WO2011092457A1

WO2011092457A1 - Proximity of particles

Info

Publication number: WO2011092457A1
Application number: PCT/GB2011/000083
Authority: WO
Inventors: Dmitry Gakamsky
Original assignee: Edinburgh Instruments Limited
Priority date: 2010-01-26
Filing date: 2011-01-24
Publication date: 2011-08-04
Also published as: GB201001191D0

Abstract

A method for determining the proximity of at least two fluorescence particles that can be excited using radiation of the same wavelengths but which emit fluorescence at different wavelengths, the method comprising: exciting a sample volume of known dimensions that includes the first and second particles; detecting coincidences in emission from the first and second particles within the sample volume, and using the detected coincidences as a measure of particle proximity.

Description

Proximity of Particles

Field of the Invention

The present invention relates to a method for detecting the spatial proximity between two or more particles. The particles could each be any type of atom, inorganic or organic molecule or compound made of them. The method is particularly applicable to, but not limited to, the range of particle separation between 0.01-0.5pm.

Background of the Invention

The spatial organisation of cell surface expressed proteins and lipids or intracellular components plays an important role in cell functioning, activation and signal transduction. Fluorescence Resonance Energy Transfer (FRET) allows molecular separations to be assessed, see Stryer, L. 1978, "Fluorescence energy transfer as a spectroscopic ruler" Annu. Rev. Biochem. 47:819-46. Whilst FRET is useful and commonly used, a limitation is that it can only be used to determine separations within a range set by the Forster radius (R₀). For the best current FRET donor- acceptor pairs, the value of R₀ does not exceed ca. 0.01 μπι (10nm).

The limited range of FRET means that it is unsuitable for many practical applications, such as for studying the organisation of proteins on the surface of a cell or for detecting analyte molecules by matched antibody pairs, and so other techniques have to be used. For example, optical microscopy methods can be used to assess the proximity of particles labelled by fluorescence probes and separated by distances longer than ca 0.5μιη, the lower resolution being set by the diffraction limit of the optical system. Separations in the range 0.01 - 0.5 μη\ are too long to be studied by the FRET method but are too short to be measured using the fluorescence microscopy method.

Another technique that can be used for determining proximity is cross-correlation spectroscopy. This monitors cross-correlated fluctuations in the intensities of the time dependent fluorescence emissions of two different fluorescent molecules co- present in the same confocal volume, see Bacia, K., et al 2006 "Fluorescence cross- correlation spectroscopy in living cells" Nat. Methods. 3:83-89. However, this technique is only applicable for molecules that have sufficiently high diffusion rates such that they would cross the field of view of the confocal volume in a time of less than a few milliseconds. For slowly diffusing molecules a long detection time, usually several minutes, is needed in order to confidently detect correlated movements. A problem with this is that long illumination of small membrane regions can lead to cytoskeleton movement resulting in the area of interest moving. A further problem is that cross-correlation spectroscopy cannot be used for fixed cells or other biological objects.

Summary of the Invention

According to one aspect of the present invention, there is provided a method for determining the proximity of particles with fluorescence properties (which could be particles which intrinsically exhibit fluorescence or particles labelled with a fluorescence species) that can be optically excited using radiation of the same wavelength but which emit fluorescence at different wavelengths, the method comprising: exciting a sample volume that includes the first and second fluorescence particles using an excitation source; detecting coincidences in emissions from the first and second fluorescence particles within the sample volume, and using the detected coincidences as a measure of particle proximity.

By using fluorescence particles that have spectrally overlapping absorption bands such that they can be excited using light of the same excitation wavelength but which emit fluorescence in non-overlapping spectral regions, proximity can be detected by monitoring when both particles emit a photon in response to excitation from the same light pulse. This is because such coincident emissions can only occur when the particles are both present within the illuminated volume, and so are within a defined distance of one another. Using the technique of the present invention the spatial proximity of particles across a wide range of separations can be determined, including the range 0.01 - Ο.δμηΊ. This can be done without the need for any movement of the particles. The first and second fluorescence particles may be used as labels for other particles. In this case, the method may involve labelling a first particle with the first fluorescence particle and labelling a second particle with the second fluorescence particle, whereby detecting the proximity of the first and second fluorescence particles gives a measure of the proximity of the first and second particles. The first and second fluorescence particles may be used as labels for different particles or different types of particles.

The first and second fluorescence particles may be used as labels for the same particles or the same types of particles.

The method may involve using three or more particles that are excitable using the same wavelength but emit at different wavelengths. The fluorescence particles may contain one or more dyes or inorganic material, such as a semiconductor material in the form of nanocrystals or quantum dots. As a specific example, the first and second fluorescence particles could be quantum dots.

The method may involve repetitively pulsing the excitation source to illuminate the sample volume. The method may involve detecting correlated coincidences of photons within the sample volume after every excitation pulse and using the number or rate of such detected coincidences as a measure of particle proximity.

According to another aspect of the present invention, there is provided a system for determining the proximity of at least a first and second fluorescence particle that can be excited using radiation of the same wavelength but which emit fluorescence at different wavelengths, the system comprising an excitation light source for exciting a sample volume that includes the first and second fluorescence particles; and a detector for detecting coincidences in emissions from the first and second fluorescence particles within the sample volume, wherein the detected coincidences provided a measure of particle proximity.

Brief Description of the Drawings

Various aspects of the present invention will now be described by way of example of two types of particles only:

Figure 1 shows excitation and emission spectra for two fluorescence probes, A and B conjugated to particles of the first and the second type;

Figure 2 is a schematic diagram of a possible experimental arrangement for determining particle proximity using the fluorescence probes A and B;

Figure 3(a) shows a train of excitation pulses; Figure 3(b) shows single photon detection pulses from detector D1;

Figure 3(c) shows single photon detection pulses from detector D2, and Figure 3(d) shows the correlated coincidences when a pulse is observed in both (b) and (c) after the same excitation pulse.

Detailed Description of the Drawings

Figure 1 shows the excitation and emission spectra of two fluorescence probes A and B which can be excited by incident radiation at the same wavelength using the same light source, but which emit in different spectral regions such that there is no "crosstalk" between their emissions. These are used to label particles of interest, for example proteins on the surface of a cell. Probe A is used to label a first type of particle and probe B is used to label a second type of particle. In accordance with the present invention, information on the proximity of the particles can be ascertained by detecting photons coincidently emitted by probes A and B in a well defined sample volume in response to the same excitation pulse.

Figure 2 shows a system for detecting proximity using the fluorescence probes A and B. This is a confocal fluorescence detection system. It has a light source that can be repetitively pulsed, normally a laser, for illuminating a small confocal volume of the sample in which the labelled particles are present. The laser has to emit at a wavelength that is in the excitation range of both of the fluorescence probes. The laser beam is collimated using a lens and directed onto a first dichroic beam splitter DBS1 from where it is reflected towards a focussing or objective lens 1 and then focussed onto a sample. Light emitted from the sample as a result of the excitation passes through and is collimated by the focussing lens, and then passes through the first beam splitter DBS1, which is specifically chosen so that it is reflective at the excitation wavelength, but transmits at the emission wavelengths.

Light transmitted through the beam splitter DBS1 continues to a second objective lens where it is focussed towards a pinhole. The size of the pinhole and the dimension of the focussed incident radiation in the sample chamber together determine the sample volume that is imaged. The focal length of the objectives and the diameter of the pinhole can be chosen to determine the confocal sample volume from which signals are detected. Behind the pinhole is a third objective lens that collimates the light and directs it towards a second dichroic beam splitter DBS2, which is chosen to reflect light from one of the fluorescence probes, but transmit light from the other.

Light reflected from the second beam splitter DBS2 is focused onto a first single photon counting detector D1. Light transmitted through the beam splitter DBS2 is focused onto a second single photon counting detector D2. Both the detectors are typically photomultipliers and ideally should have a low dark count rate, so that individual photons emitted by the first and second probes from within the excited volume can be reliably detected. The output of both detectors D1 and D2 is input to correlation electronics, which are able to identify when a pulse is detected coincidently by both detectors. Photon correlation is determined by the coincidence electronics using synchronization pulses from the light source and pulses produced by the detectors D1 and D2. In practice, a repetitively pulsed source is used to excite the sample. The pulse width of the source should be ideally less than the fluorescence lifetime of both probes A and B. The repetition rate of the source should be chosen such that the time between successive pulses is more than five times longer than the longest fluorescence lifetime of probe A and B. This ensures that the probability of a probe being excited by one pulse and emitting a photon only after the arrival time of the next excitation pulse is negligible. Typical fluorescence probes have lifetimes of the order of nanoseconds to tens of nanoseconds for example 1 - 30ns and hence the pulse repetition rate of the excitation source should be less than ca 200MHz to 6MHz respectively.

The sample concentration is chosen such that on average less than one particle or complex is present in the confocal volume. The photon counting rate in each detection channel is proportional to the concentration of the labelled particles. The rate of the correlated signals is proportional to the concentration of pairs of labelled probes in close proximity i.e. forming a complex. Hence, in a sample where some particles are bound/proximal and some free, the concentrations of such bound and free particles can be quantified.

The dimensions of the confocal volume determine the proximity scale. For example, a confocal volume of 1 femtolitre or 10^'12cm³ (equivalent to 1 micron cubed) would have a limit of proximity of up to 1 μιη. In this case, a sample concentration of less than 60nM would be needed to ensure only one particle is present in the confocal volume (1 Mole/litre concentration corresponds to approximately 6Ί0²³ molecules/litre). Assuming that the mean displacement of one or other of the particles over the duration of the excitation pulse is significantly smaller than the dimensions of the confocal volume, then coincidental detection of photons emitted by the A and B probes after the same excitation pulse indicates their presence at the same time interval, defined by the pulse duration, in the same place, confined by the size of the confocal volume. As will be appreciated, this does not provide a direct measure of the distance between the probes A and B, but does ascertain their joint presence in the same confocal volume, and thereby their proximity.

For particles in water solutions or cells, the molecular diffusion constant, D, is in the range of 1000-0.01

which means that for duration of the excitation pulse, typically 100ps, the mean displacement <r>, which is given by (r) = yj6DAt , is significantly smaller than the dimensions of the confocal volume, typically ca. 0.5 - 2 μηι. Therefore, two fluorescence probes A and B can only emit coincidentally if they are both present in the confocal volume at the same time. This means that the A and B labelled particles are spatially correlated, because the probability that a second particle could enter the illuminated volume within the pulse width of the excitation source is negligible. In other words, coincidental detection of photons emitted by the A and B probes after the same short excitation pulse determines their presence at the same time interval, defined by the pulse duration, in the same place, confined by the size of the confocal volume.

This new method for detecting the proximity of two or more particles can be used in both cell-free and cell-based applications. One possible example of a cell-free application is the use of antibodies or other proteins or molecules for the detection of analyte concentrations in biological samples, for example using "matched'¹ antibody pairs, in which one antibody (Abi) is labelled with fluorescence probe A and another antibody (Ab₂) is labelled with fluorescence probe B. Both antibodies bind the same analyte molecule (antigen) to produce a "sandwich" A-AbrAntigen-Ab₂-B. This will establish a certain spatial proximity between A and B fluorophores, which can be assessed by the method of the invention. The present invention can be used for high throughput screening of chemical libraries by monitoring changes in the spatial coordination of proteins or other molecules in solution or in live cells or in medical diagnostics for the determination of different analyte concentrations in biological samples and live cells. In addition, the invention can be used to study all types of association and dissociation reactions in solutions and biological objects, as well as the structure or spatial organization of luminescent atoms, ions or molecules in any type of transparent rigid solutions, where diffusion is virtually non-existant. Another field of possible applications relates to the study of the organisation of cell-surface expressed or intracellular proteins or other molecules. The method can also be used for studying kinetics formation and stoichiometry of multi- component complexes in live cells.

A skilled person will appreciate that variations of the disclosed arrangements are possible without departing from the invention. For example, whilst the invention has been described primarily with reference to determining the proximity of two particles, the proximities of more than two particles could be determined by using more than two fluorescence probes and corresponding emission detection channels. In addition, although single photon excitation has been described, two-photon excitation could also be used. Accordingly the above description of the specific embodiment is made by way of example only and not for the purposes of limitation. It is clear that minor modifications may be made without significant changes to the operation described.

Claims

1. A method for determining the proximity of at least two fluorescence particles that can be excited using radiation of the same wavelength but which emit fluorescence at different wavelengths, the method comprising: exciting a sample volume of known dimensions that includes the first and second particles; detecting coincidences in emission from the first and second particles within the sample volume, and using the detected coincidences as a measure of particle proximity.

2. A method as claimed in claim 1 comprising using the first and second particles as labels for other particles.

3. A method as claimed in claim 2 wherein the first and second particles are used as labels for different particles or the different types of particles.

4. A method as claimed in claim 3 wherein the first and second particles are used as labels for the same particles or the same types of particles.

5. A method as claimed in any of the preceding claims comprising using a pulsed excitation light source.

6. A method as claimed in claim 5 wherein the laser source provides a single photon excitation of the sample.

7. A method as claimed in 5 wherein the laser source provides a two-photon excitation of the sample.

8. A method as claimed in any of the preceding claims wherein the excited sample volume is sized so that it is less than the average separation of the particles.

9. A method as claimed in any of the preceding claims comprising counting the number of photon coincidences and counting the number of photon emissions from the first and second particles and using this to determine the concentration or percentage of particles that are physically close.

10. A method as claimed in any of the preceding claims comprising providing three or more particles excitable by the same wavelength but which emit at different wavelengths.

11. A system for determining the proximity of at least a first and second fluorescence particle that can be excited using radiation of the same wavelength but which emit fluorescence at different wavelengths, the system comprising an excitation light source for exciting a sample volume that includes the first and second fluorescence particles; and a detector for detecting coincidences in emissions from the first and second fluorescence particles within the sample volume, wherein the detected coincidences provided a measure of particle proximity.