Reduction of Luminescent Background in Ultrasensitive Fluorescence Detection by Photobleaching

Rl, Affleck; Wp, Ambrose; Jn, Demas; Pm, Goodwin; Ja, Schecker; Wu, Mei; Ra, Keller

doi:10.1021/ac9512517

Cited by 50 publications

(43 citation statements)

References 21 publications

(27 reference statements)

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“…Luminescence background arises primarily from optics, impurities in buffers [9,29,30], and from capillary and microchip substrates. The latter is especially problematic with certain types of glass, such as soda lime glass, and with plastics, and the problem is often more severe in the UV [14,21,[31][32][33][34].…”

Section: Background Luminescencementioning

confidence: 99%

“…Luminescence in the buffer can be minimized -although not removed entirely -by using ultrapure buffer salts, by working at longer wavelengths [16,24], or by photobleaching the buffer before use [29,30]. Figure 5 shows background scans of 10 mM buffer before and after photobleaching.…”

Section: Background Luminescencementioning

confidence: 99%

“…Mild photobleaching produced no effect; a 10-fold lower flowrate showed moderate lowering of the background, equivalent to about 10 pM fluorescein. Affleck et al [29] demonstrated substantial (2-56) lowering of background count rates for on-line bleaching of a variety of common buffers. However, others have reported less substantial improvements.…”

Section: Background Luminescencementioning

confidence: 99%

“…The probe volume is well-defined, although the mathematics of defining the volume from which light is collected can be complicated [54,55]. Generally speaking, the probe volumes can be on the order of 1-10 pL, with background luminescence as the most dominant background source [29]. The probe volume is well matched to the sample stream and all of the sample stream is imaged.…”

Section: Optical Arrangements: the Sheath-flow Cuvettementioning

confidence: 99%

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Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

2004

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Laser-induced fluorescence is an extremely sensitive method for detection in chemical separations. In addition, it is well-suited to detection in small volumes, and as such is widely used for capillary electrophoresis and microchip-based separations. This review explores the detailed instrumental conditions required for sub-zeptomole, sub-picomolar detection limits. The key to achieving the best sensitivity is to use an excitation and emission volume that is matched to the separation system and that, simultaneously, will keep scattering and luminescence background to a minimum. We discuss how this is accomplished with confocal detection, 90 degrees on-capillary detection, and sheath-flow detection. It is shown that each of these methods have their advantages and disadvantages, but that all can be used to produce extremely sensitive detectors for capillary- or microchip-based separations. Analysis of these capabilities allows prediction of the optimal means of achieving ultrasensitive detection on microchips.

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Section: Background Luminescencementioning

confidence: 99%

Section: Background Luminescencementioning

confidence: 99%

Section: Background Luminescencementioning

confidence: 99%

Section: Optical Arrangements: the Sheath-flow Cuvettementioning

confidence: 99%

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Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

2004

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“…These include, pulsed laser excitation coupled with time-gated detection 2 (to discriminate between fluorescence and scattering signals) and photobleaching of the sheath flow upstream of the detection volume (to remove the luminescence background from impurity species). 11,12 Although successful, these methods add a certain degree of experimental complexity. Consequently, in this communication we propose a simpler method that can potentially be used to size single particles or molecules in free solution.…”

mentioning

confidence: 99%

Single Particle Confocal Fluorescence Spectroscopy in Microchannels: Dependence of Burst Width and Burst Area Distributions on Particle Size and Flow Rate

Edel

Mello

2003

ANAL. SCI.

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This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (200 -930 nm diameter) passing through a focused laser beam at a variety of flow rates (100 -1000 nL/min). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate single-particle burst width and burst area distributions. Analysis of such distributions demonstrates that the average burst width and burst area decrease as particle size increases. In addition, both burst width and burst area (for a given particle size) are observed to decrease as volumetric flow rate is increased. The dependence of such distributions on particle size is proposed as a potential route to sizing single particles and molecules in microfluidic systems.

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Steps towards spatially resolved single molecule detection in solution

Mathis¹,

Kalusche²,

Wagner³

et al. 1997

Bioimaging

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Spatially resolved single molecule detection in solution is a prerequisite for molecule tracking and provides new opportunities in single molecule manipulation, for example in a sample flow. A detector with fast timing properties and high spatial resolution is required for these purposes. We introduce a concept for spatially resolved optical single molecule detection in an epi‐illuminated microscope using a novel kind of detector and a new algorithm in configurable hardware for intelligent data processing. The analysis is performed on a parallel hardware interface and includes burst detection. It can be extended to on‐line spatial and temporal data analysis. This detector will be used in combination with a molecular sorter where molecules are sorted in a microstructured flow device made of silicon. In a first step towards such sorting in these microstructures, we show that a reliable detection of single molecules in silicon microstructures is possible in zero‐dimensional detection volumes. The noise structure of the data is analysed in terms of Poissonian statistics and it is shown that in the smallest structure used (depth 20 μm) a signal‐to‐noise ratio of 40 is achieved.

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Reduction of Luminescent Background in Ultrasensitive Fluorescence Detection by Photobleaching

Cited by 50 publications

References 21 publications

Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

Single Particle Confocal Fluorescence Spectroscopy in Microchannels: Dependence of Burst Width and Burst Area Distributions on Particle Size and Flow Rate

Steps towards spatially resolved single molecule detection in solution

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