shown that not all amoA-carrying Thaumarchaeota are ammonia-oxidizing autotrophs [57]. The presence

of AOA at the Rya WWTP can therefore not be confirmed, and as has been suggested for other WWTPs [14, 16], AOA are most likely of minor or no importance for ammonia-oxidation at the Rya WWTP. One clone affiliated with Crenarchaea cluster 1.3. There are no cultured representatives of cluster 1.3, but spatial co-localization [58] and a relation between the abundance of cluster 1.3 and Methanosaeta-like species has been reported [42]. In other aggregate structures, such as anaerobic sludge Adriamycin mouse PI3K Inhibitor Library granules, Methanosaeta are important for structure and stability and they form dense aggregates which act as nuclei for granule formation [20]. In the activated sludge the Methanosaeta did not appear to have this function as they were mostly detected as small colonies or Mocetinostat solubility dmso single cells (Figure  11) and there was no apparent difference

in structure between flocs with high and low numbers of Methanosaeta. The lowest relative abundances of the Methanosaeta-like TRFs were observed in January and February 2004 (Figures  7 and 8). In October 2003 the two main Methanosaeta TRFs also decreased in relative abundance but it cannot be ruled out that the TRFs that appeared in those samples were also Methanosaeta (Table 4). The lowest water temperatures of the period were recorded during January and February 2004, which could have

reduced the survival or proliferation of Methanosaeta-like species and allowed other Archaea to increase. In anaerobic sludge, a decrease in Methanosaeta abundance has Adenosine been linked to granule disintegration [18, 19]. Although the flocs had high shear sensitivity and a more open structure in January and February 2004 when the Methanosaeta TRFs decreased and although there was a significant negative correlation between Methanosaeta TRFs and effluent non-settleable solids (Table 6) it cannot be concluded that the Archaea are important for the floc structure. The increased shear sensitivity and changed floc structure in January and February 2004 could be due to the reduced general microbial activity, which has been shown to decrease floc stability [5]. Furthermore, increased shear sensitivity and changed floc structure was also observed from June to August 2004, after the primary settlers were bypassed, but during this period the relative abundance of the Methanosaeta TRFs was 100%. Thus, if the composition of the Archaea community has any effect on floc structure or stability it is certainly only one of many other factors. Conclusions By sequencing and T-RFLP analysis of 16S rRNA genes and FISH we showed that Archaea were present in the activated sludge of a full-scale WWTP.

0) or exchanged by repetitive concentration/dilution using 30 kDa

0) or exchanged by repetitive concentration/dilution using 30 kDa Centricon or Microcon filters into 2-(N-morpholino)ethanesulfonic acid (MES) pH 6.5 or (2-[N-cyclohexylamino]ethane sulfonic acid (CHES) pH 9.5. Finally, the samples were concentrated to an OD802 of 80–130. CW X-band EPR measurements were performed with a Bruker ESP 300 spectrometer at room temperature using a rectangular cavity

with optical access (TE102, ER 4102ST, Bruker), using a capillary with 1 mm inner diameter. The radical cation P•+ was created via continuous illumination with white light in situ, using heat-absorbing glass and water filters. CW X-band Special TRIPLE measurements were done on the same spectrometer at 288 K. A home-built ENDOR cavity was used, similar to the one previously described (Zweygart et al. 1994),

but with a nitrogen gas cooling system. The cation radical P•+ was created in situ as described above. The data analysis was performed selleck compound using home-written routines in Matlab™, similar to the program used before (Tränkle and Lendzian 1989). In several cases, a baseline was recorded under identical conditions (with the magnetic field off-resonant and subtracted) under the assumption that possible drifts and artifacts would be the same in both cases. Q-band EPR and ENDOR measurements in frozen solution were done on a Bruker Elexsys E580 spectrometer at 80 K. For frozen solution experiments, sucrose (60%) was added to all samples. A home-built resonator was used (Silakov et al. 2007), similar to the one described previously SBI-0206965 order (Sienkiewicz et al. 1996). A Davies-type pulse ENDOR experiment (Davies 1974) was performed as described previously (Epel et al. 2006). Results X-band EPR measurements Measurements using the X-band EPR spectrometer were performed for both wild-type RCs and the four mutants, ND(L170), HE(L168), ND(M199), and HE(L168)/ND(L170), in liquid solution. In all cases, the spectrum was a single unresolved line centered at g

close to g e (see Fig. 2 for an example). Fig. 2 Comparison of CW X-band EPR spectra of light-induced P•+ in RCs from Rb. sphaeroides wild type with hepta-histidine tag (WT-H7) (red line) and from ND(L170) (blue line) at pH 8.0 For wild-type RCs at pH 8.0, the spectrum was simulated using a Gaussian before function with a linewidth ΔB pp (peak-to-peak) of 9.6 G (±0.2 G) at g = 2.0026 in agreement with published data of this radical in RCs from Rb. sphaeroides 2.4.1 (see for example Feher et al. 1975; Norris et al. 1971; Artz et al. 1997). The spectrum of the four mutant RCs at pH 8.0 were fitted yielding the same selleck chemicals llc g-value and different Gaussian linewidths. For all of the mutants, the EPR linewidth was increased relative to wild type. The linewidth is smallest for the ND(M199) mutant (10.1 G), followed by the HE(L168) mutant (10.2 G), with the ND(L170) mutant and the double mutant HE(L168)/ND(L170) having the most pronounced increase (11.0 G).

Infect Immun 1983,41(3):1212–1216 PubMed 12 Paton JC, Rowan-Kell

DNA Damage inhibitor Infect Immun 1983,41(3):1212–1216.PubMed 12. Paton JC, Rowan-Kelly B, Ferrante A: Activation of human complement by the pneumococcal toxin pneumolysin. Infect Immun 1984,43(3):1085–1087.PubMed 13. Boulnois GJ, Paton JC, Mitchell TJ, Andrew PW: Structure and function of pneumolysin, the multifunctional, thiol-activated

toxin of Streptococcus pneumoniae. Mol Microbiol 1991,5(11):2611–2616.PubMedCrossRef 14. Hammerschmidt S, Bethe G, Remane PH, Chhatwal GS: Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae. Infect Immun 1999,67(4):1683–1687.PubMed 15. Janulczyk R, Iannelli F, Sjoholm AG, Pozzi G, Bjorck L: Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function. J Biol Chem 2000,275(47):37257–37263.PubMedCrossRef 16.

Romanello V, Marcacci M, Dal Molin F, Moschioni buy AZD9291 M, Censini S, Covacci A, Baritussio AG, Montecucco C, Tonello F: Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease. Protein Expr Purif 2006,45(1):142–149.PubMedCrossRef 17. King SJ, Hippe KR, Gould JM, Bae D, Peterson S, Cline RT, Fasching C, Janoff EN, Weiser JN: Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in MLN2238 vivo and protect the airway. Mol Microbiol 2004,54(1):159–171.PubMedCrossRef 18. Holmes AR, McNab R, Millsap KW, Rohde M, Hammerschmidt S, Mawdsley JL, Jenkinson HF: The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. Mol Microbiol 2001,41(6):1395–1408.PubMedCrossRef 19. Zhang JR, Mostov KE, Lamm ME, Nanno M, Shimida S, Ohwaki M, Tuomanen E: The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. PLEK2 Cell 2000,102(6):827–837.PubMedCrossRef 20. Anderton JM, Rajam G, Romero-Steiner S, Summer S, Kowalczyk AP, Carlone GM, Sampson JS, Ades EW: E-cadherin is a receptor for the common protein

pneumococcal surface adhesin A (PsaA) of Streptococcus pneumoniae. Microb Pathog 2007,42(5–6):225–236.PubMedCrossRef 21. Lu L, Ma Y, Zhang JR: Streptococcus pneumoniae recruits complement factor H through the amino terminus of CbpA. J Biol Chem 2006,281(22):15464–15474.PubMedCrossRef 22. Hammerschmidt S, Tillig MP, Wolff S, Vaerman JP, Chhatwal GS: Species-specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif. Mol Microbiol 2000,36(3):726–736.PubMedCrossRef 23. Bergmann S, Rohde M, Chhatwal GS, Hammerschmidt S: alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol Microbiol 2001,40(6):1273–1287.PubMedCrossRef 24. Bergmann S, Rohde M, Hammerschmidt S: Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pneumoniae is a surface-displayed plasminogen-binding protein. Infect Immun 2004,72(4):2416–2419.PubMedCrossRef 25.

Also, minor errors (any false

Also, minor errors (any false VX-680 concentration result involving an intermediate result), major errors (false-resistant results) and very major errors (false-susceptible results) were calculated. Statistical analysis Bacterial load in ID broth for GPC and GNR was compared using an independent samples t-test. Results Inoculum of bacteria in ID broth after use of serum separator tubes (SSTs) In total, 134 blood cultures were included,

from 116 patients. The inoculum of GPC in ID broth was on average 3.6 × 107 CFU/ml, whereas that of GNR was 1.8 × 108 CFU/ml, which was a TSA HDAC molecular weight significant difference (95% CI between -1.7 × 108 and -1.2 × 108; P < 0.001). ID of GNR with the direct Phoenix method NSC23766 cost ID with direct inoculation was correct for 95.2% of all tested Enterobacteriaceae. One Escherichia coli strain was incorrectly identified as Salmonella choleraesuis with the direct method. One Serratia marcescens strain could not be identified with the direct method. Identification for Pseudomonas spp. was correct in 71.4%. Both errors in this group involved strains of Pseudomonas aeruginosa that were incorrectly identified as Pseudomonas fluorescens (Table 1). No errors in ID were observed

for the routine method. Table 1 Results of identification of GNR with the direct method   Total no. of strains No. of unidentified strains No. of misidentified strains ID of misidentified strains Enterobacteriaceae         E. coli 26   1 Salmonella choleraesuis K. pneumoniae spp. pneumoniae 8       S. marcescens 4 1     K. oxytoca 1       P. mirabilis 1  

    E. cloacae 1       M. morganii 1       Non-fermenters         P. aeruginosa 7   2 Pseudomonas fluorescens Antibiotic susceptibility testing (AST) of GNR Results of AST were available for 49 strains, one P. aeruginosa strain failed to grow sufficiently in the Phoenix system so no results were available for the direct method. Categorical agreement of the direct method with results of the standard method for GNR was 97.6%. After discrepancy analysis of the results of AST, this percentage rose to 99.0%, with 5 minor errors (0.7%), no major errors, the and 2 very major errors (0.3%) (Table 2). Both very major errors occurred with trimethoprim-sulfamethoxazole in Pseudomonas aeruginosa strains. Categorical agreement of the standard method after discrepancy analysis was 98.4% (table 2). One very major error occurred with trimethoprim-sulfamethoxazole. No antibiotic showed a categorical agreement of <95% (Table 3). Table 2 Agreements and errors for AST of GPC and GNR for the direct and routinely used Phoenix method   Direct vs routinely used method Direct method after discrepancy analysis Routine method after discrepancy analysis GPC (n = 84)       Categorical agreement 93.1% 95.4% 97.3% Minor errors 1.7% 1.1% 0.7% Major errors 4.2% 3.1% 0.8% Very major errors 0.9% 0.4% 1.

The amplification of the partial gap gene

for all of the

The amplification of the partial gap gene

for all of the Staphylococcus species (sequence similarity ranges from 24.3 to 96%) yield a single product of nearly 931 bp [19]. The sequence similarity of the gap sequences ranged from 24.3 to 96% [19]. In fact, in our analyses the second closest strain was S. haemolyticus (data not shown), which has a gap gene Captisol mw sequence similarity of 27% [19] with S. lugdunensis[19]. We found that among the 8 isolates positive in both PYR and ODC tests, 5 were S. lugdunensis and the other three were S. haemolyticus. This may due to S. haemolyticus being weakly positive for ODC, which is consistent with previous results [27]. Of the 5 isolates of S. lugdunensis identified in this study, 3 were obtained from wound, breast, and cervix secretions, suggesting that skin and soft tissue infections account for H 89 price a prominent number of the total infections caused by S. lugdunensis, which is consistent with previous results [17]. One isolate was from the synovial fluid of the patient with a joint infection. Frank et al. reported that this bacterium infects artificially replaced joints [28] and it accounts for 4% of all joint infections [29]. Another isolate was

from the venous blood of a newborn baby with pneumonia. Tee et al. previously reported a case of neonatal pneumonia caused by this bacterium but that case suffered from a catheter-related blood infection [8]. Consistent with previous results [13], S. lugdunensis was not isolated from any sputum cultures in this study, which may be due to inability of this bacterium to colonize the respiratory tract. Four out of the five S. lugdunensis isolates identified in this study produced β-lactamase (Table 3), which indicates an incidence of 80% that is much Rebamipide higher than the incidence in other countries [17], including 7-24% in France, 24-40% in the U.S., 12% in Spain,

and 15% in Sweden. Of note, our small number of positive isolates might have potentially biased such estimations. Only one out of the five isolates was not KPT-330 purchase resistant to the antimicrobial drugs tested, three were resistant to erythromycin, clindamycin, and penicillin, and one was resistant to cefoxitin and penicillin (Table 3). We found that the three isolates resistant to erythromycin were positive for the ermC gene but not the ermA or ermB gene; and the isolate resistant to cefoxitin was positive for the mecA gene; the later was only reported a few times in the previous studies [8, 30, 31]. We further found that the rate of antibiotic resistance of S. lugdunensis is more severe in China than in other countries and primarily presented as multi-drug resistance, again such an inference might suffer from potential bias due to the sample size of the confirmed isolates. We performed PFGE in order to determine the epidemiological characteristics of S.

Implications for practice Self-report measures of a work-related

Implications for practice Self-report measures of a work-related illness are used to estimate the prevalence of a work-related disease and the differences in prevalence between populations, such as different occupational groups representing

different exposures. From this review, we know that prevalence estimated with symptom questionnaires was mainly higher than prevalence estimated with the reference standards, except for hand eczema and respiratory disorders. If prevalence Dinaciclib was estimated with self-diagnosis questionnaires, questionnaires that use a combined score of health symptoms, or for instance use pictures to identify skin diseases, they tended to agree more with the prevalence based on the reference standard. The choice for a certain type of questionnaire depends also on the expected prevalence of the health condition in the target population. If the expected prevalence in the target population is high enough (e.g., over 20%), a self-report measure with high specificity (>0.90) and acceptable sensitivity (0.70–0.90) may be the best choice. It will reflect the “true” prevalence because it will find many true cases with a limited PF299 number Crenigacestat ic50 of false negatives. But if the expected prevalence is low (e.g., under 2%), the same self-report measure will overestimate the “true” prevalence considerably; it will successfully identify

most of the non-cases but at the expense of a large number of false positives. This holds equally true if self-report is used for case finding in a workers’ health surveillance program. Therefore, when choosing a self-report questionnaire for this purpose, one should also take into account other aspects of the

target condition, including the severity of the condition and treatment possibilities. If in workers’ health surveillance it is important to find as many cases as possible, the use a sensitive symptom-based self-report questionnaire (e.g., the NMQ for musculoskeletal disorders or a symptom-based questionnaire for skin problems) is recommended, under the condition of a follow-up including a medical examination Sclareol or a clinical test able to filter out the large number of false positives (stepwise diagnostic procedure). Although the agreement between self-assessed work relatedness and expert assessed work relatedness was rather low on an individual basis, workers and physicians seemed to agree better on work relatedness compared with the non-work relatedness of a health condition. Adding well-developed questions to a specific medical diagnosis exploring the relationship between symptoms and work may be a good strategy. Implications for research In the validation of patients’ and workers’ self-report of symptoms, signs, or illness, it is necessary to find out more about the way sources of heterogeneity like health condition, type of self-report, and type of reference standard influence the diagnostic accuracy of self-report.

Moreover the adhesiogenic power of BP is absolutely lower than th

Moreover the adhesiogenic power of BP is absolutely lower than the one of the other synthetic materials [13, 14]. On the contrary there are a few doubts about the intra-peritoneal use of BP from the biomechanical point of view. It has been demonstrated AZD6244 ic50 that the best integration is reached if they are placed pre-peritoneally with a greater incorporation strength, less adhesion area and lower adhesion scores compared with intra-peritoneal placement [15]. Given that the long-term persistence of the prosthesis is crucial, some authors stated that the BP durability

has a direct impact on the recurrence rate [16]. However durability depends on the implant intrinsic properties and also on the environment into which the BP are placed [16]. It has been demonstrated in animal models as the tensile strength is different between cross-linked and non-cross-linked meshes during the first months after the implant. However it reaches similar values after 12 months with the two kind of implants [8]. Moreover the strength of the repair sites doesn’t change over time. This might indicate that new tissue is deposited in the repair site as the scaffold is

degraded, preventing the site from weakening over time [8]. Another factor that should be kept into account in choosing which kind of BP to use is the demonstration that non-cross-linked material exhibits more favourable remodeling Fosbretabulin mouse characteristics [8]. This has a great importance when BP are used as bridging or alternatively as reinforcement. In fact discordant data have been published about the use of BP to bridge wide defects [16]. Few different non-randomized studies have been published reporting recurrence rate ranging between 100% and 0% if the prosthesis are placed respectively either as a bridge or not [16–19]. Even if high-quality comparative data about BP exist in animal models, only clinical reports of a restricted number of cases are reported for humans. Moreover only the recurrence rate is registered as outcome in almost all studies. Other data regarding the use of BP as wound classification, contamination risk/grade, associated therapy or comorbidity

are seldom reported. These data are needed to completely assess the usefulness, the efficacy Protein kinase N1 and the versatility of BP. All reported data derived by retrospective uncontrolled series of limited number of patients. The methodology is seldom reported and/or poorly described. Moreover the time to recurrence is rarely evaluated [16]. One last observation is that the different studies reported data about non-homogeneous cohorts of patients. Different surgical techniques, different surgeons’ skill and expertise in using BP and different hernia sites are often mixed together. These inconsistencies are PI3K inhibitor probably due to the poorness of cases for each single centre. No definitive evidence based conclusions could be obtained from the literature.

FA authored the manuscript EB edited the manuscript EB provided

FA authored the manuscript. EB edited the manuscript. EB provided patient care. TD was the attending physician who cared for the patient, instigated the study, edited the manuscript, and oversaw the project. All authors read and approved the final manuscript.”
“Introduction Injury is a major public health problem in terms of mortality, morbidity and disability and it has been largely demonstrated that the organisation of a regionalised EVP4593 purchase trauma System significantly decreases the deleterious impact of severe trauma on population [1, 2]. In Europe the inclusive trauma system model has gained dominance.

In this Ruboxistaurin nmr model a network of hospitals with different resources takes care of trauma patients suffering from any among the full spectrum of injuries [3]. Epidemiologic information based on the entire population in a given region and understanding the number of severely injured VEGFR inhibitor that need to be admitted to a level one hospital, is of pivotal importance in the design of an inclusive Trauma System. With this objective, methodological approaches in measuring incident rates should use large representative samples of the whole population, to offer the potential to observe data on all the people living

in a region or a nation. Trauma registries contain detailed information, but this is offset by the limitation of including only patients treated at trauma centre and already triaged as “severe” at a dedicated trauma unit. On the contrary, population-based registries have usually been recorded for many years and are

available for time periods before changes of the Healthcare system. Additionally, they contain readily available, alphanumeric-coded information and allow easy and low cost analysis. Moreover, population-based registries may be used to investigate resources consumption and evaluate costs of the system. Recently, many investigators have started to use large databases for quality assessment studies in trauma care, and these works are classified as providing “high end” Class III evidence [4–8]. The objective of this study was to perform an exhaustive analysis of severe trauma patients hospitalised in Lombardia, a mixed rural/industrial region of northern Italy. Mirabegron The hospital discharge registry, a population-based record of all hospitalised people of the country, has been used as source of data. All hospital admissions for injuries during a three years period have been included and severely injured patients have been extrapolated. This analysis may be a useful starting point for evaluating the need for resources and costs of regional Trauma System implementation. Methods Lombardia is a mixed rural/industrial region of the northern Italy, with an area of 24,000 Km [2] (9,302 square miles), with Alpi Mountains in the north and hill or flat in the south. Residents, evaluated at the end of 2010, were 9,737,074 (1,046 persons per square mile), 48.87% males, and Milano is the capital city.

1972) In addition, Arabidopsis thaliana is studied because it is

1972). In addition, Arabidopsis C59 wnt price thaliana is studied because it is widely used as one of the model organisms in plant sciences. Materials and methods Fluorescence lifetime imaging microscopy Multiphoton imaging was performed on a multiphoton dedicated Biorad Radiance 2100 MP system, coupled to a Nikon TE300 inverted microscope (Borst et al. 2003). A tunable Ti-Sapphire laser (Coherent Mira) was used as an excitation source which was pumped with a 5-Watt (Coherent) Verdi laser, resulting in excitation

pulses of ~200 fs at a repetition rate of 76 MHz. In the beam-conditioning unit (BCU), the excitation power was tuned by a pockell cell. The laser beam was collimated in the scanhead and focused by a Nikon 60x water immersion Apochromat objective lens (NA 1.2) into the sample. The fluorescence was detected by non-descanned direct detectors

(NDDs), which were coupled to the sideport of the microscope. Using this type of detection, Selleckchem BIBF 1120 the loss of fluorescence light was reduced, and 3–5 times more signal was obtained compared to internal detectors. Selleckchem VX-680 The emission light was split into two channels using a dichroic mirror filter wheel. FLIM measurements were performed by directing the fluorescence via a secondary dichroic (770DCXR, Chroma Technology Corp.) into a Hamamatsu R3809U photomultiplier, operated at 3.1 kV. Fluorescence was selected using a dichroic (FF 495—DiO2, Semrock) and 2x a bandpass filter (HQ700/75, Chroma Technology Corp). In the excitation branch, a longpass filter (RG 780 3 mm, Schott) was used for reduction of the excitation light. The multichannel-plate photomultiplier allows single photon detection at high time-resolution, with an IRF of 25 ps (van triclocarban Oort et al. 2008, 2009). The output of the detector was coupled to a Becker & Hickl single-photon-counting module (SPC 830) (Becker and Bergmann 2002). The signal

from the Hamamatsu triggers the START of the time ramping for the time-correlated single-photon-counting (TCSPC). The pulses from the Ti-Sapphire laser serve as the SYNC signal to stop the time ramping and allowing the timing of the arrival of the fluorescent photons. The time window (ADC) was set to 1,024 channels and typically fluorescence was recorded for 2 min at a photon count rate of approximately 20 kHz. The signal from the PMT is combined with the pixel clock and line predivider signals from the Biorad scanhead to create 2D lifetime images. Fluorescence decay curves were fitted to a sum of N exponentials Σaiexp(−τ/τ i ) (i runs from 1 to N), convoluted with the IRF (Digris et al. 1999, van Oort et al. 2008, 2009), which was determined from the decay of pinacyanol iodide in methanol. From these results, an average lifetime <τ> was also calculated, according to <τ> = Σa i τ i . The number of counts in the peak channels is ~100 in the fluorescence intensities images and traces.