RESUMEN
Fluorescence lifetime imaging microscopy (FLIM) is an essential tool in many scientific fields such as biology and medicine thanks to the known advantages of the fluorescence lifetime (FLT) over the classical fluorescence intensity (FI). However, the frequency domain (FD) FLIM technique suffers from its strong dependence on the reference and its compliance to the sample. In this paper, we suggest a new way to calculate the FLT by using the crossing point (CRPO) between the modulation and phase FLTs measured over several light emitting diode (LED) DC currents values instead of either method alone. This new technique was validated by measuring homogeneous substances with known FLT, where the CRPO appears to be the optimal measuring point. Furthermore, the CRPO method was applied in heterogeneous samples. It was found that the CRPO in known mixed solutions is the weighted average of the used solutions. While measuring B16 and lymphocyte cells, the CRPO of the DAPI compound in single FLT regions was measured at 3.5 ± 0.06 ns and at 2.83 ± 0.07 ns, respectively, both of which match previous reports and multi-frequency analyses. This paper suggests the CRPO as a new method to extract the FLT in problematic cases such as high MCP gains and heterogeneous environments. In traditional FD FLIM measurements, the variation in phase angle and modulation are measured. By measuring over varying DC currents, another variation is detected in the FLT determined through the phase and modulation methods, with the CRPO indicating the true FLT.
Asunto(s)
Microscopía Fluorescente , Imagen Óptica , Animales , Células Cultivadas , Linfocitos , Melanoma ExperimentalRESUMEN
OBJECTIVE: A hospital readmission is defined as an admission to a hospital within a certain time frame, typically thirty days, following a previous discharge, either to the same or to a different hospital. Because most patients are not readmitted, the readmission classification problem is highly imbalanced. MATERIALS AND METHODS: We developed a hospital readmission predictive model, which enables controlling the tradeoff between reasoning transparency and predictive accuracy, by taking into account the unique characteristics of the learned database. A boosted C5.0 tree, as the base classifier, was ensembled with a support vector machine (SVM), as a secondary classifier. The models were induced and validated using anonymized administrative records of 20,321 inpatient admissions, of 4840 Congestive Heart Failure (CHF) patients, at the Veterans Health Administration (VHA) hospitals in Pittsburgh, from fiscal years (FY) 2006 through 2014. RESULTS: The SVM predictions are characterized by greater sensitivity values (true positive rates) than are the C5.0 predictions, for a wider range of cut off values of the ROC curve, depending on a predefined confidence threshold for the base C5.0 classifier. The total accuracy for the ensemble ranges from 81% to 85%. Different predictors, including comorbidities, lab values, and vitals, play different roles in the two models. CONCLUSIONS: The mixed-ensemble model enables easy and fast exploratory knowledge discovery of the database, and a control of the classification error for positive readmission instances. Implementation of this ensembling method for predicting all-cause hospital readmissions of CHF patients allows overcoming some of the limitations of the classifiers considered individually, and of other traditional ensembling methods. It also increases the classification accuracy for positive readmission instances, particularly when strong predictors are not available.
Asunto(s)
Hospitalización , Readmisión del Paciente , Máquina de Vectores de Soporte , Predicción , Insuficiencia Cardíaca , Humanos , Curva ROC , Factores de TiempoRESUMEN
Recent developments in the field of fluorescence lifetime imaging microscopy (FLIM) techniques allow the use of high repetition rate light sources in live cell experiments. For light sources with a repetition rate of 20-100 MHz, the time-correlated single photon counting (TCSPC) FLIM systems suffer serious dead time related distortions, known as "inter-pulse pile-up". The objective of this paper is to present a new method to quantify the level of signal distortion in TCSPC FLIM experiments, in order to determine the most efficient laser repetition rate for different FLT ranges. Optimization of the F -value, which is the relation between the relative standard deviation (RSD) in the measured FLT to the RSD in the measured fluorescence intensity (FI), allows quantification of the level of FI signal distortion, as well as determination of the correct FLT of the measurement. It is shown that by using a very high repetition rate (80 MHz) for samples characterized by high real FLT's (4-5 ns), virtual short FLT components are added to the FLT histogram while a F -value that is higher than 1 is obtained. For samples characterized with short real FLT's, virtual long FLT components are added to the FLT histogram with the lower repetition rate (20-50 MHz), while by using a higher repetition rate (80 MHz) the "inter-pulse pile-up" is eliminated as the F -value is close to 1.
Asunto(s)
Artefactos , Imagen Óptica/métodos , Animales , Línea Celular Tumoral , Supervivencia Celular , Eritrosina/metabolismo , Fluoresceína/metabolismo , Colorantes Fluorescentes/metabolismo , Fotones , Ratas , Relación Señal-Ruido , Factores de TiempoRESUMEN
In fluorescence fluctuation polarization sensitive experiments, the limitations associated with detecting the rotational timescale are usually eliminated by applying fluorescence correlation spectroscopy analysis. In this paper, the variance of the time-averaged fluorescence intensity extracted from the second moment of the measured fluorescence intensity is analyzed in the short time limit, before fluctuations resulting from rotational diffusion average out. Since rotational correlation times of fluorescence molecules are typically much lower than the temporal resolution of the system, independently of the time bins used, averaging over an ensemble of time-averaged trajectories was performed in order to construct the time-averaged intensity distribution, thus improving the signal-to-noise ratio. Rotational correlation times of fluorescein molecules in different viscosities of the medium within the range of the anti-bunching time (1-10 ns) were then extracted using this method.
RESUMEN
In time-correlated single photon counting (TCSPC) systems, the maximum signal throughput is limited by the occurrence of pile-up and other effects. In many biological applications that exhibit high levels of fluorescence intensity (FI), pile-up-related distortions yield serious distortions in the fluorescence lifetime (FLT) calculation as well as significant decrease in the signal-to-noise ratio (SNR). Recent developments that allow the use of high-repetition-rate light sources (in the range of 50-100 MHz) in fluorescence lifetime imaging (FLIM) experiments enable minimization of pile-up-related distortions. However, modern TCSPC configurations that use high-repetition-rate excitation sources for FLIM suffer from dead-time-related distortions that cause unpredictable distortions of the FI signal. In this study, the loss of SNR is described by F- value as it is typically done in FLIM systems. This F-value describes the relation of the relative standard deviation in the estimated FLT to the relative standard deviation in FI measurements. Optimization of the F-value allows minimization of signal distortion, as well as shortening of the acquisition time for certain samples. We applied this method for Fluorescein, Rhodamine B, and Erythrosine fluorescent solutions that have different FLT values (4 ns, 1.67 ns, and 140 ps, respectively).
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Microscopía Fluorescente/métodos , Fotones , Relación Señal-Ruido , Eritrosina/química , Fluoresceína/química , Colorantes Fluorescentes/química , Rodaminas/química , Procesamiento de Señales Asistido por ComputadorRESUMEN
Recent developments in the field of single molecule orientation imaging have led us to devise a simple framework for analyzing fluorescence intensity fluctuations in single molecule polarization sensitive experiments. Based on the new framework, rotational dynamics of individual molecules are quantified, in this paper, from the short time behavior of the time averaged fluorescence intensity fluctuation trajectories. The suggested model can be applied in single molecule fluorescence fluctuations experiments to extract accurate expectation values of photon counts during very short integration time in which rotational diffusion is likely not to be averaged out.
RESUMEN
In the present study we introduce a Whole-Object Fluorescence Life Time (wo-FLT) measurement approach for ease and a relatively inexpensive method of tracing alterations in intracellular fluorophore distribution and in the physical-chemical features of the microenvironments hosting the fluorophore. Two common fluorophores, Rhodamine 123 and Acridine Orange, were used to stain U937 cells which were incubated, with and without either Carbonyl cyanide 3-chlorphenylhydrazon or the apoptosis inducer H(2)O(2). The wo-FLT, which is a non-imaging quantitative measurement, was able to detect several fluorescence decay components and corresponding weights in a single cell resolution. Following cell treatment, both decay time and weight were altered. Results suggest that the prominent factor responsible for these alterations and in some cases to a shift in emission spectrum as well, is the intracellular fluorophore local concentration. In this study it was demonstrated that the proposed wo-FLT method is superior to color fluorescence based imaging in cases where the emission spectrum of a fluorophore remains unchanged during the investigated process. The proposed wo-FLT approach may be of particular importance when direct imaging is impossible.
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Naranja de Acridina/química , Fluorescencia , Rodamina 123/química , Apoptosis/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Espectrometría de Fluorescencia , Coloración y Etiquetado , Células U937RESUMEN
We derive backward and forward fractional Feynman-Kac equations for the distribution of functionals of the path of a particle undergoing anomalous diffusion. Fractional substantial derivatives introduced by Friedrich and co-workers [Phys. Rev. Lett. 96, 230601 (2006)10.1103/PhysRevLett.96.230601] provide the correct fractional framework for the problem. For applications, we calculate the distribution of occupation times in half space and show how the statistics of anomalous functionals is related to weak ergodicity breaking.