WO1999012018A1

WO1999012018A1 - Luminescence detection

Info

Publication number: WO1999012018A1
Application number: PCT/GB1998/002585
Authority: WO
Inventors: Christopher Grant Morgan
Original assignee: Photonic Research Systems Limited
Priority date: 1997-09-02
Filing date: 1998-09-02
Publication date: 1999-03-11
Also published as: GB9718477D0

Abstract

A method of luminescence detection comprises: 1) effecting excitation of a label by absorption of one or more photons of the same or different energy, at least one of which is of visible or ultraviolet light, such that (i) the excitation leads to the population of one or more transient excited states having a lifetime greater than one microsecond but less than one minute, and (ii) the or at least one of the excited states is capable of detection directly of indirectly by luminescence; 2) effecting a perturbation to induce a transient change in the population of the or at least one of the excited states; and 3) detecting the fluctuation or fluctuations in concentration of the said excited state or states which result from said perturbation either directly by virtue of the luminescence of the said excited state or states or indirectly by observation of the luminescence of one or more other species to which excitation energy has been transferred.

Description

LUMINESCENCE DETECTION

The present invention relates to a method of luminescence detection.

Luminescence detection and imaging are now very widely used in a variety of areas where extremely high sensitivity is required. For example, fluorescent and phosphorescent labels are used in microscopy, in scientific and medical research, in diagnostics and assays and in many analytical applications. Radioisotopes have been used for many high sensitivity labelling procedures in the past, but increasingly fluorescent labels provide safer cost-effective alternatives. In technical reports 'fluorescence' normally refers to emission resulting from an allowed electronic transition, while 'phosphorescence' refers to emission resulting from a transition that is not favoured according to normal quantum mechanical formalism. There are variants of these emission processes, such as 'delayed fluorescence' and in the present context 'fluorescence' should be taken to include other types of emission processes consequent on electronic excitation, collectively referred to as 'luminescence' unless otherwise stated.

The sensitivity of fluorescence detection is determined primarily by the ability to reject background signals in most analytical applications. These signals result from a variety of sources including unwanted fluorescence from components of the sample other than the label of interest, elastically and inelastically scattered exciting light transmitted by optical filters and chemiluminescence.

A common approach to minimise background is the use of lifetime-resolved detection in conjunction with luminescent labels having long-lived emission. Most sources of background either are coincident with excitation or decay within nanoseconds of excitation. Examples of labels with particularly long decay times are lanthanide complexes and cryptates, ruthenium and osmium complexes, phosphorescent organic and metallo-organic molecules such as eosin derivatives and phthalocyanines. Such labels are becoming widely used in a variety of assay procedures and in high sensitivity detection schemes. For example, europium complexes are used commercially in the so-called 'DELFIA' assay while europium cryptates are used as labels in immunoassay and as donor species in lifetime-resolved detection of energy transfer to an acceptor species. The latter is the basis for a variety of homogeneous binding assays where approximation of donor and acceptor is detected by measurement of long-lived sensitised emission from the acceptor dye, which emits only short-lived fluorescence when excited directly.

Another promising development in fluorescence microscopy and assay is the use of multiphoton excitation. In this process fluorescence is excited by simultaneous absorption of two or more low energy photons, each of which individually has insufficient energy to excite the fluorescent molecule to the emissive state. The most common implementation of this process uses two-photon absorption and requires a very high power density for efficient excitation, since both exciting photons must be absorbed simultaneously. Exciting light from a laser is focused to a spot within the sample. The probability of fluorescence excitation is greater at the focal point than elsewhere because of the quadratic intensity dependence. This minimises background as the emissive region is limited in size. Multiphoton excitation achieves a similar depth-sectioning effect to confocal microscopy but w ithout the need for spatial filters. An additional advantage over conventional confocal microscopy is that excitation is low outside the focal region and hence photodamage to the bulk of the sample is minimised. The technique has other potential advantages over more conventional microscopy, including the high penetration depth of long wavelength light in samples that absorb short wavelengths strongly.

A significant limitation of many multiphoton excitation processes is the requirement for high power density which means either very high average power or more commonly very high peak power delivered as short pulses. The usual excitation sources are lasers capable of concentrating emission into pulses of sub-picosecond duration, but these are extremely expensive at present. The requirement for very high excitation power density results from the need for two or more photons to be absorbed simultaneously, since excitation involves a 'virtual' intermediate energy state.

Multiphoton excitation can be achieved in some cases by a sequential absorption of photons. An intermediate state of finite lifetime produced by absorption of a photon of low energy is excited to higher energy by absorption of one or more further photons. In such a case the power density required can be very much lower than that needed for simultaneous multiphoton excitation. There are a number of methods to achieve excitation by sequential absorption of photons. In the case of sequential multiphoton excitation the intermediate excited state need not be populated by a direct absorption process, but can be indirectly populated by radiationless transfer of energy from another species. Similarly the intermediate state, excited by whatever means, can in principle be further excited by transfer of energy from another species. These processes can be very efficient in appropriate circumstances.

In the present context it is convenient to distinguish between multiphoton processes where none of the exciting photons is sufficiently energetic to excite the fluorescent species and those processes where at least one of the photons is sufficiently energetic to excite the species. Examples of the former kind have been proposed as labels for a wide variety of purposes. Some phosphors are designed to ^*up-convert' infra-red light to visible light, and are used for example in security marking, in detection screens for infra-red radiation and for optical computing. One class of up-conversion phosphor is based on an energy pooling process whereby a species (usually a lanthanide such as erbium) can be excited by sequential transfers of energy from a sensitising species (typically ytterbium) which is itself excited efficiently by absorption of the incident low energy infra-red radiation. The acceptor species in this case has a series of long-lived intermediate energy states, each of which can be populated directly by relaxation from a higher energy state able to accept energy from the donor. Applications of these phosphor particles, as well as organic labels excited by high intensity infra-red light through multiphoton absorption have been described in detail in PCT publication number WO 94/07142. In our co-pending PCT patent application number PCT/GB98/00769 we describe applications of these and related up-conversion media as components within an assay system based on transfer of energy from the multi photon-excited acceptor species to a further species bound to it.

Although the up-conversion phosphors described have many advantages for assay purposes, they are subject to some limitations. For many purposes the labels must be provided as particles ot microscopic size and the efficiency of the up- conversion process is often low lor small phosphor particles. The efficiency of the up-conversion phosphors is very sensitive to lattice structure and particularly to lattice phonon energy. This restricts the choice of host lattices, and most commonly fluoride-based heavy metal glasses aie used. Such glasses are potentially toxic and are not suited to medical applications such as for tracing blood flow etc., where otherwise the labels would be υl considerable value. There is much scope for applications of labels which can be prepared from a wide variety of organic labels of low toxicity, and which might optionally be provided in organic polymer matrices, inorganic glasses or ceramics, within liposomes, lipid droplets or micelles or as complexes with organic cryptands or other protective binding agents.

It is the objective of the present invention to increase the sensitivity of luminescence detection, e.g. in

s. in sensing and imaging and in microscopy, by the use of labels designed to achie\ e excitation by processes which involve sequential absorption of quanta of energy (I e photons of electromagnetic radiation or quantised vibrational energy such as phonons) whereof at least one of the said quanta is of insufficient energy to excite the ground state species directly.

Labels suited to use in lifetime-resolved detection and those designed for efficient multiphoton excitation sequential photon absoφtion share a common feature. This is the existence of a relatively long-lived intermediate excited state. For example, in the case of a phosphorescent organic molecule, emission is a formally forbidden process from the excited triplet state to the ground state. In the case of lanthanide complexes and cryptates a long-lived intermediate excited state of the lanthanide is produced by an energy transfer process from a sensitising ligand. In the case of sequential multiphoton excitation, a long-lived intermediate excited state is required to give a reasonable probability of absoφtion of a further photon before deactivation.

The intermediate long-lived excited state can make the luminescent species susceptible to perturbations. For example, long lived triplet states are often quenched by collisional energy transfer to oxygen. This susceptibility to quenching can be a problem with long-lived labels, and is normally overcome by providing a protective microenvironment (e.g. encapsulation in a glass or polymer or binding within a macromolecule such as cyclodextrin or a cryptand). In the context of the invention described the long lifetime of the excited state provides a time window in which the state can be perturbed and this perturbation monitored. Very long lived states with lifetimes of minutes to hours are not advantageous however because it is difficult to protect them from quenching species satisfactorily, particularly when they are provided as near-colloidal sized particles for applications in labelling. In addition it is most efficient to detect perturbations electronically by a repetitive lock-in process and in some circumstances very long-lived states are slow to equilibrate between perturbations.

According to a first aspect of the present invention there is provided a method of luminescence detection comprising:

1 ) effecting excitation of a label by absoφtion of one or more photons of the same or different energy, at least one of which is of visible or ultraviolet light, such that (i) the excitation leads to the population of one or more excited states having a lifetime greater than one microsecond but less than one minute, and

(ii) the or at least one of the excited states is capable of detection directly or indirectly by luminescence

2) effecting a perturbation to cause a transient change in population of the or at least one of the excited states by virtue of

(a) increasing or relaxing a quantum mechanical limitation on emission from the intermediate,

(b) promoting the intermediate to a non-luminescent or weakly luminescent higher excited state from which it may lose energy by radiationless deactivation or by transfer of energy to a further state or species which might or might not be luminescent, or

(c) promoting the intermediate to a luminescent higher excited state; and

3) detecting the fluctuation or fluctuations in concentration of the said excited state or states which result from said perturbation either directly by virtue of the luminescence of the said excited state or states or indirectly by observation of the luminescence of one or more other species to which excitation energy has been transferred.

Preferably the perturbation is effected periodically. According to a second aspect of the present invention there is provided a method of luminescence detection comprising:

1 ) effecting excitation of a label by absoφtion of one or more photons of the same or different energy, at least one of which is of visible or ultraviolet light, such that

(i) the excitation leads to the population of one or more transient excited states having a lifetime greater than one microsecond but less than one minute, and

2) effecting a periodic perturbation to induce a transient change in the population of the or at least one of the excited states; and

In the method of the second aspect of the invention, the transient change is preferably effected by virtue of

(a) increasing or relaxing the said quantum mechanical limitation on emission from the intermediate, (b) promoting the intermediate to a non-luminescent or weakly luminescent higher excited state from which it may lose energy by radiationless deactivation or by transfer of energy to a further state or species which might or might not be luminescent, or

(c) promoting the intermediate to a luminescent higher excited state;

According to a third aspect of the present invention there is provided a luminescence detection system comprising:

1 ) a label means which is capable of excitation by absoφtion of one or more photons of the same or different energy, at least one of which is of visible or ultraiolet light, such that

(i) the said excitation process leads to the population of one or more transient excited states having a lifetime greater than one microsecond but less than one minute,

(ii) the or at least one of the said excited states is capable of detection directly or indirectly by luminescence, and

(iii) the or at least one of the excited states is capable of undergoing a transient change in population by virtue of

(c) promoting the intermediate to a luminescent higher excited state;

2) a source of visible and/or ultraviolet light to excite the said label;

3) a perturbation means which can induce said transient change, and

4) a detection means whereby the fluctuation or fluctuations in concentration of the said excited state or states which result from the application of the said perturbation means can be monitored either directly by virtue l the lummescence of the said excited state or states or indirectly by observation of the luminescence of one or more other species to which excitation energy has been transleπed.

According to a fourth aspect of the present invention theie is provided a luminescence detection system comprising:

1) a label means which is capable of excitation by absoi ption of one or more photons of the same or different energy, at least one of which is ol \ isible or ultraviolet light, such that

(i) the said excitation process leads to the population ol one or more excited states having a lifetime greater than one microsecond but less than one minute, and

(ii) the or at least one of the said excited states is capable oi^' detection directly or indirectly by luminescence.

2) a source of visible and/or ultraviolet light to excite the said label: 3) a perturbation means for providing a periodic perturbation to induce a transient change in the population of the said long-lived intermediate or intermediates; and

4) a detection means whereby the fluctuation or fluctuations in concentration of the said long-lived excited state or states which result from the application of the said perturbation means can be monitored either directly by virtue of the luminescence of the said excited state or indirectly by observation of the luminescence of one or more other species to which excitation energy has been transferred.

The mechanisms (a)-(c) by virtue of which the transient change occurs are those which intrinsically affect the luminescence efficiency of the label and/or its ability to transfer excitation energy to another luminescent species.

The lifetime of the excited species as employed in the invention is from one microsecond to one minute. This has the advantage of providing a time window within which the state can be perturbed.

The lifetime of excited states may be measured by various means well known in the art including luminescence measurements in the time domain following pulsed excitation or frequency domain measurements of phase shift and/or demodulation of luminescence using modulated excitation.

Suitable labels include organic molecules having long-lived triplet states capable of luminescence and inorganic or organometallic substances including semiconductor colloids.

Preferably the label is provided as, in or bound to a particle or surface. Most preferably the agent is provided as microscopic particles of dimensions less than 5 microns. For some applications smaller particles are advantageous in that they do not readily settle from dispersions. Such particles, which might for example be as small as lOnm or less offer a high surface area-to-volume ratio which can be useful for binding ligands such as recognition molecules (e.g. antibodies, oligonucleotides, lectins, biotin etc) and/or other coatings (e.g. polymers to limit particle aggregation.). Small particles with a high surface-to-volume ratio are particularly suited to assays where energy transfer to or from a bound ligand is to be detected by luminescence.

The said label can be an organic molecule which preferably is provided in a matrix to protect the luminescent species from quenching by external agents. Preferably the matrix should be capable of enhancing the probability of electronic transitions required for the sequential excitation process to be achieved. Preferably the matrix is such as to increase the lifetime of intermediate states required for the excitation process.

Preferably the matrix is such that surface modification techniques can be used to facilitate attachment of ligands to it to facilitate or impede binding to other materials.

The essence of the invention is the detection of a perturbation in a luminescent species induced by absoφtion of energy by a long-lived intermediate. Such a perturbation can be periodic and the resulting response can be detected through the luminescence of the label or associated species using lock-in detection or equivalent techniques well known in electronics and signal processing. An appropriate perturbation might be a thermal fluctuation for example, which alters the rate of radiationless deactivation of the relatively long-lived intermediate. Alternatively, the excited state concentration might be perturbed by the absoφtion of radiation. The important feature is that the application of the perturbation means should cause a fluctuation in the luminescence or sensitising efficiency of the label by virtue of interaction with a relatively long-lived excited intermediate and that this fluctuation, which normally would be cyclical, is detected. [Background luminescence would normally not be markedly sensitive to the perturbation to which the label is designed to respond. Thus, luminescence from the label or associated species sensitised by the label would fluctuate in a characteristic manner capable of detection in the presence of a substantial background of unperturbed luminescence. It is an important puφose of the invention to allow such discrimination because background (e.g. luminescence arising from materials other than the label of interest) commonly sets a limit to the sensitivity with which a label can be detected.

A further important feature of the invention is an improvement in assay measurements based on detection of proximity between species influenced by the presence of an analyte. A common example of such an assay involves binding between the analyte and recognition molecules such as antibodies, lectins, oligonucleotides, biotin, proteins such as avidin and streptavidin etc. One such format is a so-called 'sandwich assay' where two species labelled with recognition molecules to the analyte form a ternary complex in the presence of analyte, and hence are brought into proximity. The essential feature of the assay is to detect and quantify such proximity. A typical method to detect analyte-mediated complex formation uses fluorescence energy transfer where an excited 'donor' species can transfer excitation energy efficiently to an 'acceptor' only when both are in close proximity (e.g. bound together). As a result of the transfer of energy the 'acceptor' species is excited and luminesces in a characteristic wavelength region. One limitation of this approach is the difficulty of exciting the 'donor' species without simultaneously exciting the 'acceptor' to some extent. This limits the dynamic range of the measurement since the sensitised 'acceptor' luminescence is detected against a background of directly excited luminescence. The present invention provides a means to minimise and potentially to avoid this problem because perturbations in the 'donor' excited state level will be mirrored by fluctuations in the energy transfer efficiency, and hence will appear in the sensitised 'acceptor' emission, but not in the directly excited emission. Detection of such fluctuations in the acceptor emission will distinguish sensitised luminescence both from directly excited luminescence from the 'acceptor' and from sources of background associated with luminescence from contaminants in the sample.

In another type of assay based on luminescence quenching the present invention also offers advantages For example, an analyte might be detected on the basis of its ability to increase, 01 more usually to decrease the interaction between a label and a quenching species (which might quench by any mechanism not limited to luminescence energy transfei from the label). In the preferred embodiment of such an assay the label and quencher are pre-bound and the analyte acts to displace the quencher competitively, to

e a bond linking label and quencher or otherwise to convert the quenching species into another species that is inefficient in quenching luminescence from the label Pi elcrably the extent of quenching in the absence of analyte is substantial, and ldcalK there is little or no detectable luminescence from the quenched label, but considerable luminescence when the quencher has been released or modified by the action ol the analyte. Such an assay is normally limited by the presence of background luminescence from contaminants, which frequently requires that the assay be conducted m a so-called heterogeneous format where contaminants can be washed away befoie measuicment. The present invention improves sensitivity of such assays by allowing the luminescence from the label to be distinguished readily from background, and this m turn avoids the need to remove contaminating luminescent materials and theietoie allows a so-called homogeneous assay to be performed. Homogeneous

formats are preferred because of the reduced number of steps involved and the ιmpιo\ ed compatibility with automation for applications such as high-throughput screening programmes.

Examples of quenching mechanisms appropriate to the present invention are given in 'Modulation of the photokiminescence of semiconductors by surface adduct formation: an application of inorganic photochemistry to chemical sensing' by Aurthur E. Ellis et al (J Chemical Education , Vol. 74 No. 6, June 1997) which shows how surface-bound species such as Lewis acids and bases can affect the luminescence of semiconducting materials. Apart from assay mechanisms where the surface association of a quenching species is influenced by an analyte, the analyte might chemically or enzymatically convert a quenching species to a non-quenching species or vice-versa. One example is given in the cited paper where oxygen binds to a surface-associated cobalt complex on a semiconductor affecting the luminescence thereof. Other examples not only applicable to semiconducting luminescent labels might be the enzyme-catalysed conversion of a colourless material to a coloured substance able to quench luminescence by an energy transfer or the enzymatic reduction of a quenching species such as a nitroxide spin label in association with an otherwise luminescent molecule. Many other examples of similar processes will be apparent to those familiar with the design of luminescence assays.

A further feature of the invention is an alternative method to detect proximity between labelled species. This is based on the ability of a suitable label to respond to a thermal perturbation. Such a perturbation can be produced by absoφtion of radiation (e.g. a fast laser pulse) by an absorbing species which rapidly converts the excitation energy to thermal energy. In the immediate neighbourhood of the absorbing species the local temperature will be higher than that of the bulk medium following such a perturbation, and this can be sensed by a locally bound label. Distant labels however will not sense a significant perturbation because the thermal effects will decrease as a function of distance from the absorbing species. The distance range that can be covered by such a detection scheme will depend on the physical size and specific heat of the absorbing species (e.g. particle size if an absorbing particle is used) and on the magnitude and duration of the thermal perturbation. As a rough guide, for a particulate absorber a similar mass of water surrounding the particle to that of the particle will be substantially perturbed following an essentially instantaneous energy input (as from a fast laser pulse) and the perturbation is likely to decrease exponentially with distance from the surface. Consequently, only surface-bound species will be significantly affected by the perturbation process. The invention will be further described way of example only with reference to Figs. 1 to 6 of the accompanying drawings.

As a fust example to illustrate the principles of the invention and without limitation to the scope thereof the apparatus of Figure 1 is illustrated.

In this example the label is a luminescent phosphor particle (A) which can be excited by

lolet light and which emits visible luminescence. The quantum yield of the phosphoi is decreased when near infra-red light is adsorbed by it. A suitable phosphor m this context would be zinc sulphide activated with metal ions of copper and lead and provided with a thin coating (e.g. of polymer) so as to avoid degradation by aqueous media A continuous source of ultra-violet light (B) is optically filtered to isolate the

elength range required using a filter (C) and excites both the sample and background luminescence. The sample is further excited by a source of pulsed or modulated mtia-red light (D) which causes a periodic fluctuation in the detected luminescence orn the sample but not from the background. The fluctuating signal is optically filtcied to isolate the luminescence of the phosphor and to block scattered ultra-violet and miia-red light by a filter (E) and detected by a photodetector (e.g. a photomultipher) ( I ) connected to a lock-in amplifier (G) provided with a reference signal (I) s\ nchtonιsed to the pulsed or modulated source (D) and which is connected to a data processing device such as a computer (H) The diagram shows unfocused illumination and omits details of lenses etc. but is to be understood that focused light might be used to advantage for excitation and/or perturbation with infra-red radiation, for example where it is desired to localise the detection of signal to a particular region of the sample Such localisation might be advantageous for example in detection of surface binding during assays, to achieve a confocal effect in microscopy or flow cytometry or to minimise interference from walls of a sample container such as a microplate well It should also be understood that a variety of alternative optical formats can be used for the invention. It will be appreciated that in the example given the ultra-violet light source need not be continuous but could be pulsed or modulated and observations made at defined times or phases during the lifetime of the excited phosphor, for example before and after a pulse of infra-red light. There are many permutations inciuding but not limited to the use of pulsed or modulated exciting light and/or infra-red light, variation of phasing between pulsed or modulated excitation and perturbation sources, lock-in homodyne and heterodyne methods with modulated or gated detectors (including the application of imaging detection directly or using modulated or gated detection) and these methods are included within the scope of the invention.

In another embodiment of the invention other labels as described below are used and the perturbation means is used to deliver a fluctuating thermal or ultrasonic perturbation. As an illustrative example (Figure 2) a label (A) is excited with a light source of visible or ultraviolet light (B) filtered by an optical filter (C) and modulated in intensity at a first frequency fl by an optical modulator (D). Exciting light is incident on the sample (E) which is bound to a surface in an aqueous medium at the focus of an ultrasonic transducer (F). The transducer is energised in pulses at a pulse frequency f2. Luminescence from the sample is filtered by an optical filler (G) to reject scattered exciting light and detected by a photodetector (H) connected to a signal processor (I) which is also fed with reference signals at fl and f2 from the drive electronics (J) and (K) for the sources of excitation and perturbation respectively. The signal processor performs the mathematical filtering required to isolate the components of the detected signal resulting from the interaction between the modulated excitation and the modulated perturbation. This analysis method is used because the action of the ultrasonic transducer might cause a repetitive fluctuation in refractive index of the sample or surrounding medium which might in turn impose a modulation on the detected optical signal unrelated to the perturbation of the label. Problems such as this can be avoided by double modulation schemes taking advantage of the fact that long-lived species will not reproduce high frequency fluctuations but will reproduce slower fluctuations. Simultaneous detection at sum (fl +1 ) and difference frequencies (fl-f2) can be used to detect and compensate for unwanted background modulation effects, as discussed in our co-pending PCT patent application number PCT/GB98/00769 which is incoφorated herein by reference. Of course a simpler detection scheme as in the previous example might suffice in many cases, or conversely more elaborate multiple modulation methods could be used in applications where background fluctuations are especially troublesome. It will be understood that the experimental geometry shown is for puφoses of illustration only. It might be advantageous to combine or separate optical paths using for example dichroic beamsplitters, and multiple detection channels might be used to make measurements at several wavelengths and/or modulation frequencies or phases. Such measurements would be particularly advantageous for parallel detection of several distinguishable labels in a given measurement.

Examples of sequential multiphoton excitation where one of the photons is sufficiently energetic to excite the transition of interest will now be given by way of example and not limitation. A typical example, illustrated in Figure 3(a), involves excitation of a molecule by an allowed transition from a ground state to an excited single state. A percentage of the excited molecules will undergo intersystem crossing to the relatively long-lived triplet state. From this state the excited singlet can once more be populated by a variety of processes. A thermal re-excitation is possible in some cases, giving rise to Ε-type' delayed fluorescence, so named because it is common in dyes of the eosin family. An alternative mechanism is possible where the excited triplet interacts collisionally with a second excited triplet, giving rise to an excited singlet and a ground state species (Figure 3(b)). This is 'P-type' delayed lluorescence, so-named because it can be seen in pyrene derivatives. For the puφoses ol^' creating a label able to be excited by multiphoton absorbance via the P-type mechanism, it is convenient to a bind two chromophores covalently within a single molecule so that their interaction is highly likely. In order to increase the sensitivity of detection of delayed luminescence from labels such as the above the invention provides a method of perturbation of the long- lived triplet state and means to sense this perturbation. An appropriate perturbation for a label emitting E-type delayed fluorescence is a thermal population of a vibrational excitation which will alter the probability of reverse intersystem crossing. This can be achieved by a pulsed or fluctuating thermal energy source, or indirectly by absoφtion of photons of visible or infra-red light from a pulsed or modulated source by an absorbing species closely coupled to the emitting species. For example, a non- fluorescent dye of very short excited state lifetime can be conjugated to the emitter, or associated within a matrix. Absoφtion of light by the dye malachite green has been used previously to induce thermal perturbations in macromolecules for research purposes ("The efficiency of Malachite Green, free and protein-bound, as a photon-to- heat converter", Indig, G.L., Jay, D.G. and Grabowski. J.J.. Biophysical Journal (1992), Vol. 61, No. 3, pp. 631-638). The combination of an absorbing dye and a thermally-responsive luminescent dye is particularly appropriate as a label for microscopy. If a microscopic sample is illuminated with a focused beam of exciting light as well as a fluctuating (pulsed or modulated) focused beam of radiation to induce thermal perturbation of the luminescence, the detected fiuctuating signal will result from the common region of interaction between the focused beams. In a conventional epi-illumination geometry, this will bias signal collection to the common focal point giving a similar depth-sectioning capability to that of confocal microscopy. Alternatively, beams can be focused to a common point without sharing a common optical axis, so that the region of overlap is well defined. A label such as this in the context of the present invention allows spatial localisation of detected signals without the need for confocal optics and is therefore useful both for assay technology (e.g. to localise detection to a defined region such as a capture surface) and as a highly detectable label for microscopy. It will also be appreciated that in either case the label might be used as an energy donor in a luminescence energy transfer system, with observation of fluctuating luminescence in a wavelength region characteristic of the energy acceptor. The label described might be a molecule containing a combination of absorbing dye and luminescent dye conjugated together, or might be a particulate label such as a nanoparticle of polymer or glass (e.g. sol-gel inorganic or organo-metallic polymer) containing absorbing species and luminescent label or labels. For some applications the absorbing species might be a particle of carbon or metal in association with a coating or adsorbate of luminescent agent, either alone or bound within a coating layer (e.g. of organic polymer, glass etc).

An example of a temperature-sensitive label which emits delayed fluorescence (3(c)) is acridine yellow. This molecule immobilised in a saccharide glass has been suggested for use in optical thermometry ("Delayed fluorescence Optical Thermometry", Fister, J.C., Rank, D. And Harris, J.M., Analytical Chemistry (1995), Vol. 67. No. 23, pp. 4269-4275). In the context of the present invention however it is more appropriate to immobilise the molecule in an insoluble matrix such as an organic polymer or a glass that can be prepared by sol-gel methods under mild conditions compatible with encapsulation of organic molecules without decomposition. Excitation of the acridine yellow molecule by absoφtion of a first photon leads to the excited singlet state which is capable of fluorescence and of intersystem crossing to the triplet state. The triplet state is lower in energy than the excited singlet, but can be excited thermally to a higher vibrational level from which reverse intersystem crossing is possible to repopulate the excited singlet. Alternatively, the triplet can be deactivated radiationlessly, or can emit long wavelength phosphorescence. The thermal re-excitation to the excited singlet state gives rise to a long-lived delayed fluorescence. Since the rate of relaxation between vibrationally excited states is very fast relative to the rate of intersystem crossing, the population of the vibrationally excited states of the triplet is determined by the Boltzmann distribution, and can be considered to be at thermal equilibrium. Increase in temperature increases the population of excited vibrational levels of the triplet, leading to a higher rate of reverse intersystem crossing and hence an increase in the emission of delayed fluorescence and a corresponding decrease in phosphorescence emission. Consequently, measurement of the ratio of emissions at wavelengths corresponding to delayed fluorescence and phosphorescence can be used as a measure of temperature. The dye is said to show a change in ratio between the luminescent emissions with a sensitivity of 4.5% per °C This type of dye is useful for the purposes of the present invention because non-specific quenching of the long-lived triplet state (e.g. by oxygen) will affect both delayed fluorescence and phosphorescence emission equally, and will not alter the ratio of emissions of each. In addition, if a temperature fluctuation is imposed on the label, then the consequent fluctuations in emissions of delayed fluorescence and phosphorescence will be in antiphase to one another. Measurement in two wavelength regions chaiacteπstic of the delayed fluorescence and phosphorescence will allow emission I mm the label to be distinguished from any long-lived background emission which might also be temperature sensitive, but will not demonstrate the abovementioned con elation

To illustrate the invention furthei

w ay of example only, Figure (4) shows a scheme for detection of thermal pertuibation of emission for the purposes of high sensitivity detection. In this case a pan ol detection channels are shown for simplicity and a serial measurement is assumed, but it is to be understood that an imaging detector or a pair of such detectors could equally be used to affect the measurement of samples in parallel. The figure shows a heat source (A) which is a Peltier heat pump which is driven with a biased alternating cunent supply from the generator (B) The Peltier unit has a temperature sensor (C) hich is used m a feedback circuit to control a DC bias from the generator, so as to maintain a known average temperature irrespective of changes in the surroundings Superimposed on this average temperature is a periodic fluctuation about the mean as the heat pump alternately supplies heat to and abstracts heat from the surroundings. A thermally conductive microplate (D) containing a number of samples to be analysed for the presence of label (e.g. a label based on acridine yellow in an appropriate solid matrix) is placed on the Peltier unit A light source (E) illuminates one of the samples through a filter (F) which selects appropriate wavelengths tor excitation and directed to the samples via a dichroic mirror (G) and lens (H) Luminescence from the sample is collected by the lens and split into two wavelength regions characteristic of delayed fluorescence and phosphorescence by a second dichroic mirror unit (I) equipped with bandpass filter after which the emissions are each detected by detectors (J) and (K), which might for example be photomultipliers. The output from the detectors is passed to a pair of phase sensitive detectors (L) and (M) which are supplied with reference signals from the generator controlling the thermal perturbation via a dual phase control circuit (N) controlled by a computer (O). The computer acts as a data acquisition and control device and implements correlated ratiometric measurements from the two detection channels. For simplicity, sample positioning means are not shown in the Figure. In an implementation using imaging detection the detectors would preferably be CCD cameras imaging the sample plate through wavelength selective optics, which might use the dichroic splitter shown or might alternatively use separate imaging optics and bandpass filters for each camera. CCD cameras could be used in a mode where the cameras integrate images out of phase with one another, but with each phase locked to the reference signal from the generator (B). An image processing computer would then perform any ratiometric calculations or other data manipulations or reductions as desired. Alternatively phase-sensitive detection could be implemented directly using for example intensified gated CCDs or other detector capable of phase-sensitive imaging.

A further example of the use of the invention in context of a thermal perturbation is shown in Figure (5) for illustration only. Figure (5) shows application of the principle to a homogeneous binding assay for the detection of analytes based on the ability of the analyte to mediate binding between a label and a surface as is well known in immunology, clinical assay and molecular biology for example. A bead (A) of organic polymer, glass, ceramic or other material which might optionally be porous to increase surface area is provided, containing a high concentration of an absorbing species such as malachite green, which is capable of absorbing visible or near infra red light in appropriate spectral regions and rapidly converting the energy thereof to thermal energy. The bead is coated on its surface with a first recognition molecule (B) which might for example be an antibody capable of binding to an analyte to be detected. A thermally-sensitive label (C) which might also be provided on or in a protective matrix is also provided with a second recognition molecule (D) capable of binding to the analyte so as to bind the label in close proximity to the bead (A) through the intermediacy of the said analyte (an example of a so-called 'sandwich' assay format). A light source (E) is filtered by a filter (F) to isolate the required wavelengths to excite the label. A further light source and filter combination (G) provides an intense beam of light absorbed by the dye in bead (A). This light source is capable of being pulsed or modulated, controlled by a drive circuit (H). As a consequence of absorption of the intense periodically fluctuating light by the bead, a thermal fluctuation is produced which is most marked in the immediate neighbourhood of the bead, but which decays very rapidly as a function of distance from the surface. Luminescent labels (C) which are bound to the surface will be perturbed by the thermal fluctuation to a much greater degree than those which are not so bound and are at a greater distance from the surface. Emitted luminescence is detected by one or more photodetectors. The Figure shows two detection channels, (I) and (J), as might be used for example if the label were a dye such as acridine yellow where measurement oϊ both delayed fluorescence and phosphorescence can be advantageous as described earlier. Each detector is equipped with suitable optical filters to isolate spectral regions of interest and the signals from the detection channels are fed to an electronic processing unit (K) provided with a reference input from the drive electronics energising the light source (G). Optionally the light source (E) can be pulsed or modulated in which case a further reference signal representative of this fluctuation is also provided to the processing unit (though for simplicity the means to achieve this modulation or pulsed operation and the said reference signal are not shown in the Figure). Pulsed or modulated operation can be useful in further reducing background contributions to luminescence, for example by the use of heterodyne, homodyne, cross-correlation or equivalent detection techniques, which are well known. This implementation of the invention provides a means of homogeneous assay in that the proximity between a surface and a label is detected without the need to separate unbound label prior to measurement. It will be appreciated that the bead (A) of the invention could equally be replaced by an extended surface and focused excitation optionally could be used. Although a homogenous format is described, the invention would be equally applicable if a separation and washing step were to be included to remove unbound label in the event that this was deemed advantageous in the context of a given assay. It will equally be understood that a variety of optical formats could be used including but not limited to the use of dichroic beamsplitters to separate or combine excitation and or detection channels so as to direct signals in a common path and/or from the sample, and light-guides could also be employed if this is convenient in the application. Where long-lived luminescence is to be detected (e.g. phosphorescence), pulsed excitation might be used in conjunction with time delayed and gated detection. An equivalent frequency domain measurement might be conducted using modulated excitation with measurements of phase- shift/demodulation of emission as is well known in the art. Suitable pulsed light sources for excitation are light -emitting diodes such as the ultra-bright blue diodes supplied by Nichia, Panasonic and others. Such diodes can be pulsed in nanoseconds at megahertz rates. It is advantageous to cool the diodes in which case it is possible to drive much higher peak current through the device than would be tolerated by an uncooled diode. Some such diodes emit near ultra-violet radiation, especially when overdriven, and these are useful to excite a wide range of luminescent labels. The blue diodes emitting around 470nm supplied by Nichia are well suited to excitation of ruthenium complexes such as those with bipyridine, bathophenanthroline and related ligands which show temperature-sensitive luminescence with long (microsecond) decay times. Synchronisation of an excitation pulse with a thermally-perturbing pulse, with measurements made at one or more delay times provides a sensitive means to detect the thermal perturbation of the invention.

Another means of perturbing the rate of reverse intersystem crossing is to excite the vibrational levels of the triplet manifold directly using an infra-red laser source. This approach requires a relatively high power density of infra-red light because the lifetime of the vibronically excited levels is short relative to the rate of intersystem crossing, and in addition the absoφtion cross-section of the triplet is rather small for vibrational excitation. An alternative and more efficient approach is to excite the triplet state to the second excited triplet using a photon of visible light. Reverse intersystem crossing from this second excited triplet can then activate delayed fluorescence. Repopulation of the excited singlet via the second excited triplet state (Figure 3(c)) is known to occur in rubrene for example, and has been called " B-type' delayed fluorescence. A related phenomenon is seen with some substituted anthracene derivatives where the opposite process (thermal reexcitation of an excited singlet to a second excited triplet state) is thought to be responsible for thermal 1\ -activated fluorescence quenching

\s explained in an earlier example one means of energy input to perturb long- lived excited states is in the form of ultrasonic vibrations, which can optionally be focused to maximise the effect at a given point or surface. Pulsed ultrasound can perturb thermally sensitive species by inducing a periodic thermal perturbation and in some cases by vibrational activation of the label or host lattice. Focused ultrasound pro\ ides a method of increasing selectivity of luminescence perturbation and could be used to detect species bound to a surface in the presence of species free in solution for example This can be exploited for homogeneous assay puφoses where an analyte induces binding of a species to a surface where it is detected as a result of enhanced perturbation in the ultrasound field. Apart from the localisation of energy at the focus of an ultrasonic transducer other effects can enhance perturbation at surfaces. For example the impedance mismatch between a liquid and solid medium will result in energs deposition at the interface in a sound field. This will occur even in an unfocused beam and might be sensed by labels bound to particles as a result of the presence of analyte. Similarly, ultrasonic vibrations can interact with surface plasmon states and might influence luminescence quenching processes for luminescent species bound to surfaces having such states (e.g. thin metal films, colloidal metal particles and the like). Excited triplet states are often populated by energy transfer from a higher energy triplet donor (Figure 3(e)) rather than by intersystem crossing from an excited singlet state. This is a highly efficient process and is particularly useful to populate excited triplet states of molecules which do not undergo efficient intersystem crossing (e.g. molecules with very high quantum yields of fluorescence). Aromatic ketones such as benzophenone are widely used for such purposes in photochemical research. The donor species can be chemically linked to the molecule of interest if an intramolecular sensitisation is required, or alternatively can be provided within a matrix containing the acceptor species.

The rate of energy transfer from a triplet donor to an acceptor is not usually very sensitive to donor energy unless the energy gap between donor and acceptor is small. Under these circumstances a back-transfer process from the acceptor to the donor can operate to reduce the overall sensitisation efficiency. A similar phenomenon is seen where a triplet donor of lower energy than the acceptor triplet can be vibrationally excited and thereby populate the acceptor level. In appropriate circumstances the processes described can result in a marked temperature dependence of the sensitised excitation of an acceptor species. An example of this is given by the fluorescence of lanthanide complexes, e.g. complexes of terbium which can show large temperature sensitivity with some complexing ligands ('Getting excited about lanthanide complexation chemistry' Parker, D. and Williams, J.A.G. in J. Chem. Soc. Dalton Trans., 1996, 3613-3628).

The perturbation methods described are well suited to use with labels where the triplet or other long-lived state of the label is populated by sensitisation from another excited triplet. Back-transfer from the acceptor to the donor can be activated by a thermal fluctuation or by excitation of the acceptor to a higher energy triplet from which transfer of energy to the donor can occur efficiently. Alternatively a triplet energy donor of lower energy than the excited state of the acceptor can be provided and a thermal or other perturbation can be used to modulate energy transfer from vibrationally -excited states of the donor to the lower vibrational levels of the acceptor state.

An unconventional approach to reach the intermediate excited triplet state, is shown in Figure (3 (f)) by direct excitation from the ground state singlet. This usually is a very inefficient process. Whereas a typical singlet-singlet transition has a molar extinction coefficient in the range 10,000-100,000, the corresponding values for a singlet-triplet excitation would be much less than unity in many cases. For a sequential two-photon excitation proceeding from ground state to excited triplet followed by re-excitation to the higher triplet, the efficiency is the product of the excitation efficiencies. The triplet-triplet absoφtion of visible radiation is a fully- allowed transition and can be very efficient. Overall the sequential two-photon excitation is inefficient relative to a conventional single photon absoφtion, but it is still several orders of magnitude more probable than a simultaneous two-photon transition. Consequently the process can be excited by a light source or sources of only modest power. This is true also for a multiphoton process proceeding by triplet- triplet annihilation. Here the overall excitation probability is the product of two low probability events, but even so this is still much more efficient overall than a typical simultaneous two-photon absoφtion event.

The energies required to excite the singlet-triplet transition are typically quite low, so that a semiconductor laser emitting in the far red-near infra red is adequate in many cases. For example rubrene, which as stated earlier can be re-excited by triplet- triplet absorption, has a first excited triplet energy greater than lOOOnm and can be populated by near infra-red absoφtion. Perylene, which can undergo excitation by triplet-triplet annihilation, has a first excited triplet energy of c. 800nm. In fact there are very many luminescent labels with triplet energies in ranges easily accessible to inexpensive semiconductor laser sources. The triplet-triplet transitions usually occur in the visible spectral region and have high extinction coefficients. These transitions can be pumped by a variety of sources such as inexpensive light-emitting diodes, lasers and conventional light sources such as arcs or flashlamps.

Although the probability of a singlet-triplet transition is normally very low, this depends markedly on environment and nature of substituents. This leads to the possibility of tailoring the environment to enhance the excitation probability. Heavy atom substituents such as bromine and iodine are often effective in enhancing singlet- triplet absoφtion.

Although the discussion so far has concentrated on isolated molecules, in many cases it is desirable for labels to be prepared as microparticles. For example, fluorescent polymer microspheres are widely used in cell biology as markers for cell surface components and as intracellular tracers. The environment within a particle is favourable for long-lived luminescence emission because many quenching processes are suppressed. The environment can also be tailored to enhance the probability of the desired transitions. For example, a partially halogenated polymer can be used to enhance single-triplet excitation efficiency for a fluorescent specifies contained within.

Microscopic particles can also be prepared from inorganic species, either from bulk media (e.g. using a colloid mill or related technology) or alternatively by direct formation, for example by so-called 'sol-gel' techniques. These and similar techniques are very well known. By such means it is possible to prepare microscopic particles of phosphor materials. An alternative method of encapsulation is direct synthesis of the inorganic species within the interior of a liposome or microemulsion by prior entrapment of a soluble and impermeable agent within the structure and diffusion of a reactive species into the structure to affect a chemical reaction with the trapped material. By this means very small and uni orm particles can be prepared and these can sometimes have useful optical and electronic properties not seen in bulk phases. Inorganic semiconducting species prepared by such methods are potentially useful as labels for the present invention.

Man> inorganic species are well suited to the invention, particularly materials based on ions of zinc and/or cadmium in combination with ions of sulphur, selenium and or tellurium and appropriate dopants to modify luminescence behaviour where appropriate. As an example, it is known that certain phosphorescent inorganic species are excited w ith ultra-violet light and emit visible luminescence but are quenched when exposed to infra-red light. Phosphors based on zinc sulphide activated with certain metal ions such as copper and lead for example are efficiently quenched by near infra-red uidiation (see 'Luminescence in Inorganic Solids' by Paul Goldberg - New York London Academic Press 1996) the mechanism is thought to involve excitation of bound excitons into non-luminescent states.

In ordei to protect inorganic phosphors such as zinc sulphide from hydrolysis or other unw anted reactions it is often necessary to microencapsulate the material or to coat the phosphor with a thin layer of a resistant material. Such coating is also advantageous I^'oi the puφoses of binding recognition molecules such as antibodies, lectins peptides and oligonucleotides to the particle for applications in imaging, assay and sensing I here are many coating methods known including adsoφtion of polymers waxes or surfactants, and these materials can carry functional groups suitable for binding to other materials. Alternatively an inorganic material can be used to form a protective layer. For example, oxide coatings such as silica can be deposited by a variety of methods. Subsequently such coatings can be functionalised by reaction with organic molecules such as silanes. For example amino and thiol groups can be bound to a surface having exposed hydroxyl groups by interaction with aminopropyl triethoxysilane and mercaptopropyl triethoxysilane respectively.

Although the discussion of the invention has centred on applications where the label is detected directly by virtue of its luminescence, there are also applications where the excited label can transfer energy radiationlessly or radiatively to an acceptor species which luminesces as a result of this excitation. The transfer of energy might be from triplet levels of the label to triplet or other long-lived levels of an acceptor or might be triplet-singlet transfer from the label to the acceptor. Alternatively, if the label shows delayed fluorescence as is the case for acridine yellow for example, the transfer might be from the excited singlet of the label to a singlet state of the acceptor, for example by a dipole-dipole mechanism. Any fluctuation in the long-lived state of the label will also result in a corresponding fluctuation in luminescence from the acceptor as a result of the said energy transfer. Energy transfer from a donor species to an acceptor species is a sensitive function of distance between the two, and hence can be used to detect association between a donor and acceptor. This process has been used for a wide variety of assay puφoses. One example is to detect analyte- mediated binding in 'sandwich' assays where donor and acceptor are each labelled with a recognition molecule such as an antibody and the analyte can affect formation of a complex as a result of binding both donor and acceptor species simultaneously. In the context of the of the present invention, it is advantageous to sense the fluctuation in the luminescence of the acceptor species consequent on a perturbation of the donor because this avoids confusion of acceptor excitation via other possible mechanisms (e.g. direct excitation of acceptor by light used to excite donor label) and also allows lock-in detection to minimise background. Various examples of assay procedures can be readily devised using pulsed or modulated excitation and/or perturbation means and time-gated or phase-sensitive detection of fluctuations in luminescence. Equally, homodyne, heterodyne or cross-correlation detection schemes can be readily implemented, based on the predictable pattern of luminescence emission consequent on interactions between a pulsed and/or modulated excitation means and a pulsed and/or modulated perturbation means in the label. A simple example for illustration only is given figure 6. A label means (A) shown here as, in or bound to a bead bears a first recognition molecule (B). This bead preferably is between 5-50 nm in diameter. An acceptor species (C), labelled with a second recognition molecule is bound to the first bead in proportion to the amount of analyte (D). An excitation means (E) and a perturbation means (F) simultaneously illuminate the sample and the energies thereof are absorbed by the bead but preferably not by the acceptor species. A control circuit (G) controls the fluctuations of the excitation means and/or the perturbation means and provides synchronising signals (H) and (I). The sensitised emission of the acceptor and optionally emission characteristic of the donor are detected by detector/optical filter combinations (J) and (K). The emission from the donor might have a component useful as an internal standard, for example to correct for absoφtion of exciting or emitted radiation by components of the sample, and is therefore optionally detected. The signals from the detectors are passed to a signal process or (L) which also receives the synchronising signals (H) and (I). This performs any required calculations to extract the fluctuating signal components due to the sensitised emission.

It will be appreciated that there are a large number of measurement formats based on binding or competition between labelled species and analytes or analogues thereof and these are well described in the literature. Examples without limitation of recognition molecules which can be used are antibodies and fragments thereof, lectins, oligonucleotides and derivali\ es and analogues thereof, proteins and glycoproteins such as avidin, streptavidin. poK saccharides, sugars, and lower molecular weight species such as biotin.

Some examples of possible formats for energy transfer assay based on long- lived luminescence from upcon\ crsion phosphors are given in our co-pending PCT patent application number PCT/GB98/00769 and these can be readily adapted to the present invention. For measurements over distances where radiationless energy transfer is inefficient (i.e. lOnm or more ) radiative energy transfer (i.e. emission of a photon by a donor species and absorption of the photon by a proximate acceptor species) can be used. This approach is described in our co-pending U.K. patent application 9811481.

Claims

1. A method of luminescence detection comprising:

1) effecting excitation of a label by abso╧åtion of one or more photons of the same or different energy, at least one of which is of visible or ultraviolet light, such that

(i) the excitation leads to the population of one or more excited states having a lifetime greater than one microsecond but less than one minute, and

(ii) the or at least one of the excited states is capable of detection directly or indirectly by luminescence;

(c) promoting the intermediate to a luminescent higher excited state; and 3) detecting the fluctuation or fluctuations in concentration of the said excited state or states which result from said perturbation either directly by virtue of the luminescence of the said excited state or states or indirectly by observation of the luminescence of one or more other species to which excitation energy has been transferred.

2. A method as claimed in claim 1 wherein the perturbation is effected periodically.

3. A method of luminescence detection comprising:

1) effecting excitation of a label by absorption of one or more photons of the same or different energy, at least one of which is of visible or ultraviolet light, such that

4. A method as claimed in claim 3 wherein the transient change is effected by virtue of

(a) increasing or relaxing the said quantum mechanical limitation on emission from the intermediate,

(c) promoting the intermediate to a luminescent higher excited state;

5. A method according to any one of claims 1 to 4 wherein the label means is

excited to the said excited state by means of transfer of energy from the excited triplet

state of a sensitising agent.

6. A method according to any one of claims 1 to 4 wherein the label means is one

that gives rise to thermally-activated delayed fluorescence.

7. A method according to any one of claims 1 to 4 wherein the label means is one

that contains two or more moieties that can be excited giving rise to excited triplet

species capable of deactivation by triplet-triplet annihilation.

8. A method according to any one of claims 1 to 4 wherein the excited state is a

triplet state and the perturbation means acts to further excite the triplet leading to

population of a higher excited state capable of giving rise to delayed luminescence.

9. A method according to any one of claims 1 to 4 wherein the excitation of the

label directly populates an excited triplet state.

10. A method according to any one of claims 1 to 4 wherein the said label is a

luminescent species that is quenched by abso╧åtion of red or infra-red light, and wherein the said perturbation is effected by a source of such light.

1 1. A method according to claim 10 wherein the said label is an inorganic species.

12. A method according to claim 11 wherein the said inorganic species is a

luminescent material based on zinc and/or cadmium ions in association with ions of

sulphur, selenium and/or tellurium.

13. A method according to claim 12 wherein the said inorganic species is doped

with ions of at least one transition metal.

14. A method according to claim 13 wherein the said inorganic species is zinc

sulphide doped with ions of copper and/or lead.

15. A method according to any one of claims 1 to 4 wherein the said label is used

as an energy donor species to detect proximity between the said label and another

acceptor species capable of accepting energy efficiently from the label when closely

associated with it but not otherwise.

16. A method according to claim 15 wherein the said acceptor species luminesces

as a result of said transfer of energy and wherein the application of the said perturbation to the donor label results in a fluctuation of the said transfer of energy,

said fluctuation being detected in at least a wavelength region characteristic of the

luminescence of the acceptor species.

1 7. A method as claimed in any one of claims 1 to 4 which is an assay to detect an analyte able to influence the degree of association between the label means and

another luminescent species able to accept energy from the said label with consequent

emission of characteristic luminescence when in close proximity (e.g. bound) thereto

but not otherwise, the assay comprising at least the steps of:

contacting a medium possibly containing the said analyte with the said label and the

said luminescent species under conditions suitable for promoting interaction

therebetween,

effecting the excitation of said label, effecting the perturbation to cause a fluctuation in the excitation state of said label,

and

measuring said characteristic luminescence of the other luminescent species to detect

a related fluctuation resulting from the perturbation in transfer of energy between the

said label and the luminescent species.

18. A method according to any one of claims 1 to 4 wherein the said label means

is one that has characteristic luminescence in at least one wavelength region whereof

the emission efficiency is sensitive to temperature, and wherein the said perturbation induces a fluctuation in temperature that is detected by measurement of said

luminescence or other luminescence sensitised by transfer of energy from the said

luminescent species.

19. A method according to claim 18 wherein the said temperature fluctuation

sensed by the said label results from abso╧åtion of radiation by an absorbing species in

close proximity (e.g. bound) to the said label.

20. A method according to claim 19 wherein the said label means and the said

absorbing species are associated within or bound to a solid particle.

21. A method according to any one of claims 18 to 20 wherein the said label or

particle bearing said label additionally bears binding ligands for the pu╧åose of

binding to complementary species.

22. A method according to claims 19 wherein the proximity between said label

and said absorbing species is able to be influenced by an analyte and the possible

presence of said analyte is detected by a process comprising at least the steps of:

said absorbing species under conditions suitable for promoting interaction between

them,

effecting the excitation of said label ,

applying a localised thermal perturbation by input of radiation absorbed by the

absorbing species, and

detecting the influence of the said perturbation on the proximate label by

measurement in a wavelength region characteristic of luminescence from the said

label.

23. A method according to any one of claims 1 to 4 wherein said transient change

is effected by at least one source of ultrasonic vibration.

24. A method according to claim 23 wherein at least a part of the energy of the

said ultrasonic vibration is absorbed by an absorbing material in close proximity (e.g.

bound) to the said label.

25. A method as claimed in any one of claims 1 to 4 which is an assay to detect an

analyte able to influence the degree of association between the label means and

another species able to quench luminescence of the said label when in close proximity

(e.g. bound) but not otherwise comprising at least the steps of:

contacting a medium possibly containing the said analyte with the said label and the said other species under conditions suitable for promoting interaction therebetween,

effecting the excitation of said label,

effecting the perturbation to cause a fluctuation in the excitation state thereof, and

measuring characteristic luminescence of the said label to detect the fluctuation

therein.

26. A method according to claim 25 wherein the said quenching species is

associated with the said label so as to quench emission therefrom and the action of the analyte is to decrease the degree of association between the label and the quenching

species resulting in enhanced luminescence from the label.

27. A luminescence detection system comprising:

1) a label means which is capable of excitation by absorption of one or more photons of the same or different energy, at least one of which is of visible or ultraviolet light, such that

(a) increasing or relaxing a quantum mechanical limitation on emission from the intermediate.

(c) promoting the intermediate to a luminescent higher excited state;

2) a source of visible and/or ultraviolet light to excite the said label; 3) a perturbation means which can induce said transient change, and

4) a detection means whereby the fluctuation or fluctuations in concentration of the said excited state or states which result from the application of the said perturbation means can be monitored either directly by virtue of the luminescence of the said excited state or states or indirectly by observation of the luminescence of one or more other species to which excitation energy has been transferred.

28. A luminescence detection system comprising:

(i) the said excitation process leads to the population of one or more excited states having a lifetime greater than one microsecond but less than one minute, and

(ii) the or at least one of the said excited states is capable of detection directly or indirectly by luminescence.

2) a source of visible and/or ultraviolet light to excite the said label;

3) a perturbation means for providing a periodic perturbation to induce a transient change in the population of the said long-lived intermediate or intermediates; and