Semiconductor laser-induced fluorescence detection in picoliter volume flow cells

Larson, Anders P.; Ahlberg, Henrik; Folestad, Staffan

doi:10.1364/ao.32.000794

Cited by 24 publications

(17 citation statements)

References 18 publications

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“…The process of signal generation and photon burst detection is covered in detail in two seminal papers by Mathies et al [3,4], in the paper by Larson [5], and in the excellent review by Goodwin et al [6] and references cited therein. The strategy that emerges from this work is to pump the molecules hard (i.e., at high intensity, just below saturation) and adjust the transit time so that it is slightly greater than the average time to photobleach the fluorophore.…”

Section: The Fluorescence Signalmentioning

confidence: 99%

“…In the practice of CE, the transit time is determined by the product of analyte velocity and the distance the analyte travels through the probe volume. The analyte velocity is normally fixed by the process of optimizing the separation [5], unless the zone velocity is changed during peak measurement [7], and the probe volume (which will be discussed in detail below) is normally fixed by other optical considerations. As a practical matter also, it is usually difficult to find data on photobleaching (or other photophysical constants), although examples exist (e.g., [8]).…”

Section: The Fluorescence Signalmentioning

confidence: 99%

“…In tightly focused systems, other sources of noise such as laser pointing stability and vibration may come into play. Many lasers also have broadband background, either from plasma discharge (gas lasers) or from autofluorescence (diode lasers) that can also be scattered into the detector [5,22].…”

Section: Background Sourcesmentioning

confidence: 99%

“…We show this below in Figure 3, but it is nearly always a safe assumption that the source of excess noise is flicker noise from the laser, especially when using the most common sources, i.e., gas lasers. This type of excess noise is nearly always absent when using diode lasers [5,24]. Figure 3 shows several emission spectra from a typical sample flowing through the capillary at the chosen laser power of 500 mW.…”

Section: Background Sourcesmentioning

confidence: 99%

“…This scatter can be the largest contribution to background. The capillary can be tilted relative to the excitation beam to minimize scatter; close to the Brewster angle, the scatter is much less [5,22,24,32]. A slit or pinhole in the emission path also rejects scatter by confining the image to the center of the capillary.…”

Section: Illumination Opticsmentioning

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: The Fluorescence Signalmentioning

confidence: 99%

Section: The Fluorescence Signalmentioning

confidence: 99%

Section: Background Sourcesmentioning

confidence: 99%

Section: Background Sourcesmentioning

confidence: 99%

Section: Illumination Opticsmentioning

confidence: 99%

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

2004

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Construction of a light‐emitting diode fluorescence detector for high‐performance liquid chromatography and its application to fluorometric determination of l‐3‐hydroxybutyrate

Chen

Lee

Huang

2011

Biomedical Chromatography

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This study employed a new light source, a light-emitting diode (LED), for fluorescence detection of high-performance liquid chromatography to measure the concentration of trace constituents in biological fluids. Using l-3-hydroxybutyrate ( l-3HB) as a tested trace compound, the function of the new system was compared with that of the current commercially available model. A detailed schematic diagram of the path of the detection rays in the LED detector is given. A voltage-stabilizer for the drive circuit was designed with an input of 10 V and an output of 8 V, and another voltage regulator was used to maintain a constant 8 V. Then the regulator was used to set the output voltage for the LED at 2.8 V by two external resistors. Replacing the xenon lamp with LED, this system provided higher photon density and a narrow spectrum at a wavelength of 491 nm. At room temperature (22.1°C), the average temperature of six places in the chamber of LED detector was 22.1°C compared with 51.1°C in the xenon detector. The spectra of the excitation light sources were measured. Compared with the xenon lamp, approximately 1.32 times higher excitation intensity was obtained by the LED source. The accuracy of detection of l-3HB in 50 μL of rat serum was 99.85-100.85%, and the intra-day and inter-day precision values were within 8.99 and 13.90%, respectively. The limit of detection of l-3HB was approximately 0.73 µM (signal-to-noise ratio 3). The sensitivity of the proposed LED detector was comparable to that of traditional fluorescence detectors using xenon arc lamps; however, the cost and operating temperature of LED lamps were far lower. This assay system could be further used to detect trace constituents in various samples.

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Practical time‐gated luminescence flow cytometry. I: Concepts

2007

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The method of time-gated detection of long-lifetime (1-2,000 ls) luminescence-labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 ls), scatterings, and other prompt stray light (Hemmila and Mukkala, Crit Rev Clin Lab Sci 2001;38:441-519). The application of such techniques to flow cytometry is highly attractive but there are significant challenges in implementing pulsed operation mode to rapid continuous flowing sample to achieve high cell analysis rates (Leif R, Vallarino L, Rare-earth chelates as fluorescent markers in cell separation and analysis, In: Cell Separation Science and Technology, ACS Symposium Series 464, American Chemical Society, 1991, pp 41-58; Condrau et al., Cytometry 1994;16:187-194; Condrau et al., Cytometry 1994;16:195-205; Shapiro HM, Improving signals from labels: Amplification and other techniques, In: Practical Flow Cytometry, 4th ed., Wiley, New York, 2002, p 345). We present here practical approaches for achieving high cell analysis rates at 100% detection efficiency, using time-gated luminescence (TGL) flow cytometry. In particular, we report that new-generation UV LEDs are practical sources in TGL flow cytometry. Spatial effects of longlived luminescence from the target fluorophore in a fast-flowing sample stream have been investigated; excitation and detection requirements in TGL flow cytometry were theoretically analyzed; two practical approaches, a triggered model and a continuous flow-section model, were considered as a function of flow speed, sizes and relative positions of the excitation/detection spots, label lifetime, excitation pulse duration/intensity, and detection duration. A particular configuration using LED excitation to detect europium dye-labeled targets in such a system has been modeled in detail. In the triggered model, TGL mode is confined to a low repetition rate (<1 kHz) and engaged only while a target particle is present in the excitation zone. In the flow-section model, TGL mode is engaged continuously at high repetition rates to permit much higher cell arrival rates. The detection of 5.7-lm europium calibration beads in a UV LED-excited TGL flow cytometer has been shown to be feasible with a calculated signal-to-background ratio up to 11:1. ' 2007 International Society for Analytical Cytology

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Semiconductor laser-induced fluorescence detection in picoliter volume flow cells

Cited by 24 publications

References 18 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

Construction of a light‐emitting diode fluorescence detector for high‐performance liquid chromatography and its application to fluorometric determination of l‐3‐hydroxybutyrate

Practical time‐gated luminescence flow cytometry. I: Concepts

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