Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

FemZone trial: a randomized phase II trial comparing neoadjuvant letrozole and zoledronic acid with letrozole in primary breast cancer patients.

BACKGROUND: The objective of this prospectively randomized phase II trial (Trial registration: EUCTR2004-004007-37-DE) was to compare the clinical response of primary breast cancer patients to neoadjuvant therapy with letrozole alone (LET) or letrozole and zoledronic acid (LET + ZOL). METHODS: Patients were randomly assigned to receive either LET 2.5 mg/day (n = 79) or the combination of LET 2.5 mg/day and a total of seven infusions of ZOL 4 mg every 4 weeks (n = 89) for 6 months. Primary endpoint was clinical response rate as assessed by mammogram readings. The study was terminated prematurely due to insufficient recruitment. We report here on an exploratory analysis of this data. RESULTS: Central assessment of tumor sizes during the treatment period was available for 131 patients (66 LET, 65 LET + ZOL). Clinical responses (complete or partial) were seen in 54.5% (95% CI: 41.8-66.9) of the patients in the LET arm and 69.2% (95% CI: 56.6-80.1) of those in the LET + ZOL arm (P = 0.106). A multivariate model showed an OR of 1.72 (95% CI: 0.83-3.59) for the experimental arm. CONCLUSION: No increase in the clinical response rate was observed with the addition of ZOL to a neoadjuvant treatment regimen with LET. However a trend towards a better reponse in the LET + ZOL arm could be observed. This trend is consistent with previous studies that have investigated the addition of ZOL to chemotherapy, and it may support the evidence for a direct antitumor action of zoledronic acid.

Anti-thyroid peroxidase antibodies are associated with the absence of distant metastases in patients with newly diagnosed breast cancer.

BACKGROUND: The presence of thyroid peroxidase antibodies (TPOab) are reported to be associated with improved outcome among breast cancer patients. We evaluated the correlation between TPOab and diagnostic parameters among newly diagnosed breast cancer patients. METHODS: Three hundred and fourteen newly diagnosed patients with breast cancer, diagnosed and treated in Bethesda Essen between January 2002 and June 2006, were included in this study; 258 (82.2%) without TPOab (≤100 IU/mL) and 56 (17.8%) with TPOab (>100 IU/mL). Blood analysis was performed to measure serum levels of carcinoembryonic antigen (CEA), cancer antigen 15-3 (CA-15-3), free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH) and TPOab by radioimmunoassay. Data regarding age, tumor size, grading, TNM classification, receptor status, lymph node, and distant metastases were collected and analyzed from patient reports. Statistics were performed using Pearson's χ2-test and logistic regression analysis. RESULTS: There were no incidences of distant metastasis among 56 patients with TPOab, whereas 17 (6.6%) of 258 cases without TPOab displayed distant metastases (p=0.04). Logistic regression showed an inverse association of TPOab with CA-15-3 and CEA levels (p<0.001, respectively). Both groups, with and without TPOab, revealed no significant differences with respect to age, tumor size, grading, TNM classification, fT3, fT4, and receptor status. TPOab positive patients had higher TSH levels (2.55±3.58), compared to TPOab negative cases (1.20±1.15) (p<0.001). CONCLUSIONS: TPOab occurrence is associated with significantly lower frequency of distant metastases in breast cancer. TPOab level inversely correlates with the conventional tumor markers CA-15-3 and CEA.

Mental strain and annoyance during cognitive performance in different traffic noise conditions.

In built-up areas, an increasing number of persons are affected by road traffic noise while performing mental work. This experimental study focused on annoyance and mental strain due to various noise scenarios. A total of 102 healthy, young persons (51 women, 51 men, aged 18-31 years) were randomly assigned to one of five experimental conditions determined by traffic flow (even, lumped) and traffic composition (20%, 40% heavy vehicles). While exposed to noise they performed a grammatical reasoning and a mathematical processing task. Performance and mental strain were not affected by any of the five noisy conditions. Individuals with high noise sensitivity were partially more annoyed and performed less than persons with low sensitivity. STATEMENT OF RELEVANCE: The present study provides information about mental strain due to tasks with different cognitive demands and the role of noise sensitivity in various traffic noise conditions. The results show that measures aiming at the reduction of the proportion of heavy vehicles should additionally consider particular traffic flow.

Impairing effects of noise in high and low noise sensitive persons working on different mental tasks.

OBJECTIVE: There is no information of mental strain in noise sensitive persons working under moderate levels of noise. The aim of the study was to determine relevant dimensions of impairing effects differentiating between noise sensitive and insensitive persons. METHODS: 56 participants (27 men, 29 women; 18-31 years old) were classified into 28 low and 28 high noise sensitive persons. They worked either on a grammatical reasoning task (GRT) or a mental arithmetic task (MPT) under realistic road traffic scenarios [LAeq 55 dB(A)]. Afterwards they rated their annoyance and subjective mental strain. RESULTS: A multivariate analysis of variance provided significant effects for the individual noise sensitivity (P < 0.01) and the kind of task being performed (P < 0.01). To determine to what extent the rating scales lead to a differentiation of the four groups, a 2-factorial discriminant analysis was carried out subsequently. Results showed, that psycho-physiological effort and emotional strain differentiated best between the noise sensitivity groups and psycho physiological effort and focus on tasks (attention) separated the tasks. CONCLUSION: Noise sensitive persons are more distracted by noise than insensitive persons. Furthermore the results suggest that noise sensitive subjects do not only evaluate a noisy situation as more annoying but experience higher levels of strain than insensitive persons.

Lowering of resting core temperature during acclimation is influenced by exercise stimulus.

The decrease in resting core temperature (T (co)) and its relation to the reduced physiological strain during heat acclimation was analysed with rectal temperature data measured in three groups of eight semi-nude persons (6 males, 2 females) who were acclimated for 15 consecutive days to dry, humid and radiant heat, respectively, with equivalent WBGT (33.5 degrees C), by performing 2-h treadmill work. A fourth group followed the same protocol for 12 days in a neutral climate. After acclimation, both resting T (co), prior to heat exposure, and final T (co), measured at the end of work, were significantly reduced. The reduction in final T (co) increased with decreasing ambient water vapour pressure and was higher for the data pooled over the heat conditions (0.46 +/- 0.31 degrees C) than in the neutral climate (0.21 +/- 0.25 degrees C), whereas resting T (co) declined similarly in the heat (0.20 +/- 0.25 degrees C) and the neutral environment (0.17 +/- 0.23 degrees C). The lowering of resting and final T (co) after heat acclimation showed a significant correlation (r = 0.67) and regression analysis showed that 37% of the average reduction in final T (co) was attributable to the lowering of resting T (co). The same analysis was applied after extending the database by short-term series of clothed persons (17 females, 16 males) acclimated at 29.5 and 31.5 degrees C WBGT for 5 days. A significant correlation was found between the lowering of resting and final T (co) (r = 0.57) that did not depend on climatic conditions and gender, although the reduction in resting T (co) was significantly smaller for females (0.06 +/- 0.22 degrees C) than for males (0.21 +/- 0.23 degrees C). It is concluded that the lowering of resting core temperature contributes to the reduced physiological strain during heat acclimation. Similar effects under neutral conditions point to the exercise stimulus as a probable explanation.

Factorial validity of the noise sensitivity questionnaire.

The Noise Sensitivity Questionnaire (NoiSeQ) aims at the measurement of global noise sensitivity as well as the sensitivity for five domains of everyday life namely 'Leisure', 'Work', 'Habitation', 'Communication' and 'Sleep'. The present investigation examined the factorial validity of the NoiSeQ to determine whether the items of the NoiSeQ cover the different factors as assumed. The analysis was done using the method of Confirmatory Factor Analysis (CFA). The linear structural model took into consideration only the scales of the NoiSeQ for which reliability could be demonstrated, namely, 'Sleep', 'Communication', 'Habitation' and 'Work'. The linear structural model presumed that each of the 28 items has a relation only to one corresponding factor. Furthermore, the model allowed for correlations between the four factors. The data base encompassed 293 persons. Parameter estimation was based on the General Least Square method. The data was checked with respect to the occurrence of multivariate outliers, deviation from multivariate normality and existing collinearities. The data met the overall requirements of a CFA. The evaluation of model fit was based on the relative chi2 -value, the Root Mean Square Error of Approximation, the Goodness of Fit Index, the Adjusted Goodness of Fit Index and the Root Mean Square Residual. All fit indices indicated an acceptable match of the model. As the postulated structure of the NoiSeQ was consistent with the empirical data, the classification of the items as well as the claimed interdependencies between the scales can be maintained. The regression weights of all items as well as the correlations between the latent variables were statistically significant. The estimated reliability of the latent variables took values of >/=0.84. The findings generally justified the conclusion that there is no urgent need to modify the four scales of the NoiSeQ thus, indicating the factorial validity of the NoiSeQ.

The reliability of the noise sensitivity questionnaire in a cross-national analysis.

Noise sensitivity is regarded as a relevant predictor for annoyance reactions. Since many studies have focused on noise sensitivity at an international level, the present analysis was conducted to detect national peculiarities concerning noise sensitivity. Using the approach of the generalizability theory, reliability of the noise sensitivity questionnaire was analyzed taking into consideration relevant facets assumed to contribute to the measurement error. A total of 126 individuals from seven European countries participated in this study. The reliability coefficients for the global noise sensitivity score ranged from 0.90 to 0.91. It was determined that the translated questionnaires are comparable.

The development of the noise sensitivity questionnaire.

The existing questionnaires for determining the noise sensitivity of individuals provide information only about global noise sensitivity, although empirical data suggest that measuring noise sensitivity for different situations in daily life might be more logical. Therefore, the "Noise-Sensitivity-Questionnaire" (NoiSeQ) was developed to measure global noise sensitivity as well as the sensitivity of five domains of daily life, namely, leisure, work, habitation, communication, and sleep. The assessment of the measurement characteristics was based on the Generalizability (G) theory. The results of the G-study (N=66) proved that a single application of the questionnaire is sufficient for determining an individual's noise sensitivity. Furthermore, the ratings are age and gender independent. The subsequently conducted Decision (D)-study (N=288) provides information on the reliability of NoiSeQ. If the questionnaire is used for measuring global noise sensitivity, the reliability (relative and absolute G-coefficient) reaches a value above 0.90. According to ISO 10075-3, the questionnaire satisfies the precision level 1 "accurate measurement" in this case. The G-coefficients for all the subscales exceed the lower limit 0.70, with the exception of subscale leisure, which did not prove satisfactory. However, this subscale can reach a reliability of more than 0.70 if additional items are included. The validity of the instrument was proven for the subscales habitation (N=72) and work (N=72). In both the studies, the participants were asked to rate the annoyance in the presence of several rail and traffic noise scenarios. The subjects were characterized as low and high noise sensitive according to their sensitivity values obtained from NoiSeQ. In conclusion, a significant difference in annoyance rates was observed between the low and high noise sensitive groups for both the subscales habitation and work. This data support the validity of NoiSeQ.

Clinical response by palpation during primary systemic therapy with four dose-dense cycles doxorubicin and docetaxel in patients with operable breast cancer: further results from a randomised controlled trial.

Primary systemic therapy (PST) allows the observation of tumour response under treatment, but little is known regarding the typical course of clinical response during such therapy. The aim of this study is to support decision making in case of insufficient clinical response. Tumour response was assessed by palpation at different times in 436 patients with operable breast cancer from the dose-dense biweekly therapy arm of the GEPARDUO phase III trial. The predictive value of clinical response for pathologic complete response (pCR), prognostic models to assess the prognosis and individual courses of clinical response were investigated. Sensitivity and positive predictive value were low, but comparatively highest after the 3rd cycle. The predictive value of clinical response by palpation for pCR was subsequently limited. The majority of patients (68.1%) experienced a consistent decrease in tumour size during PST. The results indicate that decisions about further treatment should take place at the earliest after the 3rd cycle or 6 weeks of dose-dense PST.

Doxorubicin with cyclophosphamide followed by docetaxel every 21 days compared with doxorubicin and docetaxel every 14 days as preoperative treatment in operable breast cancer: the GEPARDUO study of the German Breast Group.

PURPOSE: Dose-dense and sequential administration of cytotoxic drugs are current approaches to improve outcomes in patients with early-stage breast cancer. METHODS: This phase III study investigated 913 women with untreated operable breast cancer (T2-3, N0-2, M0) randomly assigned to receive either doxorubicin 50 mg/m2 plus docetaxel 75 mg/m2 every 14 days for four cycles with filgrastim support (ADOC), or doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2 every 21 days followed by docetaxel 100 mg/m2 every 21 days for four cycles each (AC-DOC). The primary end point was the incidence of pathologic complete (invasive and noninvasive) response (pCR) in the breast and axillary nodes. Secondary end points were predictors for pCR, clinical response, rate of breast conservation, and safety. RESULTS: A pCR was achieved in 94 patients (10.6%), but the likelihood was significantly greater with AC-DOC (14.3%; n = 63) than with ADOC (7.0%; n = 31) (odds ratio, 2.22; 90% CI, 1.52 to 3.24; P < .001). Independent predictors of attaining a pCR included the use of sequential therapy, high tumor grade, and negative hormone receptor status. The response rates detected by palpation and by imaging were significantly higher with AC-DOC (85.0% and 78.6%, respectively) than with ADOC (75.2% and 68.6%, respectively; both P values < .001). The rate of breast-conserving surgery was 63.4% for AC-DOC and 58.1% for ADOC (P = .05). Predominant grade 3/4 toxicities were leucopenia (AC-DOC, 74.2%; ADOC, 53.7%) and neutropenia (AC-DOC, 66.4%; ADOC, 44.7%) but were infrequently associated with fever (AC-DOC, 4.6%; ADOC, 3.1%). CONCLUSION: Sequential AC-DOC is more effective at inducing pCR than dose-dense ADOC as preoperative treatment for patients with operable breast cancer.

Dose-dense biweekly doxorubicin/docetaxel versus sequential neoadjuvant chemotherapy with doxorubicin/cyclophosphamide/docetaxel in operable breast cancer: second interim analysis.

Timing of systemic treatment in primary operable breast cancer is subject to extensive investigation, suggesting that pathologic complete remission (pCR) might improve survival in this setting. The German Adjuvant Breast Cancer Group previously demonstrated the feasibility of a dose-dense biweekly schedule of 4 cycles doxorubicin 50 mg/m2 and docetaxel 75 mg/m2 (ddAT) +/- tamoxifen in the neoadjuvant setting to yield a pCR of 9.7% (Gepardo trial). Patients assigned to ddAT received prophylactic granulocyte colony-stimulating factor support (5 micro g/kg days 5-10). The current study (GeparDUO) was designed to assess whether the pCR rate, including no viable invasive and preinvasive tumor cells, achieved with ddAT was equivalent to sequential administration of doxorubicin/cyclophosphamide followed by docetaxel (AC-DOC) over 24 weeks in primary operable breast cancer. From June 1999 to September 2001, 913 patients were enrolled in this trial. In total, 395 patients randomized before August 1, 2000, were included in the second interim analysis. Safety data were available from 369 patients (ddAT, n = 191; AC-DOC, n = 178) demonstrating that toxicity of both regimens was tolerable. Grade 3/4 neutropenia occurred in 39.8% of patients receiving ddAT and in 69.3% of patients treated with AC-DOC. Efficacy data were available in 378 patients. A pCR occurred in 14.8% of the primary breast tumors. According to the recommendations of the data monitoring committee, recruitment to the study was halted as of September 2001 (n = 913/1000) due to the significant difference in pCR rates observed between the treatment arms. Surgery was documented in 380 patients. Breast conservation was possible in 288 cases (75.8%). The application of both schedules is safe and feasible in an outpatient setting. Although, results obtained from this interim analysis are encouraging, caution is recommended until the results obtained show statistical difference in pCR.