Ein Fluorometer. Apparat zur Messung von Fluoreszenzabklingungszeiten

Gaviola, E.

doi:10.1007/bf01776683

Cited by 105 publications

(36 citation statements)

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“…2) (5,7,8,10,13,14). As a result of the synchronous and near synchronous detections the detector signal is a DC one depending on the introduced phase step and observation time and an AC signal varying slowly ($100 kHz) at the difference frequency, respectively.…”

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“…Scattering solution of metal powder, milk, or a light reflector mirror having zero retardance or a fluorophore of known lifetime introduced in the sample position can serve as reference. The unknown lifetime can be determined from the relative phase delay and relative demodulation of the sample as compared to the reference (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)14).…”

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“…In each case the output of the detector is a sinusoid containing the same phase and modulation information as for the detected signal (fluorescence or scattering) (3,5). At the excitation side, modulated are either the power supply of the light source (for LED, arc lamps) or its emitted light (for lasers, halogen lamps) with accusto optic modulators (AOM), Pockels-cells, earlier with Kerr-cells (5,14), and even with simple mechanical shutters (Becquerel's phosphoroscope) (1,3). At the detection side, the gain of the detector (photomultiplyer gain, or image intensifier gain before CCD cameras in imaging) is modulated.…”

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“…At the detection side, the gain of the detector (photomultiplyer gain, or image intensifier gain before CCD cameras in imaging) is modulated. These arrangements have two important aspects: (i) The first is the "lock-in" principle of detection-that is, detection in synchrony or with a fixed phase with the excitation-of short time intervals originally realized by Fizeau (Paris, 1849) in a "homodyne" experiment (14), when he measured the returning time of light via visual observation of the light source through a rotating cogs-wheel-serving as for both the "master" and "slave" signal generators-from which the determined the speed of light. (ii) The second is the need for an appropriate reference for the excitation profile, that is, an emitter having a known lifetime put precisely at the point of observation.…”

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À la Fizeau in flow: Pulse shape‐assisted fluorescence lifetime

Bene

Szöllősi

2014

Cytometry Pt A

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Key term fluorescence resonance energy transfer FLUORESCENCE lifetime measurements are utilized more and more lately in molecular and cell biology partly due to its applicability for monitoring molecular dynamics and discrimination of molecular species, partly due to the commercial availability of microscopes and fluorimeters capable for fluorescence lifetime detection (1,2). Fluorescence lifetime is the average time an excited fluorophore spends in the excited state prior to returning to the ground state, via emission of light or other, non-radiative ways of de-excitation. Its widespread utility rests on the property that in addition to fluorescence, many other processes involving the excited fluorophore may exist which take place on the same time scale and may compete with fluorescence in the de-excitation and consequently may influence fluorescence lifetime (3-5). To name a few, these processes are: internal conversion, vibrational relaxation, intersystem crossing, F€ orster resonance energy transfer (FRET), dynamic quenching, solvent relaxation, charge transfer, and photolysis (photobleaching); and most of these processes involve interactions of the excited fluorophore with its local environment (1,3). For this reason fluorescence lifetime can be used to monitor locally the concentrations of ions and molecules, electric field and polarity, temperature, viscosity, and refractive index (1-3,6-9). By spanning a much larger range than the wavelength of the emitted fluorescence, it is more amenable for multiplexing than wavelength (8,10). It can be used as a contrast parameter, for example, for discriminating between emitters having overlapping emission spectra (9). It is also a state parameter, meaning that it is independent of conditions of excitation such as wavelength, intensity, and polarization of the exciting light. Based on its independence of concentration and due to the fact that it does not necessitate calibration, it is a more sensitive indicator of FRET than the intensity alone (3,11). It can be changed only physically-termed radiative decay engineering-by influencing the mode structure of the random vacuum field fluctuations responsible for the stimulation of spontaneous emission, for example, by placing a metal mirror in the vicinity of the fluorophore or by embedding the fluorophore in a cavity resonator (12,13).The detection of fluorescence lifetime rests on the "drag" and attenuation imposed on the reemission process, the fluorophore acting as a low pass filter, by cutting and smoothing out sharp features of the original time variation of the exciting light ( Fig. 1) (3). This feature mathematically is expressed by the operation on the time profile of excitation and pulseresponse of fluorophore-called convolution-leading to a shift to the right on the time-axis and demodulation for the time profile of fluorescence.Instead of using short excitation pulses the fluorescence lifetime can also be measured by a long lasting observation of fluorescence, which is periodically modulated at a frequency ($...

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À la Fizeau in flow: Pulse shape‐assisted fluorescence lifetime

Bene

Szöllősi

2014

Cytometry Pt A

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“…Autofluorescence is typically a short-lived phenomenon with excited-state lifetimes measured in nanoseconds for most common autofluorophores (2,4), and substances differing by less than a nanosecond in fluorescence lifetime can be resolved using time-resolved fluorescence (TRF) techniques. A sinusoidal modulation of the excitation source induces a phase (u) delayed modulation in fluorescence intensity from which fluorescence lifetime can be determined using u and modulation frequency parameters (5).…”

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High intensity solid‐state UV source for time‐gated luminescence microscopy

2006

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Background: The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the timedomain can selectively resolve probes with long fluorescence lifetimes (s > 100 ls) against short-lived fluorescence to deliver greatly improved signal-to-noise ratio (SNR). A novel time-gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime. Methods: A commercial Zeiss epifluorescence microscope was adapted for TGL operation by fitting with a time-gated image-intensified CCD camera and a highpower (100 mW) UV LED. Capture of the luminescence was delayed for a precise interval following excitation so that autofluorescence was suppressed. Giardia cysts were labeled in situ with antibody conjugated to a europium chelate (BHHST) with a fluorescence lifetime >500 ls. Results: BHHST-labeled Giardia cysts emit at 617 nm when excited in the UV and were difficult to locate within the matrix of fluorescent algae using conventional fluorescence microscopy, and the SNR of probe to autofluorescent background was 0.51:1. However in time-gated luminescence mode with a gate-delay of 5 ls, the SNR was improved to 12.8:1, a 25-fold improvement. Conclusion: In comparison to xenon flashlamps, UV LEDs are inexpensive, easily powered, and extinguish quickly. Furthermore, the spiked emission of the LED enabled removal of spectral filters from the microscope to significantly improve efficiency of fluorescence excitation and capture. q

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Type II Photooxygenation Reactions in Solution

Gollnick

1968

Advances in Photochemistry

374

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Ein Fluorometer. Apparat zur Messung von Fluoreszenzabklingungszeiten

Cited by 105 publications

References 0 publications

À la Fizeau in flow: Pulse shape‐assisted fluorescence lifetime

À la Fizeau in flow: Pulse shape‐assisted fluorescence lifetime

High intensity solid‐state UV source for time‐gated luminescence microscopy

Type II Photooxygenation Reactions in Solution

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