GB2609456A - An apparatus and method for calculating a number of photons associated with bound fluorescent molecules - Google Patents

An apparatus and method for calculating a number of photons associated with bound fluorescent molecules Download PDF

Info

Publication number
GB2609456A
GB2609456A GB2111103.4A GB202111103A GB2609456A GB 2609456 A GB2609456 A GB 2609456A GB 202111103 A GB202111103 A GB 202111103A GB 2609456 A GB2609456 A GB 2609456A
Authority
GB
United Kingdom
Prior art keywords
photons
fluorescent molecules
polarisation
count
angle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
GB2111103.4A
Other versions
GB202111103D0 (en
Inventor
Jiang Yonggang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Advanced Instr Ltd
Original Assignee
Advanced Instr Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Advanced Instr Ltd filed Critical Advanced Instr Ltd
Priority to GB2111103.4A priority Critical patent/GB2609456A/en
Publication of GB202111103D0 publication Critical patent/GB202111103D0/en
Publication of GB2609456A publication Critical patent/GB2609456A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N2021/217—Measuring depolarisation or comparing polarised and depolarised parts of light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Molecular Biology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Apparatus for quantifying binding of fluorescent molecules to drug molecules, comprises; control circuitry to cause a tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecules, the fluorescent molecules comprising bound fluorescent molecules and unbound fluorescent molecules; photon counting circuitry to count, within a dynamic range of the photon counting circuitry: a first number of photons emitted from the fluorescent molecules at the polarisation angle; and a second number of photons emitted from the fluorescent molecules at a depolarisation angle different to the polarisation angle; calculation circuitry to calculate, based on comparison between the first and second number of photons, a number of photons associated with the bound florescent molecules. The intensity of the light source is such that the first and second number of photons are within the dynamic range of the counting circuitry.

Description

AN APPARATUS AND METHOD FOR CALCULATING A NUMBER OF PHOTONS ASSOCIATED WITH BOUND FLUORESCENT MOLECULES
The present invention relates to an apparatus and method for quantifying binding of fluorescent molecules to a drug molecule.
In order to determine efficiency of drug production, drug molecules need to be quantified Obtaining accurate counts of small numbers of drug molecules can be challenging In some configurations of the present techniques there is provided an apparatus for quantifying binding of fluorescent molecules to a drug molecule, the apparatus comprising: control circuitry to regulate an intensity of a tuneable light source and to cause the tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule, the fluorescent molecules comprising bound fluorescent molecules bound to the drug molecule and unbound fluorescent molecules free from the drug molecule, wherein in response to absorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale; photon counting circuitry comprising a photomultiplier tube, the photon counting circuitry and the photomultiplier tube configured to operate in a photon counting mode to count photons within a dynamic range of the photon counting circuitry, wherein the photon counting circuitry is configured to count: a first number of photons that are emitted From the fluorescent molecules and have passed through a first polarisation filter allowing transmission at the polarisation angle; and a second number of photons that are emitted from the fluorescent molecules and have passed through a second polarisation filter allowing transmission at a depolarisation angle different to the polarisation angle; and calculation circuitry configured to calculate, based on a comparison between the first number of photons and the second number of photons, a number of photons associated with the bound florescent molecules, wherein the control circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range.
In some configurations of the present techniques there is provided a method for quantifying binding of fluorescent molecules to a drug molecule, the method comprising: controlling a light source to regulate an intensity of a tuneable light source and to cause the tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule, the fluorescent molecules comprising bound fluorescent molecules bound to the drug molecule and unbound fluorescent molecules free from the drug molecule, wherein in response to adsorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarisers from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale; counting, using photon counting circuitry and a photomultiplier tube configured to operate in a photon counting mode to count photons within a dynamic range of the photon counting circuitry: a first number of photons that are emitted from the fluorescent molecules and have passed through a first polarisation filter allowing at the polarisation angle; and a second number of photons that are emitted from the fluorescent molecules and have passed through a second polarisation filter allowing transmission at a depolarisation angle different to the polarisation angle; and calculating, based on a comparison between the first number of photons and the second number of photons, a number of photons associated with the bound florescent molecules, wherein controlling the light source comprises regulating the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range.
The present techniques will be described further, by way of example only, with reference to embodiments thereof as illustrated in the accompanying drawings, in which: Figure 1 schematically illustrates an apparatus according to various configurations of the present techniques, Figure 2 schematically illustrates an apparatus according to various configurations of the present techniques, Figure 3 schematically illustrates an apparatus according to various configurations of the present techniques, Figure 4 schematically illustrates an apparatus according to various configurations of the present techniques, Figure 5 schematically illustrates an apparatus according to various configurations of the present techniques, Figure 6A schematically illustrates the detection of photons by an apparatus in photon counting mode according to various configurations of the present techniques; Figure 6B schematically illustrates the detection of photons by an apparatus in integration mode according to various configurations of the present techniques; Figure 7A schematically illustrates the emission of polarised and depolarised fluorescence from an unbound fluorescent molecule according to various configurations of the present techniques, Figure 7B schematically illustrates the emission f polarised and depolarised fluorescence from a bound fluorescent molecule according to various configurations of the present techniques; Figure 8A schematically illustrates counts of photons emitted from unbound fluorescent molecules that are polarised at the polarisation angle; Figure 8B schematically illustrates counts of photons emitted from unbound fluorescent molecules that are polarised at the depolarisation angle; Figure 8C schematically illustrates counts of photons emitted from bound fluorescent molecules that are polarised at the polarisation angle, Figure 8D schematically illustrates counts of photons emitted from bound fluorescent molecules that are polarised at the depolarisation angle; Figure 9 schematically illustrates counts of photons from both bound and unbound fluorescent molecules at the polarisation angle and at the depolarisation angle, and Figure 10 schematically illustrates a sequence of steps carried out to calculate a number of photons associated with bound fluorescent molecules.
In some configurations there is provided an apparatus for quantifying binding of fluorescent molecules to a drug molecule. The apparatus comprises control circuitry to regulate an intensity of a tuneable light source. The control circuitry is configured to cause the tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule. The fluorescent molecules comprise bound fluorescent molecules which are bound to the drug molecule and unbound fluorescent molecules which are free from the drug molecule. In response to absorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale. The apparatus further comprises photon counting circuitry comprising a photomultiplier tube. The photon counting circuitry and the photomultiplier tube are configured to operate in a photon counting mode to count photons within a dynamic range of the photon counting circuitry. The photon counting circuitry is configured to count: a first number of photons that are emitted from the fluorescent molecules and have passed through a first polarisation filter allowing transmission at the polarisation angle; and a second number of photons that are emitted from the fluorescent molecules and have passed through a second polarisation filter allowing transmission at a depolarisation angle different to the polarisation angle. The apparatus further comprises calculation circuitry configured to calculate, based on a comparison between the first number of' photons and the second number of photons, a number of photons associated with the bound florescent molecules. The control circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range.
The sample contains a drug molecule (or a quantity of drug molecules) of interest and a quantity of fluorescent molecules. The choice of fluorescent molecule is not particularly limited and any fluorescent molecule that can bind to the drug of interest can be chosen. Fluorescent molecules can be chosen that bind either directly to the drug molecule or indirectly, via an intermediate molecule that binds to the drug of interest. Fluorescent molecules are any molecules that adsorb light (or other electromagnetic radiation) and that subsequently emit a light as fluorescence at a different (e.g. longer) wavelength. The incident light is linearly polarised light which is polarised at a polarisation angle. The choice of polarisation angle is not important and is defined as a reference angle. If the fluorescent molecules were stationary then any absorbed light would be emitted as fluorescence with a same polarisation as the incident light (in this case, the fluorescence would be polarised at the polarisation angle). However, fluorescent molecules are constantly in motion and, as a direct result, the polarisation angle of the fluorescence changes from being polarised at the polarisation angle at which the light was absorbed to being in a state of unknown polarisation. The rate of motion of the fluorescent molecules and, hence, the timescale over which the polarisation of the emitted fluorescence deviates from the incident polarisation is based on the size of the fluorescent molecules. Typically, a larger fluorescent molecule, or a fluorescent molecule that is bound to another molecule, will have a slower rate of motion and the polarisation of the emitted fluorescence will deviate from the incident polarisation over a longer timescale resulting in fluorescence that is more likely to be polarised in a direction that is aligned with the fluorescence. Hence, bound fluorescent molecules emit fluorescence that deviates from the incident polarisation over a different timescale to unbound drug molecules. Specifically, bound fluorescent molecules cause light to be depolarised on a bound-depolarisation timescale (the timescale over which the absorbed light deviates from the incident polarisation for fluorescent molecules bound to the drug of interest) that is different from an unbound-depolarisation timescale (the timescale over which the absorbed light deviates from the incident polarisation for fluorescent molecules that are not bound to the drug of interest). By performing measurements of the emitted fluorescence from the bound and unbound fluorescent molecules a quantity of bound fluorescent molecules and, hence, a quantity of the drug of interest can be determined.
Typically, measurements to quantify the bound and unbound fluorescent molecules are made by using sensors that produce an analogue approximation of an integrated number of photons that arrive at a sensor. The inventors have realised that highly accurate measurements can be made by using photon counting circuitry comprising photomultiplier tubes that are configured to operate in a photon counting mode. Operating a photomultiplier tube in a photon counting mode results in a digital output providing a quantification of a discrete (integer) number of photons. However, incorporation of a photomultiplier tube into the photon counting circuitry will not always result in a meaningful measurement from a photomultiplier tube. Typically, a number of photons that are emitted from the fluorescent molecules will be large and will fall outside of a dynamic range of the photon counting circuitry. The dynamic range is the range of photons that can be counted by the photon counting circuitry. If a large number of photons are received by the photon counting circuitry outside of the dynamic range, then the photon counting circuitry will saturate and will output a maximum value that is limited by the dynamic range of the photon counting circuitry. In order to overcome this problem the apparatus is provided with a tuneable light source and control circuitry to regulate an intensity of the light that is emitted by the tuneable light source. The control circuitry regulates the intensity of the light source to ensure that the number of photons fluoresced by the bound fluorescent molecules and the unbound fluorescent molecules is within (i.e., does not exceed) the dynamic range of the photon counting circuitry. In this way, the photon counting circuitry is able to produce an accurate count of a number of photons emitted by both the bound fluorescent molecules and the unbound fluorescent molecules.
In order to quantify the number of bound and unbound fluorescent molecules the photon counting circuitry is configured to perform two measurements. In the first measurement the photon counting circuitry counts a first number of photons that have passed through a first polarisation filter. The first polarisation filter is configured to allow transmission of the photons at the polarisation angle, i.e., the angle at which the incident light is polarised. This does not require that the first polarisation filter is aligned parallel to the polarisation angle. All that is required is that the first polarisation filter is not orthogonal to the polarisation angle. Hence, the first count includes photons aligned at the polarisation angle which will be a combination of photons that have been emitted prior to depolarisation (typically these will be emitted from the bound fluorescent molecules which move slower than the unbound fluorescent molecules and hence, cause absorbed light to be depolarised at a slower rate), and a subset of photons that have depolarised and are in an unknown polarisation state (typically these will be emitted from the un-bound fluorescent molecules which move faster than the bound fluorescent molecules and, hence, are more likely to emit light in an unknown polarisation state). The photon counting circuitry also performs a second measurement to count photons that are emitted by the fluorescent molecules and that pass through a different (e.g. second polarisation filter). The second polarisation filter is configured to allow transmission of photons that are polarised at a depolarisation angle. The depolarisation angle is different from the polarisation angle of the incident light. Hence, the second polarisation filter will allow transmission of photons that are orthogonal to the polarisation angle. Hence, photons counted in the second measurement will include photons orthogonal to the polarisation angle, i.e., that have depolarised from the incident polarisation angle and are in an unknown polarisation state (typically these will be emitted from the un-bound fluorescent molecules which move faster than the bound fluorescent molecules and, hence, are more likely to emit light in an unknown polarisation state). By comparing these measurements the number of photons that are associated with the bound fluorescent molecules can be determined.
The apparatus can be provided with means to control the light source and in some configurations the apparatus further comprises the tuneable light source and one Or more neutral density filters aligned between the tuneable light source and the sample. Neutral density filters modify the intensity of light that passes through them to reduce all wavelengths (within an operating range of the neutral density filter) by a same amount. Each neutral density filter is aligned between the light source and the sample such that the light emitted from the tuneable light source passes through the one or more neutral density filters to reduce the amount of light that reaches the sample.
In some configurations the one or more neutral density filters comprises at least one of a single continuous neutral density filter; and one or more discrete neutral density filters. The continuous neutral density filter is a neutral density filter that is variable across the range of wavelengths such that an amount by which the continuous neutral density filter reduces the light can be adjusted. Each one or more discrete neutral density filter reduces the amount of light that is transmitted by a fixed amount. Each of the one or more discrete neutral density filters can be configured to reduce the amount of light transmitted by a same amount or by different amounts. By using different combinations of the one or more neutral density filters a range of different reductions in the amount of light that reaches the sample can be provided.
In some configurations the control circuitry regulates the tuneable light source using a linear regulated current control method. The linear regulated current control method provides a stable output current to control the intensity of light output from the tuneable light source. In particular, by controlling a value of the stable output current, the intensity of light that is output can be varied. When used in combination with the one or more neutral density filters, this method provides an efficient and cost effective solution for controlling the tuneable light source such that the first number of photons and the second number of photons, emitted by the bound and unbound fluorescent molecules, fall within the dynamic range of the photon counting circuitry.
The intensity of the tuneable light source is set such that the counted photons fall within the dynamic range of the photon counting circuitry. In some configurations the control circuitry is configured to regulate the tuneable light source such that the number of counts Falls within a linear response range of the dynamic range. The linear response range is a range in which a count rate of the photon counting circuitry responds linearly to the input number of photons. In some configurations the control circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons falls within 1 to 6 million counts per second. This range coincides with a typical range over which the counts fall within the linear range of photon counting circuitry and provides for a range over which high accuracy can be maintained.
The polarisation angle can be any angle that is different from the depolarisation angle. The amount of light of a given polarisation angle that passes through a polarisation filter is proportional to coskS) where S is the angle between the given polarisation angle and the angle at which the polarisation filter is oriented. Hence, if the incident light is decomposed into light of intensity that is polarised at the polarisation angle and light that of intensity Jo that is orthogonal to the polarisation angle then light passing through a polarisation filter aligned at an angle S to the polarisation is given by: Ii = Ipcos2(3) + 1ocos2(3+7c/2), (1) where 3 is measured in radians, and light passing through a polarisation filter allowing transmission of light at an angle different to the polarisation angle, i.e., at a polarisation filter aligned at an angle 5+5 to the polarisation angle, is given by 12 = Ircos2(3+6) + locos2(9+5+7c/2), (2) different to the polarisation angle. Equations (1) and (2) are a pair of linear equations for the unknowns 1p and Jo. Hence, by counting the photons II passing through the first filter and the photons T2 passing through the second filter, a measurement of the number of photons orthogonal and parallel to the polarisation angle can be obtained by solving the linear equations. In some configurations the polarisation angle is substantially orthogonal to the depolarisation angle. This configuration results in the smallest overlap between photons measured in the first count and photons measured in the second count and provides for a particularly efficient apparatus.
In some configurations the first number of photons and the second number of photons are counted sequentially. The first and second number of photons are counted sequentially in that a first count is made of a first number of photons. Then, subsequent to the first count, a second count is made of the second number of photons. In this way only a single photomultiplier tube is required to perform the first count and the second count. In this way a particularly cost effective apparatus can be produced.
In particular, in some configurations the photomultiplier tube comprises a polariser configured to rotate; and the photon counting circuitry is configured to count the first number of photons with the polariser oriented at the polarisation angle to provide the first polarisation filter and to count the second number of photons with the polariser oriented at the depolarisation angle to provide the second polarisation filter.
These configurations correspond to the case of S = 0 and 6 = it/2 in equation (1) and equation (2). In which case Ii is a direct measure of Ip and 12 is a direct measure of Aligning the polarisation filter in this way reduces the amount of post processing required by the calculation circuitry.
k some configurations the photomultiplier tube is a first photomultiplier tube comprising the first polarisation filter oriented at the polarisation angle; the photon counting circuitry comprises a second photomultiplier tube configured to operate in the photon counting mode, the second photomultiplier tube comprising the second polarisation filter oriented at the depolarisation angle; and the photon counting circuitry is configured to count the first number of photons using the first photomultiplier tube and the second number of photons using the second photomultiplier tube. As in the case of the rotating polarisation filter these configurations correspond to the case of S = 0 and 6 = rr./2 in equation (1) and equation (2). In which case Ii is a direct measure of Ip and 12 is a direct measure of 10. In addition, by providing two photomultiplier tubes, there is no requirement for a rotating polariser, resulting in a more reliable system, The first and second photon counts can be carried out sequentially, i.e., one of the first photon count and the second photon count can be carried out before the other of the first photon count and the second photon count. In some configurations the photon counting circuitry is configured to count the first number of photons and the second number of photons in parallel. In particular, the first photon count is carried out at a same time or at substantially the same time (e.g. during an overlapping period) as the second photon count. This provides for a shorter total measurement time and increases the potential throughput of the apparatus.
In some configurations the photomultiplier tube comprises a detector and is switchable between the photon counting mode and an integration mode; and the photomultiplier tube is configured to: when operating in the photon counting mode, amplify a current output from the detector to enable detection of a single photon; and when operating in the integration mode, measure an integrated current output from the detector in response to a plurality of photons striking the detector. The photon counting mode counts discrete instances of photons striking the detector to produce a count that identifies a discrete (integer) number of photons that have hit the detector. In contrast, and when operating in integration mode, the photomultiplier tube outputs an analogue signal that is approximately proportional to a total number of photons incident on the detector. The quantity of photons incident on the detector can then be inferred based on the settings of the photomultiplier tube. However, in integration mode it is not possible to obtain a precise indication of a discrete (integer) number of photons incident on the detector. Due to the possibility of plural simultaneous photon strikes and the non-linear response of the detector, the charge on the detector associated with N photons is not necessarily equal to N times the charge associated with one photon and, hence, the photomultiplier tube provides only an indication of the number of photons from the charge.
In some configurations a number of fluorescent molecules included in the sample is larger than an expected number of molecules of the drug molecule. Whilst the actual number of molecules of the drug molecule is not known a-priori, it is typically possible to estimate how many molecules may be expected, for example, based on a mass of the molecules in the sample. By providing a greater quantity of fluorescent molecules than the quantity of drug molecules there is a greater chance that each of the drug molecules will end up bound to a fluorescent molecule. In this way, a more accurate quantification of the number of fluorescent molecules can be obtained In some configurations the sample is a single sample of the drug molecule. However, in some configurations the apparatus further comprises a plurality of receptacles each configured to hold a different one of a plurality of samples of the drug molecule, wherein the sample is one of the plurality of samples In this way an apparatus can be provided to support a number of samples.
In some configurations the photon counting circuitry is configured to perform the first photon count and the second photon count for each of the plurality of samples sequentially. In particular the photon counting circuitry is configured to perform the first photon count and the second photon count for one of the plurality of samples before performing the first photon count and the second photon count for a next sample of the plurality of samples. In some configurations the photon counting circuitry is provided with a single photomultiplier tube and performs each of the first photon count and the second photon count sequentially, i.e., one of the first photon count and the second photon count is performed for a sample and then the other of the first photon count and the second photon count is performed for the same sample before the next sample is considered. In alternative configurations the photon counting circuitry is provided with a first photo multiplier tube and a second photomultiplier tube and the photon counting circuitry is configured to perform the first photon count and the second photon count for a particular sample in parallel before moving on to perform the first photon count and the second photon count for a next sample. In such configurations, it is the samples that are considered sequentially and not the first and second photon counts themselves.
In some configurations, the photomultiplier tube is one of a plurality of photomultiplier tubes each associated with a corresponding receptacle of the plurality of receptacles, and the photon counting circuitry is configured to: perform a corresponding first photon count for each of the corresponding receptacles in parallel; and perform a corresponding second photon count for each of the corresponding receptacles in parallel. In some configurations the plurality of photomultiplier tubes are provided as a single photomultiplier tube per receptacle such that one of the first photon count and the second photon count is performed for each of the plurality of samples in parallel then separately, either before or after the one of the first photon count and the second photon count, the other of the first photon count and the second photon count is performed for each of the plurality of samples in parallel. In other configurations a first photomultiplier tube is provided for each receptacle and a second photomultiplier tube is provided for each receptacle. In this way the first photon counts and the second photon counts for each of the plurality of receptacles can all be performed in parallel This approach provides for a particularly rapid quantification of the drug molecule in each of the plurality of samples.
In some configurations, the plurality of receptacles comprise a control receptacle for containing a quantity of the unbound fluorescent molecules, wherein the photon counting circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range based on a control photon count of the control receptacle The regulation can be performed by first performing a first and second photon count for the control receptacle and by adjusting the light intensity of the tuneable light source such that the photons counted in the first photon count and the second photon count fall within the dynamic range of the photon counting circuitry. This illumination intensity can then be used for each measurement of each of the plurality of receptacles.
In some configurations the photon counting circuitry is configured to count the first number of photons and the second number of photons for a counting duration longer than the unbound-depolarisation timescale and longer than the bound-depolarisation timescale. By providing a counting duration longer than each of the depolarisation timescales a more accurate representation of the total amount of photons can be obtained.
The fluorescent molecules can be selected as any fluorescent molecules that are suitable for binding to the drug molecules. In some configurations the bound fluorescent molecules emit fluorescence over a fluorescence timescale and the unbound fluorescent molecules emit over the fluorescence timescale, and the fluorescence timescale is longer than the unbound-depolarisation timescale and shorter than the bound-depolarisation timescale. In other words, the fluorescent molecule is selected as a molecule that, when unbound, is likely to emit the majority of photons before the photons have the opportunity to depolarise. However, as the bound-depolarisation timescale is longer than the fluorescence timescale the majority of photons emitted by the bound-fluorescent molecules are likely to be emitted after the polarisation of the photons has changed from the polarisation angle. Hence, by choosing a fluorescent molecule for which the fluorescence timescale is shorter than the bound-depolarisation timescale and longer than the unbound-depolarisation timescale an improved measurement accuracy can be obtained.
Various methods can be used to compare the first photon count and the second photon count. In some configurations the comparison is based on a ratio of a weighted difference of the first number of photons and the second number of photons and a weighted sum of the first number of photons and the second number of photons For example the photons polarised at the polarisation angle can be given by the number of photons associated with the bound fluorescent molecules IB plus half the number of photons associated with the unbound fluorescent molecules 112/2 and the photons polarised at the depolarisation angle is given by half the number of photons associated with the unbound fluorescent molecules 11212 Hence, the first photon count and the second photon count can be rewritten as Ii = IBcos2(9) + Iu/2, (3) and 12 -TBCOS2(8+6) iu/2 (4) Therefore by combining a weighted combination of the first number of photons Ii and the second number of photons 12 it is possible to isolate values of 1LT and I. In some configurations the calculation circuitry is configured to perform the comparison to determine a proportion of photons associated with the bound fluorescent molecules R = (11-612)/ (11+612), (5) where G is a grating factor to account for variation in the polarisation filters. In particular, assuming that each polarisation filter is identical G=1, and that the angles are orthogonal (9-0, corresponding to the first polarisation filter being aligned to the polarisation angle, and 6= 7t/2, corresponding to the second polarisation filter being orthogonal to the first polarisation filter) in equations (3) and (4). Then R=Ia/(Ia+k). (6) Hence, by combining the first photon count and the second photon count in this way a proportion of the photons associated with the bound fluorescent molecule can be determined.
k some configurations there is provided a method for quantifying binding of fluorescent molecules to a drug molecule, the method comprising: controlling a light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule, the fluorescent molecules comprising bound fluorescent molecules bound to the drug molecule and unbound fluorescent molecules free from the drug molecule, wherein in response to adsorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarisers from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale; counting, using photon counting circuitry and a photomultiplier tube configured to operate in a photon counting mode: a first number or photons that are emitted From the fluorescent molecules and have passed through a first polarisation filter allowing transmission at the polarisation angle; and a second number of photons that are emitted from the fluorescent molecules and have passed through a second polarisation filter allowing transmission at a depolarisation angle different to the polarisation angle; and calculating, based on a comparison between the first number of photons and the second number of photons, a number of photons associated with the bound florescent molecules. In such configurations the light source is a fixed light source outputting light with a fixed intensity or with variable intensity. In some such configurations the light source is selected, a-priori, to be of an appropriate intensity such that accurate counts of the photons can be achieved by the photon counting circuitry. Advantageously, this method benefits from the improved accuracy of the photon counting circuitry without the requirement for the additional control step of tuning the light source Particular configurations will now be described with reference to the accompanying drawings Figure 1 schematically illustrates an apparatus 10 according to various configurations of the present techniques. The apparatus 10 is provided with control circuitry 16 to regulate intensity of a tuneable light source 22 and to cause the tuneable light source 22 to illuminate a sample 18. The sample contains a quantity of fluorescent molecules and a drug molecule (or a plurality of drug molecules). The fluorescent molecules comprise bound fluorescent molecules which are bound to the drug molecule and unbound fluorescent molecules which are free from the drug molecule. In response to absorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale. The light from the tuneable light source 22 is polarised, for example, by causing the light to be transmitted through a polarisation filter 24. The apparatus 10 is further provided with photon counting circuitry 14. The photon counting circuitry 14 is provided with a photomultiplier tube 20 that is configured to operate in a photon counting mode to count photons within a dynamic range of the photon counting circuitry 14. In particular, the photon counting circuitry 14 is configured to perform a count of a first number of photons that are emitted from the fluorescent molecules and have passed through a first polarisation filter and to count a second number of photons that have passed through a different polarisation filter (i.e., a second polarisation filter). Each of the photon counts corresponds to a measurement of light polarised at a different polarisation angle. The apparatus 10 is also provided with calculation circuitry 12 to calculate, based on a comparison between the first number of photons and the second number of photons, a number of photons that are associated with bound fluorescent molecules in the sample.
Figure 2 schematically illustrates the apparatus 10 according to various configurations of the present techniques. In addition to the components described in relation to figure 1, the apparatus 10 is further provided with one or more neutral density filters 26. The one or more neutral density filters can be provided as a combination of a continuous neutral density filter and/or one or more discrete neutral density filters. The one or more neutral density filters 26 work, in combination with the tuneable light source 22, to regulate an amount of light incident on the sample 18 such that the emitted photons, from the bound and unbound fluorescent molecules, are within a dynamic range of the photon counting circuitry 14. The apparatus 10 is further provided with a rotating polarisation filter 28 and, in such configurations, the photon counting circuitry 14 is arranged to perform a count of the first number of photons either before or after performing a count of the second number of photons. In this way, the photon counting circuitry performs the count of the first number of photons and the count of the second number of photons sequentially. Once the photon counting circuitry 14 has counted the first number of photons and the second number of photons, these counts are passed to the calculation circuitry which compares the measurements to determine a number of photons associated with the bound fluorescent measurement. This number can be output to an external device for storage, display, or further analysis.
Figure 3 schematically illustrates the apparatus 10 according to various configurations of the present techniques. In addition to the components described in relation to figure 1 and figure 2, the photon counting circuitry 14 is provided with a first photomultiplier tube 34 (PMT) and a second photomultiplier tube 30. Furthermore, a first polarisation filter 36 and a second polarisation filter 32 are provided. The first polarisation filter 36 is positioned between the sample 18 and the first photomultiplier tube 34 such that emitted bound fluorescence and the emitted unbound fluorescence from the sample 18 is transmitted through the first polarisation filter 36 and is incident on the first photomultiplier tube 34 which performs a count of the first number of photons. The second polarisation filter 32 is positioned between the sample 18 and the second photomultiplier tube 30 such that emitted bound fluorescence and the emitted unbound fluorescence from the sample 18 is transmitted through the second polarisation filter 32 and is incident on the second photomultiplier tube 30 which performs a count of the second number of photons. The first polarisation filter 36 is aligned to allow transmission of light at the polarisation angle, i.e., the angle at which light passing through the polarisation filter 24 is polarised. The second polarisation filter 30 is aligned to allow transmission of light at a depolarisation angle. The depolarisation angle is different to the polarisation angle. Whilst the first polarisation filter 36 is illustrated as being aligned with the polarisation filter 24 it would be appreciated by the person skilled in the art that the first polarisation filter could be aligned at any angle that allows transmission of a portion of the light polarised at the polarisation angle of the polarisation filter 24. Furthermore, whilst the second polarisation filter 32 is illustrated as being orthogonal to the polarisation angle, it would be appreciated by the skilled person that the second polarisation filter 32 could be provided at any angle that is different from the first polarisation filter 36. Although, by providing the first polarisation filter 36 aligned at a same angle as the polarisation filter 24, and the second polarisation filter 32 aligned orthogonal to the polarisation filter 24 a particularly efficient apparatus can be provided. An indication of the count of the first number of photons and the second number of photons are provided to the calculation circuitry 24 which compares the measurements to determine a number of photons associated with the bound fluorescent measurement. This number can be output to an external device for storage, display, or further analysis.
Figure 4 schematically illustrates a configuration 40 in accordance with the present techniques. The configuration is provided with an apparatus 42 which is provided with control circuitry, photon counting circuitry, and calculation circuitry as described in relation to any of figures 1 to 3. In addition, the configuration 40 is provided with a plurality of receptacles 54, each receptacle 52 of the plurality of receptacles holds a corresponding sample of a plurality of samples. The sample comprises a drug molecule and a quantity of fluorescent molecules as described in relation to figure 1. Each of the samples within the plurality of receptacles 54 is illuminated by a tuneable light source 46 under control of the apparatus 42. Light emitted from the tuneable light source 46 passes through one or more neutral density filters 48 which can take the form of any of the neutral density filters described with reference to figure 2 and is polarised by transmission through a polarisation filter 50. The polarised light is absorbed by samples contained in each of the plurality or receptacles which, in turn, emit fluorescence comprising fluorescence from both bound and unbound fluorescent molecules. The apparatus 42 is provided with a plurality of photomultiplier tubes 44 to allow the apparatus 42 to perform counts of each of the samples in a number of the plurality of receptacles in parallel. In the illustrated embodiment the apparatus is provided with four sets of photomultiplier tubes 44(A), 44(B), 44(C). Each of the sets of photomultiplier tubes 44(A), 44(B), 44(C) can be a single photomultiplier tube or a pair of photomultiplier tubes.
The sets of photomultiplier tubes 44(A), 44(B), 44(C) can be provided as either single photomultiplier tubes 44 or pairs of photomultiplier tubes. Each of the photomultiplier tubes are provided as a single photomultiplier tube. The photomultiplier tube 44(A) is configured to count the first number of photons from the sample in receptacle 52(A) in parallel to the photomultiplier tube 44(B) counting the first number of photons from the sample in receptacle 52(B) and the photomultiplier tube 44(C) counting the first number of photons in the sample in receptacle 52(C). Subsequently, the photomultiplier tube 44(A) is configured to count the second number of photons from the sample in receptacle 52(A) in parallel to the photomultiplier tube 44(B) counting the second number of photons from the sample in receptacle 52(B) and the photomultiplier tube 44(C) counting the second number of photons in the sample in receptacle 52(C). The first counts can be performed (in parallel) either before or after the second counts are performed (in parallel).
In alternative configurations the photomultiplier tubes 44(A), 44(B), 44(C) are each provided as two photomultiplier tubes, i.e., a first photomultiplier tube and a second photomultiplier tube, the pair of photomultiplier tubes 44(A) are configured to count the first number of photons from the sample in receptacle 52(A) and to count the second number of photons from the sample 52(A) in parallel with each other and in parallel to the photomultiplier tube 44(B) counting the first number of photons from the sample in receptacle 52(B) and the second number of photons from the sample in receptacle 52(B), and the photomultiplier tube 44(C) counting the first number of photons in the sample in receptacle 52(C) and the second number of photons in the sample in receptacle 52(C). In the such alternative configurations, a total of six measurements are made in parallel, two for each receptacle 52.
In the illustrated configuration the apparatus 42 is configured to treat receptacle 52(A) as a control receptacle holding a control sample. The apparatus 42 regulates the intensity of the tuneable light source 46 such that the first number of photons and the second number of photons are within the dynamic range of the photon counting circuitry based on a control photon count of the control receptacle 52(A). The control photon count may be performed before, or in parallel to the photon counts associated with the receptacle 52(B) and the receptacle 52(C). Whilst the illustrated embodiment shows 3 sets of photomultiplier tubes 44 it would be readily apparent to the skilled person that any number of sets of photomultiplier tubes 44 could be provided within the apparatus 42.
Figure 5 schematically illustrates a configuration 60 in accordance the present techniques. The configuration 60 is similar to the configuration 40 described in relation to figure 4 and, for conciseness, similar components with same reference numerals will not be described again The configuration 60 differs from the configuration 40 in that the apparatus 56, which is provided with control circuitry, photon counting circuitry, and calculation circuitry as described in relation to any of figures Ito 3 comprises a single photomultiplier tube 58. The single photomultiplier tube 58 is used to perform a count of a first number of photons associated with a first receptacle 52(A) and to count a count of a second number of photons associated with the first receptacle 52(A) sequentially. In particular the photomultiplier tube 58 is used to count the first number of photons either before or after the photomultiplier tube 58 is used to count the second number of photons. Once the photomultiplier tube 58 has been used to count the first number of photons and the second number of photons associated with the first receptacle 52(A), the apparatus 56 moves on to count a first number of photons and a second number of photons associated with the receptacle 52(B) and, subsequently the apparatus 56 moves on to count a first number of photons and a second number of photons associated with the receptacle 52(C). In alternative configurations the photomultiplier tube 58 could be modified to include a first photomultiplier tube and a second photomultiplier tube. In which case, the apparatus is able to count the first number of photons and the second number of photons for each sample in parallel A control receptacle 52(A) is provided in the plurality of receptacles 54, the apparatus 56 counts the first number of photons and the second number of photons associated with the control receptacle and, based on the number of photons that are counted, regulates the intensity of the tuneable light source 46 such that the first number of photons and the second number of photons are within the dynamic range of the photon counting circuitry based on a control photon count of the control receptacle 52(A). For example, if the first number of photons and the second number of photons that are counted for the control receptacle fall outside of an upper limit of the dynamic range of the photon counting circuitry then the apparatus regulates the tuneable light source to reduce the amount of light that is incident on the control samples. The control photon count may be performed before, or after the photon counts associated with the receptacle 52(B) and the receptacle 52(C) and may be performed a number of times throughout the counting procedure Figures 6A and 6B schematically illustrate the difference between photon counting circuitry' operating in a photon counting mode and photon counting circuitry operating in an integration mode. In figure 6A the detector 62 is responsive to a single photon 64 striking the detector 62 of photon counting circuitry operating in a photon counting mode. The photon counting circuitry is configured such that there is large gain associated with the detector such that the single photon hitting the detector causes a large output from the detector 62. Hence, for each photon 64 striking the detector a discrete change in the total charge that has been output from the detector 62 over time can be seen. In particular, photon strikes can be observed at times to 64, ti 66 and b 68. In figure 6B the photon counting circuitry is operating in integration mode. The gain associated with the detector 62 is much lower and photons striking the detector slowly build up a total integrated charge. The charge from the detector 62 can then be read to determine an indication of an approximation of a total number of photons that have struck the detector 62.
Figures 7A schematically illustrates the emission of fluorescence from an unbound fluorescent molecule 70 over a period of time. The fluorescent molecule has been illuminated with photons that are polarised at a polarisation angle. There are two timescales associated with the emission of fluorescence from the unbound fluorescent molecules: the fluorescence timescale 744 and the unbound-depolarisation timescale. As previously discussed, the fluorescence timescale is the timescale over which the fluorescent molecules emit photons. The unbound-depolarisation timescale is the timescale over which photons absorbed by the unbound fluorescent molecule depolarise due to the motion of the fluorescent molecule. The graph 74 shows, as a percentage of the total photons emitted as fluorescence at a given time after absorption over a range of times up to the fluorescence timescale 744, the percentage of photons polarised at the polarisation angle 740 and the percentage of photons polarised at the depolarisation angle 742 (which, in the illustrated embodiment is orthogonal to the polarisation angle). When the time after absorption is short, the photons have had less time to depolarise and a greater percentage of the photons are polarised at the polarisation angle 740. When the time after absorption is longer, the photons have had a longer time to depolarise and an equal percentage of the photons are emitted at the depolarisation angle and at the polarisation angle. Hence, the graph 74 shows an increase over time in the percentage of photons that are emitted at the depolarisation angle 742 and a drop off in the percentage of photons that are emitted at the polarisation angle 740. The unbound fluorescent molecule 70 moves rapidly and, as a result, the unbound depolarisation timescale is short relative to the fluorescence timescale 744. Hence, a measurement of a photon that has been emitted from an unbound fluorescence molecule, over the fluorescence timescale, is approximately equally likely to be a photon polarised at the polarisation angle and a photon polarised at the depolarisation angle.
Figure 7B schematically illustrates the same information for a bound fluorescent molecule 78 that is bound to a drug molecule 72. In this situation, the timescales associated with the emission of photons as fluorescence are the fluorescence timescale 764, which is the same as in the case of the unbound fluorescent molecule, and the bound fluorescent timescale. The drug molecule 72, to which the fluorescent molecule is bound, acts to anchor the fluorescent molecule 78 such that its motion is much slower. Hence the bound-fluorescence timescale is much longer than the unbound fluorescent timescale. As a result fluorescence emitted from the bound fluorescent molecule 78 depolarises from the polarisation angle much slower and, as a result, a greater percentage of the photons that are emitted from the fluorescent molecule are emitted polarised at the polarisation angle 760. As a result, and as can be seen in the graph 76, a percentage of photons emitted as fluorescence from the bound fluorescent molecule 78 that are polarised at the polarisation angle 760 is much higher than the percentage of photons emitted as fluorescence from the unbound fluorescent molecule at the depolarisation angle 762. Hence, fluorescence that is emitted from bound fluorescent molecules can be distinguished from fluorescence that is emitted from unbound fluorescent molecules.
Figures 8A to 8D schematically illustrate the counts of photons emitted from bound and unbound fluorescent molecules including a first count of photons that have passed through a first polariser at the polarisation angle (i.e., the angle of the polarised light used to illuminate the fluorescent molecules) and a second count of photons that have passed through a second polariser at the depolarisation angle, which in the illustrated embodiment is orthogonal to the polarisation angle. Figure 8A illustrates counts of photons emitted from unbound fluorescent molecules that are polarised at the polarisation angle. As the unbound-depolarisation timescale (the timescale over which photons absorbed by the unbound fluorescent molecule depolarise due to the motion of the unbound fluorescent molecule) associated with the unbound fluorescent molecules is short (typically of the order of a nano-second), relative to the fluorescence timescale (the timescale over which the fluorescent molecules emit photons), the unbound fluorescent molecule is equally likely to emit photons at the polarisation angle and at the depolarisation angle. Hence, a number of counts associated with the unbound fluorescent molecules at the polarisation angle is seen to increase linearly and reach a count cl over a time Tl. Similarly, and as illustrated in figure 8B a number of counts associated with the unbound fluorescent molecule at the depolarisation angle is seen to increase linearly and reach a count cl over a time Tl.
Figures 8C and 8D schematically illustrate the counts of photons emitted from bound fluorescent molecules over time. As the depolarisation timescale (the timescale over which photons absorbed by the bound fluorescent molecule depolarise due to the motion of the bound fluorescent molecule) associated with the bound fluorescent molecules is long (typically of the order of tens or hundreds of nano-seconds), relative to the fluorescence timescale (the timescale over which the fluorescent molecules emit photons), the bound fluorescent molecule is more likely to emit photons near the polarisation angle than near the depolarisation angle. Hence, a number of counts associated with the bound fluorescent molecules at the polarisation angle is seen to increase linearly and reach a count c2 over a time T 1. Similarly, as the bound fluorescent molecule is unlikely to emit photons near the depolarisation angle then, as illustrated in figure 8D, the fluorescence counts from the unbound molecules will be negligible (zero). The relative sizes of cl and c2 are therefore dependent on the number of unbound fluorescent molecules and the number of bound fluorescent molecules. In a sample with no bound fluorescent molecules, c2 will be zero. In a sample where all the fluorescent molecules are bound, cl will be zero.
In alternative configurations the counts of photons emitted from bound and unbound fluorescent molecules include a first count of photons that have passed through a rotating polariser that is aligned at the polarisation angle (i e., the angle of the polarised light used to illuminate the fluorescent molecules) and a second count of photons that have passed through the rotating polariser aligned at the depolarisation angle. It would be readily apparent to the skilled person that this alternative configuration would result in the same counts of photons as illustrated schematically in figures 8A to 8D.
Figure 9 schematically illustrates the total first photon count and the total second photon count resulting from the counts schematically illustrated with reference to figures 8A to 8D. The total first photon count is the sum of the photons emitted as fluorescence from the bound fluorescent molecule at the polarisation angle (which in this case is equal to cl and is illustrated in figure 8A) and the photons emitted as fluorescence from the unbound fluorescent molecule at the polarisation angle (which in this case is equal to c2 and is illustrated in figure 8C). As can be seen the total first photon count increases linearly to a count of cl+c2 over the time Ti. Similarly, the total second photon count, which is a measure of photons emitted as fluorescence from the bound fluorescent molecules at the depolarisation angle (which in this case is equal to cl and is illustrated in figure 8B) and the photons emitted as fluorescence from the unbound fluorescent molecules at the depolarisation angle (which in this case is zero and is illustrated in figure 8D). As can be seen, the second photon count increases linearly to a count of c I over a time TI. Hence, the number of photons associated with the bound fluorescent molecules can be determined from the difference in the counts. If El = cl+c2 is the first photon count and F2 = cl is the second photon count then the difference H -F2 = c2 is the count of the number of photons that are emitted from the bound fluorescent molecules. Alternatively, by dividing the difference of the total first photon count and the total second photon count by a sum of the total first photon count and the total second photon count a fraction of the total photon count associated with the bound fluorescent molecules can be obtained. In this case (F l-F2)/(F I +F2) = f2/(2*cl+c2). It is noted that 2*cl is the total number of photons detected from the unbound fluorescent molecules and c2 is the total number of photons detected from the bound fluorescent molecules. Hence, the equation (Fl-F2)/(F1+F2) can be used to determine the fraction of bound fluorescent molecules providing a quantification of the drug molecule.
Figure 10 schematically illustrates a sequence of steps carried out in various configurations of the present techniques. At step 5100 the tuneable light source is controlled to illuminate a sample containing the fluorescent molecules and the drug with light that is polarised at a polarisation angle, where the intensity of the tuneable light source is regulated such that the number of counted photons falls within a dynamic range of the photomultiplier tube. Flow then proceeds to step S102 where a first number of photons that are emitted from the fluorescent molecules and that have passed through a first polarisation filter allowing transmission at a polarisation angle are counted Flow then proceeds to step S104 where a second number of photons that are emitted from the fluorescent molecules and that have passed through a first polarisation filter allowing transmission at a depolarisation angle, different to the polarisation angle, are counted. Flow then proceeds to step S106 where, based on a comparison between the first number of photons and the second number of photons, a number of photons associated with the bound fluorescent molecules are counted.
In brief overall summary there is provided an apparatus and method for calculating a number of photons associated with bound fluorescent molecules. The apparatus comprises control circuitry to cause a tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule, the fluorescent molecules comprising bound fluorescent molecules and unbound fluorescent molecules. The apparatus further comprises photon counting circuitry to count, within a dynamic range of the photon counting circuitry: a first number of photons emitted from the fluorescent molecules at the polarisation angle; and a second number of photons emitted from the fluorescent molecules at a depolarisation angle different to the polarisation angle. The apparatus is also provided with calculation circuitry to calculate, based on a comparison between the first number of photons and the second number of photons, a number of photons associated with the bound florescent molecules.
In the present application, the words -configured to..." are used to mean that an element of an apparatus has a configuration able to carry out the defined operation.
In this context, a "configuration" means an arrangement or manner of interconnection of hardware or software. For example, the apparatus may have dedicated hardware which provides the defined operation, or a processor or other processing device may be programmed to perform the function. "Configured to-does not imply that the apparatus element needs to be changed in any way in order to provide the defined operation.
Although illustrative embodiments have been described in detail herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes, additions and modifications can be effected therein by one skilled in the art without departing from the scope and spirit of the invention as defined by the appended claims. For example, various combinations of the features of the dependent claims could be made with the features of the independent claims without departing from the scope of the present invention.

Claims (20)

  1. CLAIMS1. An apparatus for quantifying binding of fluorescent molecules to a drug molecule, the apparatus comprising: control circuitry to regulate an intensity of a tuneable light source and to cause the tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule, the fluorescent molecules comprising bound fluorescent molecules bound to the drug molecule and unbound fluorescent molecules free from the drug molecule, wherein in response to absorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale; photon counting circuitry comprising a photomultiplier tube, the photon counting circuitry and the photomultiplier tube configured to operate in a photon counting mode to count photons within a dynamic range of the photon counting circuitry, wherein the photon counting circuitry is configured to count: a first number of photons that are emitted from the fluorescent molecules and have passed through a first polarisation filter allowing transmission at the polarisation angle; and a second number of photons that are emitted from the fluorescent molecules and have passed through a second polarisation filter allowing transmission at a depolarisation angle different to the polarisation angle; and calculation circuitry configured to calculate, based on a comparison between 25 the first number of photons and the second number of photons, a number of photons associated with the bound florescent molecules, wherein the control circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range.
  2. 2. The apparatus of any preceding claim, further comprising the tuneable light source and one or more neutral density filters aligned between the tuneable light source and the sample.
  3. 3 The apparatus of claim 2, wherein the one or more neutral density filters comprises at least one of: a single continuous neutral density filter; and one or more discrete neutral density filters.
  4. 4. The apparatus of any preceding claim, wherein the control circuitry is configured to regulate the tuneable light source using a linear regulated current control method.
  5. 5. The apparatus of any preceding claim, wherein the control circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons falls within 1 to 6 million counts per second.
  6. 6. The apparatus of any preceding claim, wherein the polarisation angle is substantially orthogonal to the depolarisation angle.
  7. 7. The apparatus of any preceding claim, wherein the first number of photons and the second number of photons are counted sequentially.
  8. 8. The apparatus of any preceding claim, wherein: the photomultiplier tube comprises a polariser configured to rotate; and the photon counting circuitry is configured to count the first number of photons with the polariser oriented at the polarisation angle to provide the first polarisation filter and to count the second number of photons with the polariser oriented at the depolarisation angle to provide the second polarisation filter.
  9. 9 The apparatus of any of claims 1 to 6, wherein: the photomultiplier tube is a first photomultiplier tube comprising the first polarisation filter oriented at the polarisation angle; the photon counting circuitry comprises a second photomultiplier tube configured to operate in the photon counting mode, the second photomultiplier tube comprising the second polarisation filter oriented at the depolarisation angle; and the photon counting circuitry is configured to count the first number of photons using the first photomultiplier tube and the second number of photons using the second photomultiplier tube.
  10. 10. The apparatus of claim 9, wherein the photon counting circuitry is configured to count the first number of photons and the second number of photons in parallel.
  11. 11. The apparatus of any preceding claim, wherein: the photomultiplier tube comprises a detector and is switchable between the photon counting mode and an integration mode; and the photomultiplier tube is configured to: when operating in the photon counting mode, amplify a current output from the detector to enable detection of a single photon; and when operating in the integration mode, measure an integrated current output from the detector in response to a plurality of photons striking the detector.
  12. 12 The apparatus of any preceding claim, wherein a number of fluorescent molecules included in the sample is larger than an expected number of molecules of the drug molecule.
  13. 13. The apparatus of any preceding claim, further comprising a plurality of receptacles each configured to hold a different one of a plurality of samples of the drug molecule, wherein the sample is one of the plurality of samples.
  14. 14. The apparatus of claim 13, wherein the photon counting circuitry is configured to perform the first photon count and the second photon count for each of the plurality of samples sequentially.
  15. 15. The apparatus of claim 13, wherein the photomultiplier tube is one of a plurality of photomultiplier tubes each associated with a corresponding receptacle of the plurality of receptacles, and the photon counting circuitry is configured to: perform a corresponding first photon count for each of the corresponding receptacles in parallel; and perform a corresponding second photon count for each of the corresponding receptacles in parallel.
  16. 16. The apparatus of any of claims 13 to 15, wherein the plurality of receptacles comprise a control receptacle for containing a quantity of the unbound fluorescent molecules, wherein the photon counting circuitry is configured to regulate the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range based on a control photon count of the control receptacle.
  17. 17. The apparatus of any preceding claim, wherein the photon counting circuitry is configured to count the first number of photons and the second number of photons for a counting duration longer than the unbound-depolarisation timescale and longer than the bound-depolarisation timescale.
  18. 18. The apparatus of any preceding claim, wherein: the bound fluorescent molecules emit fluorescence over a fluorescence timescale and the unbound fluorescent molecules emit over the fluorescence timescale; and the fluorescence timescale is longer than the unbound-depolarisation timescale and shorter than the bound-depolarisation timescale.
  19. 19. The apparatus of any preceding claim, wherein the comparison is based on a ratio of a weighted difference of the first number of photons and the second number of photons and a weighted sum of the first number of photons and the second number of photons.
  20. 20. A method for quantifying binding of fluorescent molecules to a drug molecule, the method comprising: controlling a light source to regulate an intensity of a tuneable light source and to cause the tuneable light source to illuminate, with incident light polarised at a polarisation angle, a sample containing the fluorescent molecules and the drug molecule, the fluorescent molecules comprising bound fluorescent molecules bound to the drug molecule and unbound fluorescent molecules free from the drug molecule, wherein in response to adsorbing the incident light, the bound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over a bound-depolarisation timescale, and the unbound fluorescent molecules emit fluorescence that depolarises from the polarisation angle over an unbound-depolarisation timescale; counting, using photon counting circuitry and a photomultiplier tube configured to operate in a photon counting mode to count photons within a dynamic range of the photon counting circuitry: a first number of photons that are emitted from the fluorescent molecules and have passed through a first polarisation filter allowing transmission at the polarisation angle; and a second number of photons that are emitted from the fluorescent molecules and have passed through a second polarisation filter allowing transmission at a depolarisation angle different to the polarisation angle; and calculating, based on a comparison between the first number of photons and the second number of photons, a number of photons associated with the bound florescent molecules, wherein controlling the light source comprises regulating the intensity of the tuneable light source such that the first number of photons and the second number of photons are within the dynamic range.
GB2111103.4A 2021-08-02 2021-08-02 An apparatus and method for calculating a number of photons associated with bound fluorescent molecules Pending GB2609456A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB2111103.4A GB2609456A (en) 2021-08-02 2021-08-02 An apparatus and method for calculating a number of photons associated with bound fluorescent molecules

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB2111103.4A GB2609456A (en) 2021-08-02 2021-08-02 An apparatus and method for calculating a number of photons associated with bound fluorescent molecules

Publications (2)

Publication Number Publication Date
GB202111103D0 GB202111103D0 (en) 2021-09-15
GB2609456A true GB2609456A (en) 2023-02-08

Family

ID=77651341

Family Applications (1)

Application Number Title Priority Date Filing Date
GB2111103.4A Pending GB2609456A (en) 2021-08-02 2021-08-02 An apparatus and method for calculating a number of photons associated with bound fluorescent molecules

Country Status (1)

Country Link
GB (1) GB2609456A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020158212A1 (en) * 1998-04-17 2002-10-31 French Todd E. Apparatus and methods for time-resolved optical spectroscopy
US20040099813A1 (en) * 2000-12-21 2004-05-27 Christian Eggeling Method for characterizing samples of secondary light emitting particles
JP4475525B2 (en) * 2005-02-10 2010-06-09 国立大学法人京都工芸繊維大学 Biopolymer screening method by depolarization
US9347882B2 (en) * 2011-06-22 2016-05-24 Molecular Devices, Llc Dynamic signal extension in optical detection systems

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020158212A1 (en) * 1998-04-17 2002-10-31 French Todd E. Apparatus and methods for time-resolved optical spectroscopy
US20040099813A1 (en) * 2000-12-21 2004-05-27 Christian Eggeling Method for characterizing samples of secondary light emitting particles
JP4475525B2 (en) * 2005-02-10 2010-06-09 国立大学法人京都工芸繊維大学 Biopolymer screening method by depolarization
US9347882B2 (en) * 2011-06-22 2016-05-24 Molecular Devices, Llc Dynamic signal extension in optical detection systems

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. M. DUBACH ET AL: "In vivo imaging of specific drug–target binding at subcellular resolution", NATURE COMMUNICATIONS, vol. 5, 28 May 2014 (2014-05-28), XP055233186, DOI: 10.1038/ncomms4946 *

Also Published As

Publication number Publication date
GB202111103D0 (en) 2021-09-15

Similar Documents

Publication Publication Date Title
Lapp et al. A new luminescence detection and stimulation head for the Risø TL/OSL reader
CN103649726B (en) For fluorescence and the system and method for absorptance analysis
Goddard et al. Single particle high resolution spectral analysis flow cytometry
EP2381240A1 (en) Spectrometer, spectrometry, and spectrometry program
JP2012220472A (en) Spectroscopic measuring apparatus and program
CN101408503A (en) Method for automatically detecting and dynamically substracting stray light of spectrometer and spectrometer
JP6985730B2 (en) Sample analysis system, display method and sample analysis method
CN104422516B (en) Wavelength calibration method for monochromator, and spectrophotometer
WO2010054018A1 (en) Dynamic modification of shaping time in x-ray detectors
JPH06300628A (en) Method for grouping spectrum belt for data acquisition in spectrophotometer and spectral measuring device
GB2609456A (en) An apparatus and method for calculating a number of photons associated with bound fluorescent molecules
US6597439B1 (en) Method and apparatus for measurement of light from illuminated specimen eliminating influence of background light
CN1249424C (en) Atomic absorption spectrophotometer
CN107076609A (en) The response of fluorescent instrument is normalized using spectral response
US6970241B1 (en) Device for enabling slow and direct measurement of fluorescence polarization
Giauque et al. Trace element determination using synchrotron radiation
JPH06123717A (en) Fluorescent x-ray qualitative analytical method under plurality of conditions
CN102507518B (en) 12-lamp multi-channel atomic fluorescence spectrometer
CN1270174C (en) Method and appts. for atom absorbing analysis of constant magnetic field reverse zeeman effect
Becker et al. Flexible instrument for time‐correlated single‐photon counting
US10684169B2 (en) Optical analysis device using multi-light source structure and method therefor
JPH08178849A (en) Fluorometry and fluorometer
Kapri et al. Indigenous design and development of gated photon counter for low-rate photon regime
JP2522224B2 (en) X-ray fluorescence analysis method
da COSTA et al. Development and characterization of a portable total reflection X-ray fluorescence system using a waveguide for trace elements analysis