Accounting for Triplet and Saturation Effects in FCS Measurements

Davis, Lloyd M.; Shen, Guoqing

doi:10.2174/138920106777950843

Cited by 25 publications

(24 citation statements)

References 61 publications

(113 reference statements)

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“…Tripletinduced photophysical dynamics may lead to wrong fitting of diffusion times, particularly if the triplet fraction is high and the diffusion times are short (Davis and Shen 2006). Yet, this phenomenon can usually be corrected for in the fitting function, as seen in Table 2.…”

Section: Difficulties and Artifacts In Fcs Applicationsmentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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Section: Difficulties and Artifacts In Fcs Applicationsmentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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“…Alternatively, the average fluorescence count rate can be determined from the fluorescence intensity distribution of the image (Fig. 1B) In continuous-wave excitation, fluorescence is typically lost through the transition of the fluorophore to the triplet state, fractional depopulation of the singlet state, or irreversible photochemical destruction (10). By using the initial velocity approximation to analyze the experimentally observed fluorescence count rate decay ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Quantitative single-molecule imaging by confocal laser scanning microscopy

Vukojević

Heidkamp

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.fluorescence correlation spectroscopy ͉ live cells ͉ sensitivity L imited sensitivity and spatial resolution impede the usage of fluorescent microscopy for quantitative analysis of low copy numbers of biologically relevant molecules in live cells. Therefore, methodological and instrumental advancements are required. The aim of our work is to explore the benefits of integrating confocal laser scanning microscopy (CLSM) with fluorescence correlation spectroscopy (FCS) (1-4) as a platform for quantitative imaging of the spatiotemporal dynamics of cellular processes in real time.Fast scanning was suggested as a possible way to increase signal intensity in CLSM (5-8), but has not been systematically pursued. A contributing factor is that for increased scanning speed the number of detected photons becomes lower. With low photon counts, detector properties become increasingly relevant because the internal noise of the detector may considerably limit the quality of the image. Therefore, our first aim was to build an instrument for CLSM imaging with improved detection efficiency. We achieved this by introducing avalanche photodiodes (APDs) as detectors. Compared with the photomultiplier tubes (PMTs), normally used as detectors in conventional CLSM, the APDs are characterized by higher quantum and collection efficiency-Ϸ70% in APDs compared with 15-25% in PMTs; higher gain, faster response time, and lower dark current (6, 9). The considerably improved signal-tonoise ratio that was achieved by the introduction of APDs enabled the implementation of fast scanning. Fast scanning offers additional advantages: increased fluorescence yield by avoiding intersystem crossing, data collection at higher encountering frequency and from independent volumes, further significantly improving the signal-tobackground ratio (SBR) in imaging.We first demonstrate that improved SBRs enabled us to quantify the average number of molecules in the observation volume element by analyzing the image statistics, without resorting to temporal or...

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“…In section 2.5.4.5, it was shown that TFOS and also some non-ionic surfactants are very useful to prevent interfacial effects, and the use of surfactants in FCS experiments were also proposed to overcome interfacial effects in Ref. [126], but it is important to first study the interactions between the investigated molecules and the surfactants used in the buffer. In addition, in Chapter 5 the use of non-ionic surfactants significantly extends the functionality lifetime of many soluble proteins and will be hypothesized that the surfactant molecules form micelles and change the hydrogen-bond network in aqueous solution.…”

Section: Discussionmentioning

confidence: 99%

“…However, our SFCM instrument can only work on the time-scale slower than 12.5 µs and is limited by the data acquisition card for acquiring fluorescent intensity signals, so it is difficult to judge the effects of the photophysical and photochemical dynamics of fluorophores in the FCS ACF calculated from an intensity time trace data. Fortunately, two major photophysical effects, fluorescent triplet blinking and saturation, in FCS measurements have been theoretically studied and it is known how they can affect the determination of TDt from FCS [119,126]. Optical saturation can lead to a flattening top on the FCS ACF profile and thus results in a slower decay of the FCS ACF curve at larger lag times [119].…”

Section: Photophysical and Photochemical Dynamics Of Fluorophoresmentioning

confidence: 99%

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Quantitative fluorescence nanospectroscopy of nucleotide excision repair : from single molecules to cells

Zhang¹

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Accounting for Triplet and Saturation Effects in FCS Measurements

Cited by 25 publications

References 61 publications

Fluorescence Techniques to Study Lipid Dynamics

Fluorescence Techniques to Study Lipid Dynamics

Quantitative single-molecule imaging by confocal laser scanning microscopy

Quantitative fluorescence nanospectroscopy of nucleotide excision repair : from single molecules to cells

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