Radiant energy – Luminophor irradiation
Patent
1992-09-11
1995-10-17
Hannaher, Constantine
Radiant energy
Luminophor irradiation
G01N 2164
Patent
active
054593239
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the measurement of luminescence (e.g. fluorescence of phosphorescence), and more particularly to the measurement of luminescence decay times.
Reference in the following description is made to fluorescence decay times, but the description is also applicable mutatis mutandis to other forms of luminescence.
Fluorescence microscopy is very widely used in modern biology, forensic science and materials analyses, as well as in many other areas. Fluorescence is sensitive in two senses. It can be detected with very high sensitivity, and emission (and sometimes excitation) parameters are very environmentally sensitive. The environmental sensitivity leads to the use of fluorescent probes to monitor local environment (pH, oxygen, tension, concentration of important ions such as calcium, etc.). However, this sensitivity also leads to a potential ambiguity, in that fluorescence intensity depends on concentration of fluorophore, excitation intensity and quantum yield of said species. Thus, it is not possible to directly relate concentration of fluorophore to measured intensity, even where excitation intensity is constant, unless the quantum yield is also known to be constant within a sample. For many fluorescent samples, especially those studied by fluorescence imaging (e.g. microscopy), variation of quantum yield within a sample is common. Ideally one would wish to have a means of measuring not only fluorescence intensity, but also quantum yield. Generally this is a very difficult problem. However, in many circumstances one can infer fluorescence quantum yield from a measurement of fluorescence decay time. Where this is not valid, this implies a change in the radiative lifetime (the decay time in absence of all extraneous deactivating processes, which is an intrinsic property of the fluorescent species related to the fluorescence efficiency). Such changes are usually detectable, since the perturbations which change radiative decay time also influence spectroscopic properties of excitation and/or emission.
Fluorescence decay times are usually measured using one of two techniques. The most common is the time-correlated single photon counting method where fluorescence is excited using a repetitive source of short optical pulses (1). An alternative method uses phase-shift/demodulation measurements of fluorescence emitted in response to an amplitude modulated excitation source (2). In the latter technique, it is necessary to make measurements as a function of modulation frequency if the fluorescence decay is not a single exponential.
The choice between the time and frequency-domain approaches to fluorescence decay measurement is largely governed by practical considerations such as availability of equipment. However, there are clear sets of circumstances where one or other method finds favour. If the fluorescence can be excited conveniently by the output of a low cost laser (such as the green Helium Neon or Argon ion laser), then the phase shift/demodulation approach is particularly convenient, since laser output is easily modulated with a Pockels cell (3), and some lasers have built-in provision for amplitude modulation. Where laser excitation is not convenient, phase methods become more difficult. Light from an arc source can be modulated using Pockels cell, but only at the expense of very low throughput due to stringent collimation requirements. When a wide wavelength range is desired, the necessary Glan Taylor polarisers attenuate the beam further.
If an arc source is projected through a Pockels cell, a characteristic pattern is seen composed of a pair of cusps coming together at a central point to form a cross. Application of a voltage to the Pockels cell causes these cusps to move apart, so that by using a small central aperture as a spatial filter it can be arranged that the intensity of light passing through the aperture is modulated if a fluctuating voltage is applied to the device. The appearance of the projected pattern is a consequence of light passing through the modulator assembl
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Hannaher Constantine
University of Salford
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