Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

Luchowski, Rafał; Szabelski, Mariusz; Sarkar, Priyabrata; Apicella, Elisa; Midde, Krishna; Raut, Sangram; Borejdo, Julian; Gryczyński, Zygmunt; Gryczyński, Ignacy

doi:10.1366/000370210792081000

Cited by 14 publications

(11 citation statements)

References 19 publications

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“…The Eosin Y fluorescence can be excited from 350 to 500 nm and ranges between 450 and 680 nm with a sufficient intensity. Based on the reported Rose Bengal fluorescence lifetime of approximately 16 ps when dissolved in 5 M potassium iodide, 34 it is reasonable to assume that Eosin Y, another fluorescein derivative, shows similar characteristics, which we can confirm. Additionally, we compared IRFs based on Eosin Y fluorescence with IRFs measured using scattered excitation laser light and found no differences regarding the shape as well as the width.…”

Section: Flio Setup and Image Acquisitionsupporting

confidence: 75%

Impact of Macular Pigment on Fundus Autofluorescence Lifetimes

Sauer

Schweitzer

Ramm

et al. 2015

Invest. Ophthalmol. Vis. Sci.

120

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Macular pigment, which is known to have very short fluorescence decays, considerably contributes to the macular autofluorescence (AF). This study gives indirect evidence for a strong impact of MP on macular τm, although no direct measurement of MP autofluorescence lifetimes in vivo is possible at this point. Potentially, imaging the FAF lifetimes could lead to a novel methodology for the detection of macular pigment properties and pathology-induced changes in the living human retina.

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Section: Flio Setup and Image Acquisitionsupporting

confidence: 75%

Impact of Macular Pigment on Fundus Autofluorescence Lifetimes

Sauer

Schweitzer

Ramm

et al. 2015

Invest. Ophthalmol. Vis. Sci.

120

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“…The excited-state lifetime, , of Rb is 0.49 ± 0.01 ns. 3 Some reported values are 0.53 ± 0.01 ns 1 and 0.512 ns, 28 with no error estimate. We have measured the excited-state lifetime of RhB to be 2.45 ± 0.01 ns.…”

Section: Introductionmentioning

confidence: 95%

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B

et al. 2016

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It is often convenient to know the minimum amount of data needed to obtain a result of desired accuracy and precision. It is a necessity in the case of subdiffraction-limited microscopies, such as stimulated emission depletion (STED) microscopy, owing to the limited sample volumes and the extreme sensitivity of the samples to photobleaching and photodamage. We present a detailed comparison of probability-based techniques (the maximum likelihood method and methods based on the binomial and the Poisson distributions) with residual minimization-based techniques for retrieving the fluorescence decay parameters for various two-fluorophore mixtures, as a function of the total number of photon counts, in time-correlated, single-photon counting experiments. The probability-based techniques proved to be the most robust (insensitive to initial values) in retrieving the target parameters and, in fact, performed equivalently to 2-3 significant figures. This is to be expected, as we demonstrate that the three methods are fundamentally related. Furthermore, methods based on the Poisson and binomial distributions have the desirable feature of providing a bin-by-bin analysis of a single fluorescence decay trace, which thus permits statistics to be acquired using only the one trace not only for the mean and median values of the fluorescence decay parameters but also for the associated standard deviations. These probability-based methods lend themselves well to the analysis of the sparse data sets that are encountered in subdiffraction-limited microscopies. Disciplines Chemistry | Physical Chemistry CommentsThis document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication as Santra, Kalyan, Emily A. Smith, Jacob W. Petrich, and Xueyu Song. ABSTRACTIt is often convenient to know the minimum amount of data needed in order to obtain a result of desired accuracy and precision. It is a necessity in the case of subdiffraction-limited microscopies, such as stimulated emission depletion (STED) microscopy, owing to the limited sample volumes and the extreme sensitivity of the samples to photobleaching and photodamage.We present a detailed comparison of probability-based techniques (the maximum likelihood method and methods based on the binomial and the Poisson distributions) with residual minimization-based techniques for retrieving the fluorescence decay parameters for various twofluorophore mixtures, as a function of the total number of photon counts, in time-correlated, singlephoton counting experiments. The probability-based techniques proved to be the most robust (insensitive to initial values) in retrieving the target parameters and, in fact, performed equivalently to 2-3 significant figures. This is to be expected, as we demonstrate that the three methods are fundamentally related. Furthermore, methods based on the Poisson and binomial distributions have the desirable feature of providing a bin-by-bin analysis of a single fluorescence decay trace, which thus permits statistic...

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“…1 A more recent study gives 516 ps (with no error estimate). 9 The fluorescence decay of rose bengal is collected over a total of 1024 bins in a set of five experiments in which the total number of arrival times (counts) in all the bins is approximately 20, 200, 1000, 3000, and 6000, respectively. Each set of experiments was repeated 50 times in order to obtain appropriate statistics.…”

Section: Introductionmentioning

confidence: 99%

What Is the Best Method to Fit Time-Resolved Data? A Comparison of the Residual Minimization and the Maximum Likelihood Techniques As Applied to Experimental Time-Correlated, Single-Photon Counting Data

et al. 2016

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The need for measuring fluorescence lifetimes of species in subdiffraction-limited volumes in, for example, stimulated emission depletion (STED) microscopy, entails the dual challenge of probing a small number of fluorophores and fitting the concomitant sparse data set to the appropriate excited-state decay function. This need has stimulated a further investigation into the relative merits of two fitting techniques commonly referred to as "residual minimization" (RM) and "maximum likelihood" (ML). Fluorescence decays of the well-characterized standard, rose bengal in methanol at room temperature (530 ± 10 ps), were acquired in a set of five experiments in which the total number of "photon counts" was approximately 20, 200, 1000, 3000, and 6000 and there were about 2-200 counts at the maxima of the respective decays. Each set of experiments was repeated 50 times to generate the appropriate statistics. Each of the 250 data sets was analyzed by ML and two different RM methods (differing in the weighting of residuals) using in-house routines and compared with a frequently used commercial RM routine. Convolution with a real instrument response function was always included in the fitting. While RM using Pearson's weighting of residuals can recover the correct mean result with a total number of counts of 1000 or more, ML distinguishes itself by yielding, in all cases, the same mean lifetime within 2% of the accepted value. For 200 total counts and greater, ML always provides a standard deviation of <10% of the mean lifetime, and even at 20 total counts there is only 20% error in the mean lifetime. The robustness of ML advocates its use for sparse data sets such as those acquired in some subdiffraction-limited microscopies, such as STED, and, more importantly, provides greater motivation for exploiting the time-resolved capacities of this technique to acquire and analyze fluorescence lifetime data.

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Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

Cited by 14 publications

References 19 publications

Impact of Macular Pigment on Fundus Autofluorescence Lifetimes

Impact of Macular Pigment on Fundus Autofluorescence Lifetimes

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B

What Is the Best Method to Fit Time-Resolved Data? A Comparison of the Residual Minimization and the Maximum Likelihood Techniques As Applied to Experimental Time-Correlated, Single-Photon Counting Data

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