WO2021084137A1

WO2021084137A1 - Optical system and method

Info

Publication number: WO2021084137A1
Application number: PCT/EP2020/080714
Authority: WO
Inventors: Sanathana KONUGOLU VENKATA SEKAR; Stefan Andersson-Engels; Jean MATIAS; Katarzyna KOMOLIBUS
Original assignee: University College Cork, National University Of Ireland, Cork
Priority date: 2019-11-01
Filing date: 2020-11-02
Publication date: 2021-05-06
Also published as: GB201915957D0

Abstract

An apparatus and method for performing optical characterisation of luminescent particles within a sample is provided. The apparatus comprises a light source (10) configured to produce a first pump beam having a first centre wavelength and a substantially speckle-free beam profile for illuminating, along a first illumination optical (path (101), a sample to thereby produce emitted light by the interaction of the first pump beam with the sample; a first detector (70E) for detecting pump-induced emitted light from a detection region (65) within the sample along a first detection optical path (102); and a first optical filter element in the first detection optical path (102) for attenuating light at the first centre wavelength. The method comprises illuminating the sample with the pump beam having the substantially speckle-free beam profile at a detection region (65) within the sample; and detecting light emitted from the detection region of the sample produced by the interaction of the pump beam with the sample.

Description

OPTICAL SYSTEM AND METHOD

Technical Field

This invention relates generally to a method and apparatus for performing optical characterisation of particles such as up-converting nanoparticles.

Background to the Invention

Up-converting materials emit higher energy light from lower energy excitation as a consequence of sequential absorption of two or more photons. In particular, lanthanide-based up- converting nanoparticles (UCNPs) emit at several bands including visible light upon absorption of lower energy near-infrared (NIR) light. This absorption-emission process precludes background fluorescence in the detection band, while the NIR excitation reduces light scattering and attenuation and has an increased penetration depth in biological materials. This has led to significant interest in these materials for a range of biomedical applications such as bio-imaging, bio-sensing, and drug delivery where one can excite with NIR light and detect the luminescence at the up-converted band(s). A key challenge, particularly in deep tissue applications where low excitation power densities are required, is the relatively low efficiency of the up-converting process which means you don’t get much light out (luminescence) for the light you put in. This efficiency is characterised by the quantum yield (QY), f, which is defined as the ratio of the number of photons emitted to the number of photons absorbed, which is typically in the range of a few percent for UCNPs.

Increasing the QY of up-converting materials such as UCNPs is the subject of intense ongoing research. Also, the QY of the up-converting material must be accurately known or characterised in order to characterise other properties of the sample based on the measured luminescence, such as quantifying the concentration of particles. Accurate objective and standardised methods of measuring the QY are therefore essential. In addition, as up-conversion is a non-linear optical process, the non- linear power dependence of the up-conversion process and thus luminescence and QY should be taken into account in any quantitative analysis. For example, it is not sufficient measure the QY at a single excitation power, rather, accurate characterisation over a range of power densities is required.

Experimentally, measurements of QY typically involve laser excitation of the sample (typically a cuvette filled with a liquid containing the UCNPs) at the absorption band of the UCNPs, measuring the luminescence intensity at detector such as a spectrometer or avalanche photodiode (APD) and estimating the number of absorbed photons from the excitation power and absorbance of the UCNPs at the laser wavelength (which may be known or determined through absorption measurements). In practice, the measurement of QY is a complex procedure because there are various experimental factors that can effect or distort the measured QY value and which should be taken into account or compensated for to yield accurate QY measurements. These factors include, but are not limited to: transmission loss through the limited band width of optical filters used that introduce error to the luminescence signal; and contributions from particle scattering in absorption measurements that effect the determination of absorbance, and the inner filter effect of the sample that distort the estimated number of absorbed photons. In addition, recent work of M. Mousavi et al. in Phys. Chem. Chem. Phys. 19, 22016 (2017) has demonstrated the importance of considering the intensity distribution of laser beam in QY measurements of UCNPs and analysis, since a non-uniform beam profile means that UCNPs at different positions within the laser beam will experience different excitation power densities and thus yield different QY. Traditionally, integrating-sphere instruments have been used to measure absolute QY, as light emitted in all directions can be collected. An alternative approach is to use a fluorometer-based set-up in which a detector collects light emitted from a limited solid angle to measure a relative QY. This requires measurement of a reference sample (e.g. a dye) with a known QY in the same measurement set-up/geometry to calibrate the measured QY, assuming that the spatial distribution of the emission from the two samples is identical. The advantage with the fluorometer- based set-up is that the excitation beam profile can be better controlled and measured.

There is currently no complete system that can adequately characterise the QY or other optical properties of fluorophores, particularly those which exhibit a non-linear power density dependence such as UCNPs. There is a demand for a compact optical characterisation system that takes into account the non-linear optical properties of luminescent materials such as UCNPs and is capable of measuring luminescence and/or QY over a wide dynamic range of excitation power densities with a broad spectral range to cover the multiple emission bands of interest for different samples. There is also a need for standardised method or protocol for measuring QY to allow for direct comparisons between values for various samples developed by different research groups.

Aspects and embodiments of the present invention have been devised with the foregoing in mind.

Summary of the Invention

According to a first aspect of the invention, there is provided a method of optical characterisation of a sample. The method may be a method of characterising or measuring the quantum yield of a sample, or another property or optical property of a sample. The sample may be a liquid sample or a solid sample. The sample may be or comprise a liquid or solid scattering medium. The sample may comprise particles, such as luminescent particles, non-linear luminescent particles/material, fluorophores or upconverting nanoparticles. Where the sample is liquid, the particles may be dispersed in a liquid scattering medium such as a solvent. Where the sample is solid, the particles may be dispersed within a solid sample or scattering medium. The sample may be a biological sample and/or comprise biological media, e.g. biological tissue. The sample may be held in a sample cell or the sample may comprise the sample cell holding the sample. The method may comprise illuminating a sample with a pump beam having a first beam width, a first centre wavelength and a first excitation power density at a detection region within the sample. The pump beam may have a substantially speckle-free beam profile and/or a substantially smooth continuous intensity distribution at a detection region within the sample. The method may further comprise detecting light emitted from the detection region of the sample produced by the interaction of the pump beam with the first beam width with the sample. Detecting light emitted from the detection region of the sample produced by the interaction of the pump beam with the sample may comprise detecting/measuring the luminescence of the sample or particles within the sample.

The method may further comprise illuminating the sample with a pump beam having the (same) first centre wavelength, a second beam width and a second excitation power density at the detection region. The method may further comprise detecting light emitted from the detection region of the sample produced by the interaction of the pump beam with the second beam width with the sample. The method may comprise detecting light emitted from the detection region of the sample produced by the interaction of the pump beam with the respective first and second beam width with the sample.

The method may comprise deriving (e.g. at each beam width and excitation power density) a quantum yield of particles within the sample based on the detected pump-induced emitted light by performing quantum yield analysis. The method may comprise deriving (e.g. at each beam width and excitation power density) an optical property of particles within the sample based on the detected pump-induced emitted light (luminescence). For example, the method may be used for a number of applications, including but not limited to: measuring emission/absorption rates/lifetimes of particles in the sample, flow cytometry measurements of particles within the sample, monitoring a polymerase chain reaction (PCR) in the sample, and optogenetics.

Varying the beam width at the detection region gives access to a wider dynamic range of excitation power densities for the luminescence and/or quantum yield (QY) measurement. Typically, the excitation power of a pump beam can only be varied by a certain amount, which, for a single fixed beam width, provides a single range of excitation power densities. By varying the beam width, a number of ranges of excitation power densities, which may at least partially overlap, can be provided by independently varying the excitation power at the first and second beam widths. This allows the luminescence/QY of the particles to be characterised over a wider dynamic range of excitation power densities in a single system/measurement, which is significant as the luminescence/QY is a quantity that can exhibit a non-linear dependence on the excitation power density of the pump beam, particularly in up-converting materials such as up-converting nanoparticles, or any non-linear luminescent material.

The detected emitted light may be that which is emitted along a detection optical path that intersects the pump beam within the sample/sample cell at the detection region. The method may further comprise filtering out (scattered) light at the first centre wavelength or scattered light produced by the interaction of the pump beam with the sample along the detection optical path.

The respective pump beam may be produced by a light source. The light source may be or comprise a laser. The method may further comprise shaping the pump beam (having the respective first and/or second beam width) to have a substantially speckle-free beam profile and/or a substantially smooth continuous profile or intensity distribution at the detection region. Shaping the pump beam (having the respective first and/or second beam width) to have a substantially speckle-free beam profile may comprise passing the respective pump beam through a single mode optical fiber; and/or using a single mode laser as a light source producing the respective pump beam; and/or spatially filtering the respective pump beam. The method may comprise illuminating the sample with a pump beam (having the respective first and/or second beam width) having a substantially speckle-free beam profile and/or a substantially smooth continuous profile.

Speckle in the pump beam means that the power density varies in a substantially random non- uniform and uncontrolled manner across the beam profile or width at the detection region. This makes it difficult to accurately quantify or quantitatively analyse the luminescence or QY, particularly where the sample is or comprises a non-linear luminescent material such as up-converting nanoparticles. The QY is defined as the ratio of the number of emitted photons to the number of absorbed photons. The detected emitted light is proportional to the number of emitted photons. Accurate determination of the number of absorbed photons requires accurate knowledge of the excitation power density. Speckle in the pump beam leads to uncertainty in the true excitation power density at the detection region and thus inaccuracy in the QY measurement. It is then difficult to compensate for such beam profile induced distortions to the measured luminescence and QY. Speckle, by its random nature also makes it difficult to compare otherwise like-for-like measurements on samples performed using different apparatuses. By using a substantially speckle-free pump beam and/or shaping the beam to have a substantially speckle- free and/or smooth profile, the sample is excited in a more quantifiable way, fewer compensations are needed and thereby more accurate analysis of the luminescence and measurements of the QY can be made. Further, a speckle-free and substantially smooth beam profile means that any beam profile- induced distortion to the luminescence/QY value can be more easily and accurately compensated, leading to more accurate analysis of the luminescence and measurements of the QY.

The pump beam (having the respective first and/or second beam width at the detection region) may further have a uniform intensity distribution at the detection region. The method may further comprise shaping the pump beam (having the respective first and/or second beam width) to have a substantially uniform intensity distribution or beam profile at the detection region. This may comprise using one or more lenses in an illumination optical path between a light source producing the respective pump beam and the sample. Using a uniform profile pump beam means that all particles in the sample across the detection region are excited with the same intensity. This further provides for accurate quantitative analysis of the luminescence signal and measurements of the QY, particularly in materials which exhibit non-linear luminescence.

The pump beam having the respective first and second beam width may be produced by the same light source or a different light source. Where the pump beam with the respective first and second beam width is produced by the same light source, the step of illuminating the sample with a pump beam having the first centre wavelength, a second beam width and a second excitation power density at the detection region may comprise: adjusting the pump beam to have a second beam width and a second excitation power density at the detection region, and illuminating the sample with the (adjusted) pump beam at the second beam width and second excitation power density.

Adjusting the pump beam to have a second beam width may comprise adjusting and/or changing a lens arrangement in an illumination optical path between a light source producing the pump beam and the sample cell. The lens arrangement may comprise one or more lenses configured to provide a collimated beam of adjustable width. Adjusting and/or changing the lens arrangement may comprise (selectively) moving at least one of the one or more lenses into or out of the illumination optical path; and/or (selectively) directing the pump beam so as to pass through or bypass at least one of the one or more lenses.

The lens arrangement may comprise a focusing lens and a collimating lens. The focusing lens and the collimating lens may have a different focal length. The lens arrangement may comprise, in order, a first collimating lens, a focusing lens and a second collimating lens. The focusing lens and the second collimating lens may have a different focal length. Adjusting and/or changing the lens arrangement may comprise moving the focusing lens and the second collimating lens into or out of the illumination optical path; and/or directing the pump beam so as to pass through or bypass the focusing lens and the second collimating lens.

The method may further comprise: varying the excitation power density of the pump beam having the first beam width at the detection region, and illuminating the sample with the respective pump beam at a plurality of excitation power densities. The method may comprise: varying the excitation power density of the pump beam having the second beam width at the detection region, and illuminating the sample with the respective pump beam at a plurality of excitation power densities. The method may comprise detecting, at each excitation power density, emitted light from the detection region of the sample (e.g. along the first detection optical path).

Varying the excitation power density of the pump beam with the respective first and/or second beam width may comprise adjusting an optical power output of a light source producing the respective pump beam; and/or attenuating the respective pump beam. Attenuating the respective pump beam may comprise adding, removing, changing and/or adjusting one or more optical density filter elements to/from/in an illumination optical path between a light source producing the respective pump beam and the sample. The one or more optical density filter elements may attenuate the respective pump beam by a fixed or variable amount. The one or more optical density filter elements may be or comprise a neutral density filter.

Detecting pump-induced emitted light from the detection region of the sample may comprise measuring a luminescence of particles within the sample and/or measuring the intensity of the detected pump-induced emitted light. The luminescence may be proportional to the intensity of detected pump- induced emitted light.

Detecting pump-induced emitted light from the detection region of the sample may comprise detecting light emitted along a detection optical path. The detection optical path may be co-linear or non-co-linear with the illumination optical path or incident pump beam. The detection optical path may be in a reflection, transmission or side-emission (i.e. at an angle to the incident pump beam) geometry.

The method may further comprise measuring an emission spectrum of the detected pump- induced emitted light. The emission spectrum may be the emission spectrum of the particles within the sample. The detected pump-induced emitted light may be the emitted light detected along the first detection optical path, and/or emitted light along a second detection optical path that intersects the pump beam within the sample/sample cell at the detection region. The quantum yield analysis may comprise compensating the detected pump-induced emitted light, or the measured luminescence or intensity of the detected pump-induced emitted light, for a limited transmission bandwidth of one or more optical elements in a (e.g. the first) detection optical path based on the measured emission spectrum.

The pump beam may be substantially monochromatic. The method may further comprise illuminating the sample with a (separate) broadband light beam having a broad spectral content. The broadband light beam may be produced by a broadband light source. The method may comprise detecting the light transmitted through the sample to derive an absorption spectrum of the sample. The quantum yield may be derived based on the detected pump-induced emitted light and the derived absorption spectrum, optionally or preferably, the component of the derived absorption spectrum at the centre wavelength of the pump beam. Detecting light transmitted though the sample may comprise measuring a transmission spectrum of the sample. The method may further comprise illuminating an empty sample cell with the broadband light beam and detecting the light transmitted through the empty sample cell to measure a transmission spectrum of the empty sample cell. The method may comprise deriving an absorption spectrum of the sample based on the transmission spectrum of the sample and the empty sample cell. Where the sample comprises particles dispersed in a solvent, the method may comprise illuminating a reference solvent sample (e.g. held in a separate sample cell) with the broadband light beam and detecting the light transmitted through the reference solvent sample to measure a transmission spectrum of the reference solvent sample. The method may comprise deriving an absorption spectrum of the reference solvent sample based on the transmission spectrum of the reference solvent sample and the empty sample cell. The method may comprise deriving an absorption spectrum of the particles based on the absorption spectrum of the sample and empty sample cell and the reference solvent sample, e.g. subtracting the absorption spectrum of the reference solvent sample from the absorption spectrum of the sample.

The method may comprise illuminating the sample, the empty sample cell and/or the reference solvent sample with the broadband light beam and detecting the light transmitted therethrough substantially simultaneously. The method may comprise detecting the light transmitted through each of the sample, the empty sample cell and/or the reference solvent sample using a separate detector or the same detector. The method may comprise splitting the broadband light beam into multiple secondary broadband beams for illuminating the sample, reference solvent sample and/or the empty sample cell. Where the same detector is used for measuring the transmission spectrum of the sample and reference solvent sample, the method may comprise modulating at least one of the secondary broadband light beams at a frequency and deriving an absorption spectrum of the particle based on the modulated detector output.

Measuring samples and/or the empty sample cell and/or the reference solvent sample simultaneously speeds up the measurement sequence thereby reducing the chances of errors due to any changes in experimental conditions over time. By modulating one of the secondary broadband light beams and using the same detector, the detector output comprises transmission data for two or more samples which can be analysed to derive an absorption spectrum of particles in the sample faster quickly more efficiently than using separate detectors or swapping out samples in the same sample holder.

The quantum yield may be derived based on the detected pump-induced emitted light and the derived absorption spectrum, and optionally or preferably, the component of the derived absorption spectrum at the first centre wavelength of the pump beam. The quantum yield analysis may comprise compensating for or substantially removing scattering contributions in/from the derived absorption spectrum. Where the sample contains particles dispersed in a solvent, the quantum yield analysis may comprise compensating for or substantially removing contributions from the solvent in the derived absorption spectrum.

The method may further comprise imaging an intensity profile of the pump beam to determine the excitation power density and/or intensity distribution at the detection region. The quantum yield analysis may comprise compensating for a non-uniform intensity distribution at the detection region based on the measured or imaged beam profile and a rate equation describing the emission process in the sample, and/or a model describing the power density dependence of quantum yield. The model may be derived from a rate equation describing the emission process in the sample or particles within the sample.

The method may comprise illuminating the sample with a focused, collimated or diverging pump beam.

The method may further comprise replacing the sample (or sample cell holding the sample) with a reference sample (or reference sample cell holding a reference sample) having predetermined quantum yield characteristic and repeating any or all of the above method steps to derive, at each beam width and excitation power density, a quantum yield of particles within the reference sample. The method may comprise calibrating the derived quantum yield of particles within the sample using the derived quantum yield of particles in the reference sample and the predetermined quantum yield characteristics of the reference sample. The reference sample may be illuminated using a pump beam having a different centre wavelength.

According to a second aspect of the invention, there is provided a particle characterisation apparatus or system. The apparatus may be configured to perform the method of the first aspect. The apparatus may comprise a first light source configured to produce a first pump beam having a first centre wavelength for illuminating, along a first illumination optical path, a sample. The sample may be held in a sample cell. This may produce emitted light by the interaction of the first pump beam with the sample. The apparatus may further comprise a first detector for detecting pump-induced emitted light from a detection region of the sample. The first detector may detect light emitted along a first detection optical path that intersects the first illumination optical path at the detection region within the sample/sample cell. The apparatus may comprise a first optical (wavelength) filter element in the first detection optical path for attenuating light at the first centre wavelength.

The sample may be a substantially liquid sample or a substantially solid sample. The sample may be or comprise a liquid or solid scattering medium. The sample may comprise particles or luminescent particles, such as fluorophores or upconverting nanoparticles. The sample may comprise non-linear luminescent particles such as upconverting nanoparticles. Where the sample is liquid, the particles may be dispersed in a solvent. Where the sample is solid, the particles may be dispersed within the solid sample or scattering medium sample, e.g. the sample may be or comprise biological media such as tissue.

The first detection optical path may be arranged in a side emission (i.e. angled with respect to the incident pump beam), transmission or reflection geometry.

The apparatus may be used for a number of optical applications based on detection of light emitted/scattered from the sample, particular luminescence particles within the sample, including but not limited to: measuring quantum yield of particles in the sample, measuring emission/absorption rates/lifetimes of particles in the sample, flow cytometry measurements of particles within the sample, monitoring a polymerase chain reaction (PCR) in the sample, and optogenetics.

The apparatus may comprise a first lens arrangement in the first illumination optical path configured to adjust the first pump beam to have a first beam width or a second beam width at the detection region.

It will be appreciated the advantages set out with respect to the first aspect apply equally to the apparatus of the second aspect. The apparatus may be configured illuminate the sample with a focused, collimated or diverging pump beam. The apparatus may further comprise a first focusing lens in the first illumination optical path for focussing the first pump beam (to a point) within the sample/sample cell. The first light source may be configured to produce a first pump beam with a substantially speckle-free beam profile and/or a substantially smooth continuous intensity distribution at the detection region. The first light source may comprise a single mode fiber coupled to the output of a laser. Alternatively or additionally, the first light source may be or comprise a single mode laser.

The first detector may be configured to measure the luminescence intensity of the detected pump-induced emitted light. Additionally or alternatively, the first detector may be configured to measure the emission spectrum of the detected pump-induced emitted light. The first detector may be or comprise a spectrometer or a photodetector, such as photodiode or an avalanche photodiode.

The first lens arrangement may be or comprise one or more lenses configured to provide a collimated beam of adjustable width. The first lens arrangement may be or comprise one or more lenses configured to provide a collimated beam of adjustable width impinging on the first focussing lens. At least one of the one or more lenses may be moveable and/or removable to provide the collimated beam of adjustable width. Alternatively or additionally, the first lens arrangement may comprise a plurality of mirrors arranged to selectively direct the first pump beam so as to pass through or bypass at least one of the one or more lenses to provide the collimated beam of adjustable width.

The first lens arrangement may be or comprise a focusing lens and a collimating lens. The focusing lens and the collimating lens may have a different focal length. The lens arrangement may comprise, in order, a first collimating lens, a focusing lens and a second collimating lens. The focusing lens and the second collimating lens may have a different focal length. The focussing lens and the second collimating lens (or collimating lens) may be moveable into and out of the first illumination optical path to provide the collimated beam of adjustable width. Alternatively or additionally, the first lens arrangement may comprise a plurality of mirrors arranged to selectively direct the first pump beam so as to pass through or bypass the focussing lens and the second collimating lens to provide the collimated beam of adjustable width.

The first pump beam may be a single mode beam with a Gaussian beam profile. The apparatus may further comprise a first beam shaping arrangement in the illumination optical path configured to transform the first pump beam to have a substantially uniform spatial intensity distribution at the detection region. The first beam shaping arrangement may be or comprise one or more lenses, and optionally a diffraction element, to transform the beam profile using truncation, refraction and/or diffraction. The beam shaping arrangement may comprise an aperture for truncating the first pump beam. The beam shaping arrangement may comprise a pair of lenses arranged in a 4f configuration with respect to the aperture for compensating for diffraction of the beam resulting from the truncation at the aperture. The beam shaping arrangement may comprise one or more beam shaping lens configured to transform a substantially Gaussian input beam profile to a substantially uniform beam profile at the detection region.

The apparatus may further comprise a means for varying, at the first and/or second beam width, the excitation power or power density of the first pump beam at the detection region. The means for varying the excitation power density of the first/second pump beam at the detection region may comprise an adjustable power controller of the first light source; and/or one or more optical filter elements in the first illumination optical path configured to attenuate the first/second pump beam by a fixed or variable amount.

The first lens arrangement and/or the means for varying, at the first and/or second beam width, the excitation power density of the first pump beam at the detection region may be configured to provide a dynamic range of excitation power densities at the detection region of up to 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸.

The apparatus may further comprise a beam profiler in the first illumination optical path for measuring a two-dimensional spatial intensity distribution of the first pump beam at a location equivalent to the detection region. The beam profiler may be positioned between the first focusing lens and the sample/detection region. The beam profiler may be or comprise a beam-splitter that directs a percentage of the first pump beam towards a two-dimensional imaging detector.

The apparatus may further comprise a second light source for producing a second pump beam having a second centre wavelength for illuminating, along a second illumination optical path, a sample to thereby produce emitted light by the interaction of the second pump beam with the sample. The sample may be a reference sample (e.g. held in a reference sample cell). Alternatively, the first light source may be configured to produce the first pump beam and the second pump beam.

The second illumination optical path may join the first illumination optical path. The second illumination optical path may join the first illumination optical path before the first focussing lens (between the first light source and the first focusing lens) such that the first focussing lens focusses the second pump beam within the reference sample cell; or before the first lens arrangement (between the first light source and the first lens arrangement); or (where present) before the first single mode fiber (between the first light source and the first single mode fiber).

Alternatively, the second illumination optical path may join the first illumination optical path after the first focusing lens (in the direction of propagation of the pump beam), or between the first focusing lens and the sample. Where the beam profiler is present, the second illumination optical path may join the first illumination optical path between the first focusing lens and the beam profiler. In this case, the apparatus may further comprise a second focussing lens for focussing the second pump beam within the sample cell. The apparatus may further comprise a second lens arrangement in the second illumination optical path substantially identical to the first lens arrangement for adjusting the second pump beam to have a first beam width or a second beam width at the detection region. The second light source may be configured to produce a second pump beam with a substantially speckle-free beam profile. The second light source may comprise a single mode fiber coupled to the output of a laser. Alternatively or additionally, the second light source may be or comprise a single mode laser.

The first detector may be configurable to measure an emission spectrum of pump-induced emitted light from the sample. Alternatively or additionally, the apparatus may comprise a second detector for detecting the emission spectrum of pump-induced emitted light from the sample. The second detector may be configured to detect the spectrum of light emitted along a second detection optical path. The second detection optical path may intersect or join the first illumination optical path at the detection region. The first and/or second light source may be or comprise a laser. The apparatus may further comprise a broadband light source for producing a broadband light beam having a broad spectral content for illuminating, along a third illumination optical path, the sample for measuring an transmission spectrum of the sample. The third illumination optical path may join the first or second illumination optical path. The apparatus may be configurable such that the light transmitted through the sample is detectable by the first or second detector. Alternatively, the apparatus may comprise a third detector for detecting light transmitted through the sample.

The apparatus may comprise a means to modulate the first and/or second pump beam and/or broadband light beam. The means may comprise one or more optical choppers.

The apparatus may be configured to measure the transmission spectra of multiple different samples simultaneously. The apparatus may be configured to split the broadband light beam into multiple secondary beams directed to the multiple samples. Light transmitted through the multiple samples may be detected by the same of different detectors. Where the same detector (e.g. the first, second or third detector) is used to measure the transmission of multiple samples, one of the secondary broadband light beams may be modulated. This may allow transmission spectra from multiple samples to be measured using the same detector output.

The apparatus may be configured to measure/detect pump-induced light emission from the sample in more than one direction, e.g. simultaneously. The apparatus may be configured to measure pump-induced light emitted from the sample in the forward direction (e.g. in the transmission geometry) with respect to the incident pump beam, in additional to detecting light emitted at an angle to the incident pump beam. The third detector may be configured for detecting pump-induced emitted light from a detection region of the sample in the forward direction. The third detector may detect light emitted along a third/transmission detection optical path that intersects the first illumination optical path at the detection region within the sample/sample cell. The third detector may be configured to measure the luminescence intensity of the detected pump-induced emitted light. The third detector may be or comprise a spectrometer or a photodetector, such as photodiode or an avalanche photodiode. The first or second detector may be arranged and/or used to detect emission from the sample at a first (non zero) angle to the incident pump beam along the first or second detection optical path (e.g. the angle may be substantially 90 degrees or in the range between substantially 45 to 135 degrees), and the third detector may be arranged and/or used to detect emission from the sample at a second angle that is different to the first angle. The second angle may be approximately 0 degrees i.e. the forward direction or transmission geometry. Alternatively, the first or second detector may be arranged and/or used to detect the emission at both the first and second angles, e.g. simultaneously. In this case, the third detection optical path may join the first/second optical detection path. The apparatus may comprise a means to modulate the light emitted along the first/second and/or third optical detection path, so as to enable separation of the emission signals. The means to modulate the light may be configured to permit light from only one detection optical path to impinge on, or be detected by, the first/second detector at any one time. The means may comprise one or more optical choppers, e.g. positioned in the first/second and/or third detection optical path(s). Using the same detector for detecting multiple signals removes relative errors originating from any different detector sensitivity. The apparatus may be configured to measure absorption of the pump beam and pump-induced emission from the sample simultaneously. The third detector may be arranged and/or used/configured to detect light transmitted through the sample along the third detection optical detection path, and the first/second detector may be arranged and/or used/configured to detect emission from the sample along the first/second detection optical path(s). Alternatively, the first or second detector may be arranged and/or used to detect the transmitted light and the light emitted from the sample at the first angle, e.g. simultaneously. This allows QY to be determined at the pump wavelength using the same detector 70E, avoiding relative errors in the absorption and emission measurements originating any difference or drift in detectors sensitivity.

In this case, the third detection optical path may join the first/second optical detection path. The third detection optical path may comprise an optical filter to attenuate light emitted from the sample in the forward direction. The optical filter may also attenuate transmitted pump light. The optical filter may comprise a band pass filter and/or a neutral density filter. The apparatus may comprise a means to modulate the light emitted along the first/second and/or third optical detection path, so as to enable separation of the transmission and emission signals. The means to modulate the light may be configured to permit light from only one detection optical path to impinge on, or be detected by, the first/second detector at any one time. The means may comprise one or more optical choppers, e.g. positioned in the first/second and/or third detection optical path(s).

The apparatus may comprise an integrating sphere for holding the sample. The integrating sphere may comprise an excitation port for receiving the first or second pump beam and one or more detection ports for coupling to the first and/or second detector. The integrating sphere may be configured to collect light emitted from the sample in all directions.

The apparatus may be a quantum yield measurement apparatus. The apparatus may be operable to perform a quantum yield measurement of particles within the sample based on an output of the first detector.

The apparatus may be connectable to a processing device for performing a quantum yield measurement of particles within the sample based on an output from the first detector. The apparatus may comprise a processing device for receiving an output signal from the first and/or second detector and/or beam profiler, and deriving a quantum yield of particles within the sample based on the output from the first and/or second detector and/or beam profiler.

The apparatus may be operable for use in a range of optical applications based on detection of light emitted from luminescent particles, including but not limited to: optogenetics, flow cytometry, monitoring a polymerase chain reaction (PCR) within the sample.

The apparatus may further be operable to perform a dynamic light scattering measurement of particles within the sample based an output of the first detector.

Features which are described in the context of separate aspects and embodiments of the invention may be used together and/or be interchangeable. Similarly, where features are, for brevity, described in the context of a single embodiment, these may also be provided separately or in any suitable sub-combination. Features described in connection with the apparatus may have corresponding features definable with respect to the method(s), and vice versa, and these embodiments are specifically envisaged. Brief Description of Drawings

In order that the invention can be well understood, embodiments will now be discussed by way of example only with reference to the accompanying drawings, in which:

Figure 1 shows a block diagram of an apparatus according to the invention;

Figure 2 shows an example layout of an apparatus according to the invention in a first configuration; Figure 3 shows the apparatus of figure 2 in a second configuration;

Figure 4 shows another example layout of an apparatus according to the invention in a first configuration;

Figure 5 shows another example layout of an apparatus according to the invention in a first configuration;

Figure 6 illustrates a beam shaping operation;

Figure 7 shows an example an example beam shaping arrangement;

Figures 8a-8d show, respectively, an absorbance spectrum, a luminescence versus absorbed power curve, a luminescence spectrum, and a speckle-free beam profile obtain using the apparatus of figure 2; Figure 9a and 9b shows example emission spectra of a sample and reference sample suffering transmission loss through a band pass filter;

Figures 10a and 10b show a method of measuring quantum yield according to the invention;

Figures lla-e show experimentally determined quantum yield curves obtained using the apparatus and methods of the invention;

Figures 12a-12e show experimentally determined quantum yield curves obtained under different experimental conditions;

Figure 13 shows another example layout of the apparatus for measuring multiple samples;

Figure 14 shows an energy level diagram for a rare earth up-converting material system;

Figures 15a and 15b show, respectively, experimental measurements of rise and decay lifetimes of the system of figure 14;

Figure 16 shows another example layout of the apparatus for measuring luminescence in reflection geometry; and

Figure 17 shows another example layout of the apparatus for measuring luminescence in transmission geometry.

It should be noted that the figures are diagrammatic and may not be drawn to scale. Relative dimensions and proportions of parts of these figures may have been shown exaggerated or reduced in size, for the sake of clarity and convenience in the drawings. The same reference signs are generally used to refer to corresponding or similar features in modified and/or different embodiments.

Detailed Description

For most practical applications of luminescent particles such as up-converting nanoparticles (UCNPs), a sample comprising the luminescent particles is excited and the luminescence is measured and quantitatively analysed to determine or derive one or more characteristics of the sample. This typically requires accurate knowledge or characterisation of the QY of the particles, which is particularly challenging with non-linear particles such as UCNPs which requires both characterisation over a wide dynamic range of excitation powers and careful consideration of the beam profile. The invention provides a solution to this, and as such the apparatus 100 is initially described below in the context of QY measurements. However, the invention is not limited to QY measurements, but is applicable generally to luminescence measurements of luminescent particles.

Figure 1 shows a generalised block diagram of an apparatus 100 for characterising/measuring the luminescence and/or quantum yield (QY) of particles, such as fluorophores and up-converting nanoparticles (UCNPs) dispersed within a sample. In particular, the apparatus 100 is configured for performing accurate measurements of luminescence and/or QY over a wide dynamic range of excitation power densities that can be obtained quickly, to minimise errors due to any changing experimental conditions with time. The apparatus 100 is particularly well suited for optical measurements on non linear luminescent particles such as UCNPs which exhibit a non-linear dependence of the luminescence and QY on excitation power density, particularly at low excitation levels applicable for many biomedical applications.

The sample can be a liquid sample or liquid scattering medium with particles dispersed within the sample. This is typically the case when characterising QY of particles. However, it will be appreciated, that in certain applications, the sample can be a non-liquid or substantially solid sample or scattering medium with particles dispersed therein, e.g. biological tissue.

The apparatus 100 comprises one or more light sources 10 for producing one or more pump beams for illuminating, along an illumination optical path 101 (indicated by the solid arrow in figure 1), a sample or a sample held in a sample cell 60 to thereby produce emitted light by the interaction of the pump beam with the sample. The or each light source 10 is a laser that emits substantially monochromatic light at a centre wavelength _pump appropriate for the sample being measured. Where there is more than one light source 10, one or more or each light source 10 may be used to illuminate the same sample, for example to excite different absorption bands of the sample, or different samples, e.g. with different types of particles dispersed therein. In the latter case, one of the different samples may be a reference sample, such as a dye, having known properties, such as a known QY value used to calibrate the QY measurement of the other sample(s), as explained in more detail below.

A focusing lens 50 is provided in the illumination optical path 101 for focussing the pump beam to a focal point within the sample. The sample cell 60 is positioned in the focal plane of the focusing lens 50 such that the focal point of the focusing lens 50 is substantially in the centre/middle of the sample cell 60. A detector 70E is provided for detecting light emitted from the sample (indicated by the dashed arrows in figure 1) along a detection optical path 102 that intersects the illumination optical path at a detection region 65 within the sample cell 60 that coincides with the focal point of the focusing lens 50. The detector 70E may be configured to output a signal proportional to the amount or intensity of emitted light falling on detector 70E (luminescence). For example the detector 70E may be or comprise a photodetector such as an avalanche photodiode (APD) or camera. Alternatively or additionally, the detector 70E may be configured to output a signal containing the emission spectrum of the light falling on the detector 70E. For example the detector may be or comprise a spectrometer. Alternatively, a separate detector for measuring the emission spectra of light emitted along a separate detection optical path can be provided (e.g. see figure 2). One or more optical filter elements BP, such as a band pass filter, are provided in the detection optical path 102 to attenuate any scattered pump beam light while transmitting the emitted light to the detector 70E. The detector 70E and detection optical path 102 may be arranged and configured to collect and detect light emitted from a limited solid angle defined by the numerical aperture of a collimating/collecting lens, as in a conventional fluorometer-type set up, in which case measurements of a reference sample with a known QY value can be used to calibrate the measured QY yield of the sample. Alternatively, the apparatus 100 may comprise an integrating sphere 90 in which the sample cell 60 can be placed for collecting and detecting light emitted in all directions (a 4p solid angle) and measuring an absolute QY value of the sample, as is known in the art. In this case, the integrating sphere 90 comprises an illumination port for receiving the pump beam (e.g. at the intersection of the dotted circle and the solid arrow in figure 1) and one or more detection ports at a or various locations around the sphere 90 (e.g. at the intersection of the dotted circle and the dashed arrows in figure 1) for coupling the emitted light to the detector 70E.

The apparatus 100 also comprises a broadband light source 80 for producing a broadband light beam having a broad spectral content for illuminating, along a third illumination optical path 103, the sample to thereby produce emitted light by the interaction of the broadband light beam with the sample for measuring an absorption spectrum of the sample. Another detector 70T, which is a spectrometer, is provided for detecting the spectrum of light transmitted through the sample to derive an absorption spectrum from which the absorbance of the sample at the pump wavelength μ( _pump) can be determined.

In an embodiment, the or each light source 10 is configured to produce a substantially speckle- free and smooth beam profile. This is achieved by using a single mode laser, and/or by coupling the laser output to a single mode optical fiber. Both cases produce a speckle-free pump beam with an approximately Gaussian beam profile. Use of a speckle-free pump beam means that the beam profile varies in a predicable way allowing for the effects of beam-profile non-uniformity to be compensated or mitigated (see below) thereby allowing for more accurate QY measurements to be made, as will be described in more detail below.

The beam width or spot size produced by the focusing lens 50 is dependent on the center wavelength of the pump beam λ_pump focal length of the lens 50 and the width of the pump beam impinging of the focusing lens 50. The apparatus 100 further comprises a lens arrangement 20 in the illumination optical path 101 configured to adjust the beam width or spot size of the pump beam at the detection region 65 (whilst preserving the speckle-free beam profile). In an embodiment, the lens arrangement 20 comprises one or more lenses configured to provide a collimated beam of adjustable width impinging on the focussing lens 50, as will be described in more detail below with reference to figures 2-5.

In known QY measurement apparatuses, the excitation power density is varied over a certain limited range by attenuating the pump beam and/or adjusting the power output of the laser. By varying the beam width at the detection region, the pump beam power can be independently varied using known means at each beam width to extend the range of excitation power densities over which QY measurements can be made in the apparatus. Each beam width effectively generates a separate QY versus power density curve of set of QY data which can be combined to provide a QY curve with extended dynamic range of power densities. The different beam widths can be chosen such that the separate QY curves at least partially overlap in power density to produce a substantially continuous QY curve.

The apparatus 100 further comprises a beam profiler 40 in the illumination optical path for imaging the profile of the pump beam at a location equivalent to the detection region 65. The output of the beam profiler 40 provides imaging data that can be used to determine the beam width and/or intensity profile/distribution of the pump beam at the detection region 65. Additionally, the imaging data may be used to compensate the QY value for the effects of non-uniformity in the intensity distribution at the detection region (as would be the case for a Gaussian beam profile), as will be described in more detail below.

Alternatively or additionally, the apparatus 100 may further comprise a beam shaping arrangement 30 in the illumination optical path 101 to transform a non-uniform beam profile of the pump beam, e.g. Gaussian, to a substantially uniform beam profile. Such beam shaping is commonly employed in laser machining applications, and can be achieved using several known techniques, e.g. involving truncation of the pump beam or re-distribution of the pump beam intensity (e.g. using diffraction or refraction). For example, the pump beam can be truncated using an aperture to leave only the central substantially or near uniform/flat part of the beam, or specifically designed (commercially available) beam shaping lenses can be used. The latter typically requires a speckle-free Gaussian input beam. The provision of a substantially speckle-free and substantially uniform beam profile minimises beam profile-induced distortions to the QY value and therefore leads to more accurate QY measurements.

The apparatus 100 will now be described in more detail with reference to figures 2-5. Figure 2 shows an example layout of the apparatus 100. The apparatus 100 comprises a first light source 10A for producing a first pump beam 110 with a first pump wavelength λ_pump1 and a second light source 10B for producing a second pump beam 120 with a second pump wavelength λ_pump2. The first and second light sources 10A, 10B each comprise a single mode fibre coupled to the output of a diode laser (not shown) to produce a substantially speckle-free pump beam 110, 120. The lasers are driven by a power controller (not shown) for varying the current through the diode lasers and their output power. A first illumination optical path 101 receives the first pump beam 110 and comprises a focusing lens 50 (also labelled L4 in figure 2) that focuses the first pump beam 110 to a detection region 65 within a sample held within a sample cell 60. A second illumination optical path 101' receives the second pump beam 120 and comprises a focusing lens 50’ (also labelled L4’ in figure 2) that focuses the second pump beam 120 to the detection region 65. The sample cell 60 may be a standard cuvette held in a sample/cuvette holder (not shown), as is known in the art. The sample comprises particles, such as fluorophores or UCNPs, dispersed in a solvent.

The second illumination optical path 101' is essentially a replica of the first illumination optical path 101. In this example, the two focusing lens 50, 50’ are substantially identical and have the same focal length. The second illumination optical path 101' joins the first illumination optical path 101 at a position after the focusing lens 50 (i.e. in the direction of propagation of the first pump beam 110) at a first moveable mirror FM1 (and via a mirror Ml) which can be selectively moved into and out of the second illumination optical path 101' to selectively direct the second pump beam 120 to the detection region 65. (N.B. mirror Ml can be omitted if the second light source 10B is arranged such that the second pump beam crosses the first illumination optical path 101.)

A first lens arrangement 20 is provided in the first illumination optical path 101 for providing a collimated beam of adjustable width impinging on the focusing lens L4. This varies the beam width at the detection region 65. The first lens arrangement 20 comprises, in order, a first collimating lens L1, a focusing lens L2 and a second collimating lens L3. The focusing lens L2 is arranged to focus the collimated first pump beam 110 to a point between lenses L2 and L3, and the collimating lens L3 is arranged to re-collimate the diverging pump beam, as shown. The focal length, f2, of the lens L2 is different to the focal length, f3, of lens L3 (in this case with f3 < f2) resulting in a collimated first pump beam 110 exiting lens L3 which has a different width (in this example, narrower) to the collimated first pump beam 110 impinging on lens L2. For this purpose, lens L3 is positioned at a distance f2+f3 from lens L2. The second illumination optical path 101’ comprises a second lens arrangement 20’ substantially identical to the first lens arrangement 20. The second lens arrangement 20’ comprises lens L1’, L2’, L3’ which are substantially identical to lenses L1-L3.

Two different beam widths at the detection region 65 can be achieved by either including or not including lenses L2 (L2’) and L3 (L3’) in the first (second) illumination optical path 101 (101’). Figure 2 shows the apparatus 100 in a first configuration where the first (second) illumination optical path 101 (10L) includes lenses L2 and L3 (L2’ and L3’) to produce a first beam width W1 at the detection region 65. Figure 3 shows the same apparatus 100 in a second configuration where the first (second) illumination optical path 101 ( 101') does not include lenses L2 and L3 (L2’ and L3’) to produce a second beam width W2 at the detection region 65.

For a given pump power, varying the beam width between W1 and W2 provides a different power density at the detection region. The first and second illumination optical paths 101, 101' further comprise one or more optical density filters ND, ND’ to attenuate the pump power by a fixed or variable amount and adjust the excitation power density at the detection region 65. The filters ND, ND’ may be movable into and out of the first and second illumination optical path 101, 101' to selectively attenuate the first and second pump beam 110, 120. The optical density filters ND, ND’ may be or comprise a neutral density filter with a fixed or variable (e.g. a filter wheel) optical density, or a set of neutral density filters with different optical densities. For each beam width W1, W2 and optical density filter setting, the output power of the diode lasers can then be varied over a certain range using the power controllers. The combination of QY measurements taken at different beam widths W1, W2, optical density filter settings and laser output powers provides access to wide dynamic range of power densities of up to 10⁴, 10⁵ or 10⁶.

A first detection optical path 102 is provided for measuring the pump-induced luminescence (emission) signal of the sample. The first detection optical path 102 receives light emitted from particles dispersed within the sample. The intersection of the first illumination optical path 101 and the first detection optical path 102 defines the detection region 65. The first detection optical path 102 comprises a collimating lens L5 for collecting light emitted from the sample along the first detection optical path 102 and a focusing lens L8 for focusing the collected emitted light onto a detector 70E. In this example, the detector 70E is an APD configured to output a detector signal proportional to the intensity of detected emitted light. A first optical wavelength filter BP1 is positioned in the first detection optical path 102 to filter/attenuate any scattered pump light and transmit the emitted light from the sample. The first optical wavelength filter BP1 may be a band pass filter, a long pass filter, or short pass filter (or a combination of multiple optical wavelength filters), depending on the sample being measured and the emission spectrum. Optionally, a spatial filter arrangement comprising, in order, a focusing lens L6, an aperture or slit A1 and a collimating lens L7 may be positioned between lenses L5 and L8 to focus emitted light through the aperture A1 to suppress any light originating from volumes in the sample other than the detection region 65.

A pair of linear polarisers P1 and P2 may be used to make the detected emitted light substantially independent of any anisotropy in the sample. For this purpose, a first linear polariser P1 (P1') is positioned in the first (second) illumination optical path 101 (101') at a given orientation, e.g. to vertically polarise the first (second) pump beam 110 (120), and a second linear polariser P2 is positioned in the first detection optical path 102 oriented at a magic angle of 54.7 degrees with respect to the first polariser P1. The first polariser PI is preferably positioned in an expanded portion of first (second) pump beam 110 (120) as shown in figures 2 to 5, to prevent any damage of the polariser P1 due to the power density of the first (second) pump beam 110 (120).

A beam profiler 40 comprising a beam splitter BS and an imaging detector or camera 40D is positioned in the first illumination optical path 101 for imaging the beam profile of the first or second pump beams 110, 120 at the detection region 65. The beam splitter BS is positioned after (in the direction of propagation of the first/second pump beam 110, 120) the focusing lenses 50 and 50’ and directs a small fraction of the first or second pump beam 110, 120 towards to the imaging detector 40D, which is positioned at the focal point of the focusing lenses 50 and 50’. In an embodiment, the imaging detector 40D is a high resolution charge coupled device (CCD) camera that outputs imaging data containing a two-dimensional image of the intensity distribution in the beam.

A second detection optical path 102’ is also provided for measuring the pump-induced luminescence (emission) spectrum of the sample. Similar to the first detection optical path 102, the second detection optical path comprises a collimating lens L9 for collecting light emitted from the sample along the second detection optical path 102’ and a focusing lens L10 for focusing the collected emitted light onto a detector 70E1, but in this case, detector 70E1 comprises a spectrometer that outputs a detector signal containing the spectrum of detected light. A second optical wavelength filter BP2 is also positioned in the second detection optical path 102’ to filter/attenuate any scattered pump light and transmit the emitted light from the sample.

The first and second detection optical paths 102, 102’ are oriented at an angle of substantially 90 degrees to the direction of the first and second pump beam 110, 120 through the sample cell to reduce the amount of pump light reaching the detectors 70E, 70E1. However, since emission from the sample is typically isotropic, the detection geometry is not limited to the geometry shown. Other detection geometries may be used to measure QY, e.g. where the first and/or second detection optical path 102, 102 is arranged to detect forwards, and backwards, upwards or downwards emission. Further, the first and second detection optical paths 102, 102’ need not be at the same angle.

Another detector 70T is provided in a transmission geometry for detecting light transmitted through the sample cell 60 to derive an absorbance of the sample at each pump wavelength μ(λ_1ump1 or λ_Pump2)· The detector 70T can also be used to measure the power of the first or second pump beam. For this purpose, the detector 70T may be a photodetector or calibrated power meter configured to output a detector signal proportional to the intensity of the detected transmitted light.

Alternatively or additionally, the detector 70T may comprise a spectrometer for measuring the transmission spectrum of the sample to derive an absorption spectrum m(l). Alternatively, the detector 70T can be replaced with an optical fiber or fiber bundle to collect and couple in the transmitted light and send it to the detector 70E1. In this way, the absorbance of the sample at each pump wavelength can be determined in a single shot measurement. For this purpose, a broadband light source 80 (e.g. a white light source) is provided for producing a broadband light beam 130 having a broad spectral content for measuring the broadband transmission, absorption and/or scattering spectrum of the sample. A third illumination optical path 103 receives the broadband light beam 103 and comprises a collimating lens L 11 for collimating the output of the broadband source 80. The third illumination optical path 103 joins the first illumination optical path 101 at a position before (in the direction of propagation of the first pump beam 110) the focusing lens 50 at a second moveable mirror FM2 which can be selectively moved into and out of the third illumination optical path 101' to selectively direct the broadband light beam 130 to the detection region 65. Alternatively, the third illumination optical path 103 may join the second illumination optical path 101' at a position before (in the direction of propagation of the second pump beam 120) the focusing lens 50’ by suitably positioning the second moveable mirror FM2 (not shown). An iris (not shown) can be placed in the collimated beam 130 to control the beam width and power of the broadband light beam 130 at the detection region.

The apparatus 100 is connectable to a processing device (not shown), e.g. comprising a data acquisition (DAC) device and one or more processors, for receiving the various output signals from detectors 70E, 70T, 70E1, 40D, controlling the laser output power and deriving, at each power density, a QY value based on the received detector data, as will be described in more detail below.

In the illustrated examples, the output of the light sources 10 A, 10B is divergent by virtue of the single mode fiber, and the first collimating lens L1, L1’ is arranged to collimate the diverging output of the light source 10 A, 10B. However, it will be appreciated that, alternatively the collimating lens L1 (L 1') may be part of a beam expanding arrangement in the first (second) illumination optical path 101 (101’). Further, where a single mode laser is used instead of a laser and single mode fiber combination as the light source 10A, 10B such that the output of the light source 10A, 10B is collimated, the lens L1 may be omitted altogether.

Figure 4 shows another example layout of the apparatus 100 where the second illumination optical path 101' joins the first illumination optical path 101 at a position before the focusing lens 50 via mirrors Ml and FM1. In this case, focusing lens 50’ is not required - focusing lens 50 is used to focus both the first and second pump beams 110, 120 to the detection region. For example, the focusing lens 50 can be an achromatic lens with a focal length that is substantially the same for the first and second pump wavelengths λ_pump1, _pump2. However, it will be appreciated that the apparatus 100 can instead be configured such that the second illumination optical path 101' joins the first illumination optical path 101 at a position before the first lens arrangement 10 by suitably positioning the first moveable mirror FM1, thus also omitting the second lens arrangement 10’. Alternatively, the outputs of the lasers of the first and second light sources 10A, 10B may be coupled to the same single mode fiber using a suitable fiber combiner/coupler, as is known in the art. In the example layout of figures 2 to 4, the lenses L2 and L3 (L2’ and L3’) are movable, such that they can be selectively moved into and out of the first (second) illumination optical path 101 (101'). Alternatively, figure 5 shows another example layout of the apparatus 100 where the first (second) pump beam 110 (120) is selectively directed through the lenses L2 and L3 (L2’ and L3’) via a combination of mirrors M2 and M3 (M2’ and M3’) and third and fourth movable mirrors FM3 and FM4 (FM3’ and FM4’) which can be selectively moved into and out of the first (second) illumination optical path 101 ( 101'). In figure 5 the broadband light source 80 and third illumination optical path 103 have been omitted for clarity. In this way, the first (second) illumination optical path 101 (101’) is effectively altered to include or bypass the lenses L2 and L3 (L2’ and L3’). It will be appreciated that when movable mirrors FM3 and FM4 (FM3’ and FM4’) are moved out of the first (second) illumination optical path 101 (101’) the collimated first (second) pump beam 110 (120) continues through to the focusing lens 50 (50’) with an unaltered width similar to figure 3.

It will be appreciated that movement of the moveable optical elements, including the lenses (L2, L3, L2’, L3 ’), the moveable mirrors (FM1, FM2, FM3, FM4, FM1’, FM2’, FM3’, FM4’) and/or the movable optical density filters ND, ND’, may be manually effected/adjusted or automatically adjusted (motorised) depending on the optics mounts used. Any optics mount known in the art can be used that allows the moveable lenses and mirrors to move or be moved between a position out of the respective illumination optical path 101, 101' and a predefined position in the respective illumination optical path 101, 101' without having to re-adjust any optical elements in the apparatus 100 once set up. For example, they may be mounted on flip mounts, pivotable mounts, magnetic mounts, multi-position mounts, translation stages or fast change drop-in mechanics, any of which can be manually operated or motorised and controlled via an appropriate controller. Where the moveable optical elements are motorised, their movement may be controlled by the processing device.

The apparatus 100 may further include a beam shaping arrangement 30 comprising one or more lenses in the first and second illumination optical path for transforming the near Gaussian beam profile of the (speckle-free) first and second pump beams 110, 120 to a substantially uniform beam profile. This function is shown schematically in figure 6. Figure 7 shows an example beam shaping arrangement 30 based on beam truncation. An expanded input pump beam 110a with a Gaussian intensity profile is truncated by an aperture A2 which selects only a suitably flat or near-flat portion of the input beam 110a (typically the central part of the input beam 110a). Truncation or aperturing typically leads to diffraction effects at the edge of the truncated beam. Fourier optics is used to compensate for or removed these diffraction artefacts using a 4f-type configuration. A focusing lens L12 is positioned a distance equal to its focal length f12 away from the aperture A2 and focuses the truncated beam to a focal point. This is equivalent to performing a Fourier transform on the diffraction pattern of the truncated beam. A collimating lens L13 positioned a focal length f 13 away from the focal point then collimates the diverging beam. The output truncated beam 110b observed a focal length f 13 away from lens 13 (in the plane of the dotted line), which would be the detection region 65 of the sample cell 60, is the reconstructed shape of the aperture A2 without diffraction artefacts. An aperture A3 placed in the focal plane of lens L12 may be used to spatially filter light at the edges of the focussed beam which contain the diffraction artefacts. The above beam shaping arrangement may be placed between the focusing lens L4, 50. Alternatively, it can be used in the place of the focusing lens L4, 50. In this case, it will be appreciated that the sample is illuminated with collimated light of varying beam width.

In another example, a beam shaping lens may be used to transform the Gaussian profile of the first or second pump beam 110, 120 impinging on it to a substantially uniform or top-hat profile. In one example, the focusing lens 50 (50’) can be replaced with a beam shaping lens.

The apparatus 100 shown in figures 2 to 5 is based on a conventional fluorometer design. As such, the first detection optical path 102 may receive/collect light from a limited range of angles, defined by the numerical aperture of the collimating lens L5. In this case, it is common to measure the QY of a reference sample with a known or predetermined QY value in the same apparatus 100 to calibrate the measured QY of the sample, as described in the experimental section below. Alternatively, in another embodiment, the apparatus 100 comprises an integrating sphere 90 (see figure 1) in which the sample cell 60 can be placed for detecting light emitted from a 4p solid angle for measuring an absolute QY value, as is known in the art. In this case, the integrating sphere 90 comprises an illumination port for receiving the first or second pump beam 110, 120 and one or more detection ports at different locations around the sphere for coupling the emitted light to the detector 70E and/or 70E1 in the first and/or second detection optical paths 102, 102’.

Figure 13 shows an alternative layout of the apparatus 100 configured to measure the transmission spectrum of multiple samples simultaneously using the broadband light beam 130 (the first and second illumination optical paths 101, 101' and beam profiler 40 have been omitted for clarity). Here, a second beam splitter BS2 is used to direct a fraction of the broadband light beam 130 towards a second sample cell 60r. This can be used to hold a reference sample, e.g. containing only the solvent the particles in sample cell 60 are dispersed in. The second beam splitter BS2 effectively splits the broadband light beam 130 into two secondary broadband light beams 130a, 130b. The light transmitted through the second sample cell 60r is detected by the detector 70T, i.e. the same detector used to measure the transmitted light through sample cell 60. For example, the light transmitted through sample cell 60r can be sent/routed to the detector 70E via an optical fiber (not shown). An optical chopper CH is positioned in one of the secondary light beams 130a, 130b to modulate the respective secondary beam 130a, 130b at a frequency, in this case the light beam illuminating sample cell 60. The output of the detector 70T is therefore also modulated and contains the transmission spectrum for both sample cell 60 and second sample cell 60r which can be readily be extracted. Alternatively, the light transmitted through sample cell 60r can be detected by a separate detector (not shown). This layout may speed up the QY measurement sequence, as described in more detail below.

A chopper CH can also be used to modulate the first and/or second pump beams 110, 120 to study the temporal dynamics of the QY, e.g. in the time shortly after pump excitation. This may be achieved using a suitably fast APD detector 70E.

In addition to any of the embodiments described above, the apparatus 100 can further be configured to detect pump-induced light emission from the sample 60 in more than one direction simultaneously. With reference to figure 13, detector 70E can be used to detect emission from the sample along the first detection optical path 102 at an angle (in this example 90 degrees) to the incident pump beam 110, 120, and detector 70T can be used to detect emission from the sample in the forward direction or transmission geometry along a third/transmission detection optical path 102”. In this case, the transmission detection optical path 102” is essentially a replica of the first optical detection path 102 comprising the same optical elements such as lenses L5-L8 and filters BP1, P2 (not shown). It will be appreciated that the light exiting the sample along the transmission detection optical path 102” includes a large pump signal and a relatively small forward emission signal. The pump signal can be filtered out using one or more optical filters BP1. Alternatively, the same detector, e.g. detector 70E or 70T, can be used to detect both the side emission and the forward emission (not shown). An optical chopper (not shown) is positioned in the transmission detection optical path 102” and/or the first detection optical 102 to modulate the respective signal(s) such that only one signal (i.e. transmission or side emission) is detected by the detector 70T/70E at any one time. The two signals can then be temporally separated. In an example where the detector 70E is used to detect both side emission and transmission signals, the transmitted light can be directed to the detector 70E by one or more mirrors or beam splitter and/or an optical fiber. Preferably, the transmission detection optical path 102” can join the first detection optical path 102 so as to use at least some of the same optical elements. Using the same detector 70T/70E for detecting both signals removes relative errors originating from any difference or drift in detector sensitivity.

Alternatively, the above described arrangement can be used to measure the absorption and emission signal simultaneously with the same detector, e.g. detector 70E. In this case, instead of filtering out the pump signal in the transmission detection optical path 102”, the forward emission signal is filtered out or attenuated (before joining the first detection optical path 102). Here, the transmission detection optical path 102” joins the first detection optical path 102 (e.g. using a beam splitter) after the band pass filter BP1. Because the forward emission signal is small (e.g. by several orders of magnitude) compared to the transmitted pump signal, filtering the forward emission signal can be done using a neutral density filter to attenuate both the pump light and the emitted light to a level suitable for the detector 70E, making the already small emission signal negligible. In practice, this is to a level that is close to the level of light emitted along the second detection optical path 102. This allows QY to be determined at the pump wavelength using the same detector 70E, avoiding relative errors in the absorption and emission measurements originating from any difference or drift in detector sensitivity.

The lenses L1-L11 shown in the illustrated embodiments are refractive optical elements. It will be appreciated that, in principle, one or more of the lenses may be replaced with a reflective lenses, as is known in the art.

IA Quantum yield measurement

The proposed method of measuring the QY of a sample comprises illuminating a sample with the pump beam 110 having the first beam width W1 (and thus a first excitation power density) at the detection region 65, detecting the resulting light emitted from the detection region of the sample, illuminating the sample with the first pump beam having the second beam width W1 (and thus a second excitation power density) at the detection region, the detecting resulting light emitted from the detection region of the sample, and deriving a QY of particles within the sample based on the pump-induced emitted light detected at the first and second beam widths W1, W2 by performing quantum yield analysis. The measurements at the first and second beam widths W1, W2 are repeated at a plurality of pump powers by attenuating the pump beam and/or controlling the output power of the laser to yield a set of QY values over a wide dynamic range of power densities.

An example QY measurement procedure for a sample cell holding a sample containing UCNPs dispersed in a solvent is described below with reference to figure 10a. The method applies equally to other fluorophores. In step SI, the broadband transmission spectrum of the sample, a reference solvent sample (held in a separate sample cell) and an empty sample cell is measured to determine the net absorption spectrum of the UCNPs. This involves illuminating the sample, reference solvent sample, and empty sample cell with the broadband light beam 130 and detecting the light transmitted therethrough. This can be measured detector 70T, or the transmitted light can be collected and sent/routed (e.g. via an optical fiber) light to detector 70E1. In both cases, the detector is a spectrometer. The above transmission measurements are repeated for a reference sample containing reference particles, such as a dye, dispersed a reference solvent (i.e. transmission measurements are performed for the reference sample and its reference solvent sample). QY measurements of the reference sample are used to calibrate the QY measurements of the sample.

In step S2, the pump-induced luminescence emission signal and spectrum of the UCNPs in the sample is measured over a wide dynamic range of excitation power densities. This involves illuminating the samples with the first pump beam 110 at each beam width W1 and W2 and detecting the luminescence signal and emission spectra at detectors 70E and 70E1 at various pump beam powers. In an example, the pump beam power is varied at each beam width by varying the output power of the laser source 10 A and repeating the measurements with and without the optical density filter ND in place.

In step S3, the beam profile for each beam width and beam power combination is imaged in imaging detector 40D and recorded. This step may occur simultaneously with step S2. Figure 8d shows an example 2D profile of the first pump beam 110 imaged at detector 40D, demonstrating a substantially speckle-free and smooth Gaussian beam profile. In step S4, the pump beam power at each beam width and beam power combination is measured using a power meter at the position of detector 70T. In step S5, steps S2-S4 are repeated for the reference sample using the second pump beam 120.

Both luminescence emission and signal measurements can be done simultaneously, and in real time. This is significant since the emission spectrum of the particles such as UCNPs or the reference sample may change (slightly) over time.

This completes the measurement sequence for QY characterisation. The outputs of the measurement sequence are: (i) broadband transmission spectra of the sample, reference sample, reference solvents and empty sample cell; (ii) pump-induced emission spectra of the samples and reference sample at each beam width and pump power combination; (iii) pump-induced luminescence signals for the samples and reference sample at each beam width and beam power combination; (iv) beam profiles of the first and second pump beams 110, 120 at each beam width and beam power combination; and (v) beam power measurements at each beam width and beam power combination.

IB Quantum yield analysis The QY analysis procedure is described below with reference to figure 10b. The QY analysis takes all the measurement outputs of the measurement sequence as inputs and calculated various optical parameters to yield a QY curve compensated for possible distortions.

In step S6, the compensated (absolute) absorbance at the pump wavelength determined. This is used to calculate the number of absorbed photons. The total absorbance of a medium x being measured (A_x) is related to transmission by Beer-Lambert’s law as: A_x(λ) = 1n(I₀(λ)/I(λ)) (1) where I (λ) and I₀(λ) are the spectra obtained from the sample cell holding the medium and the empty sample cell, respectively. The absorbance is calculated for the sample, reference sample and reference solvents. As the sample contains UCNPs in a solvent, the net absorbance of the UCNPs is obtained by subtracting the absorbance of the reference solvent sample. Figure 8a shows an example net absorbance spectrum obtained from a sample containing UCNPs (see curve labelled A). The measured absorbance spectrum also contain contributions from light scattering that should be removed to yield accurate QY values. This is demonstrated in figure 8a, in which the absorption spectrum exhibits a strong background signal, particularly as shorter wavelengths, due to light scattering from the UCNPs. Following well known scattering laws, this scattering contribution can be removed by fitting to the background with a polynomial of the form A_SC λ) a and subtracting the resulting fit to yield the

absolute absorbance spectrum of the UCNPs in the sample . Here, a and b are fitting

coefficients that relate to the density and size of the particles. The curve labelled B in figure 8a shows an example fit to the polynomial. This procedure is repeated for the reference sample to obtain the absolute absorbance spectrum of the reference dye, The absolute absorbance of the UCNPs and

the reference dye at the respective pump wavelengths can then be determined.

In step S7, the emission spectrum is used to determine a correction factor to compensate the luminescence signal for any transmission loss due to the limited bandwidth of the optical wavelength filter(s) BP1. This is particularly important in samples or reference samples with relatively broad emission characteristics. Figure 8c shows an example emission spectrum of a sample containing UCNPs, which is centred on 800 nm. The effect of the transmission loss through filter BP1 is demonstrated in figures 9a and 9b which show the normalised emission spectrum of the reference dye and the sample compared to the filtered emission spectrum, which is the product of the normalised emission spectrum and the transmission spectrum of the filter BP1. Transmission correction factors, T_UCNP, T_Dye for the sample and the reference sample can be determined by integrating emission and filtered emission spectra and taking the ratio of the resulting values.

In step S8, the QY of the sample and reference sample is determined using the compensated absorbance of the sample and reference sample at the pump wavelength and the transmission correction factors derived in steps S6 and S7. The experimental quantum yield Φ_exp(p) (not compensated with the reference dye QY) of the UCNPs of the sample at the various power densities is obtained by taking the ratio of luminescence signal of the UCNPs L_ucnp(p) (compensated for the transmission losses through the filter BP1) to the number of photons absorbed per unit length in the middle of the sample cell 60

N_{ab s},_ucnp (p) (determined using the compensated absorbance). To compensate for the inner filter effects, the power at the middle of the sample cell 60, P_c, can be defined as:

where x refers to the medium being measured (e.g. the sample or the reference sample), P_x is the power of the pump beam after passing through the medium and (λ_pump) is the total absorbance of the

medium at the pump wavelength calculated in equation 1 (including contributions from the solvent and scattering). Figure 8b shows an example plot of the luminescence signal from a sample containing UCNPs as a function of the absorbed power at the middle of the sample cell

The number of photons absorbed by the UCNPs in the sample at the middle of the sample cell 60 is defined as:

where hc/ _pump1 is the photon energy of the first pump beam 110 and DT is the time period the measurement/exposure. The experimental QY of the sample is then obtained from:

Equivalent equations can be written for the experimental QY of the reference sample, Φ_exp,dye (p)· However, because the absolute QY of the reference sample is known and constant (Φ _abs,dye ), the QY values for the sample can be calibrated relative to the reference dye value to obtain the relative QY of the UCNPs in the sample according to:

where n_ss and n_rs are the refractive indices of the sample solvent and the reference sample solvent, respectively, accounting for the reflections losses of light passing into and out of the sample and reference sample.

Equation 5 is calculated for each beam width, optical density filter and beam power combination to obtain four sets of QY values, one for each beam width and optical density filter combination (in this case present or not present) which are combined to provide a high dynamic range QY curve.

In step S10, the relative QY values are compensated for the non-uniform beam profile using the imaging data obtained in step S3 to avoid underestimating the QY values. Figure 8d shows an example 2D profile of the first pump beam 110 imaged at detector 40D. The image data contains a 2D matrix of pixels, each pixel having a pixel value proportional to the intensity of the beam at that position (i.e. an image-based intensity matrix). The beam profile is a substantially speckle-free smooth Gaussian shape. Beam profile compensation is implemented by calculating the QY for each pixel in the beam profile using a rate equation describing the emission process in the sample. This process can use the image- based intensity matrix, or a Gaussian-profile-based intensity matrix derived from fitting to the image- based intensity matrix with a 2D Gaussian distribution function. In the below discussion only image- based compensation is discusses, as Gaussian-based compensation follows the similar method. The image is analysed in terms of its intensity matrix, G, with total power density, P, represented by Π_(p) = P(p)/Area , where P(p) is the total laser power for a given power density, and Area is the beam profile area. A compensation matrix, g was obtained by normalizing the intensity matrix G such that the total intensity summed over all pixels in the matrix is equal to 1. Each element k of g is then represented by Π_(p) = P(p)/Area, where Γ_k is an element of the intensity matrix Γ, y_k is an element of the normalised compensation matrix and Σ_k represent the sum over k. A γ_k image-based compensation matrix is created for each beam width and optical density filter combination for the samples and the reference sample to obtain relative quantum yield values for all 4 configurations, as described below.

Directly from rate equations, the quantum yield, f, for two-photon emission process at a certain power density, p, is given by:

where Φ_b and p_b are the quantum yield and power density at the balancing point. The balancing point is related to the saturation of the absorption-emission process. Because the power density varies across the beam profile, the total quantum yield, Φ_T, is by definition given by,

where, n_{e(a) k} is the total number of photons emitted (absorbed) by UCNPs along the region due to the kth-pixel, and is the energy of each photon emitted (absorbed). However, based on equation

is the power at each pixel, and P_c,k = P_cγ_k where P_c is the

total measured power (at the centre of the sample cell 60). Then,

From equation 6, the quantum yield due to a certain pixel region can be defined as,

where p_k=P_cγk/A_px and A_px is the pixel area. Noting also that, by definition, the QY at each pixel is and substituting in equation 8 gives,

Substituting equations 8, 9 and 10 into equation 7 it can be shown that the total quantum yield is

Equation 11 can be calculated for each beam width, optical density filter and beam power combination using only and p_b as fitting parameters which are valid for the whole data set. Φ_T is minimized against the Φ _rel,ucnp value for each power density point.

Any presence of speckles in the beam profile makes it more difficult to compensate for the beam profile.

II. Experimental validation To experimentally validate the apparatus and proposed method, QY measurements of two UCNP samples were performed using a setup according to figure 2. Sample 1 was water-soluble NaYF₄:Tm core UCNPs (UCNP1) and sample 2 was water-soluble NaYF₄:Tm core shell UCNPs (UCNP2), both produced from Hangzhou Fluo Nanotech Co. Ltd at a concentration of 10 mg/ml. The samples were diluted in distilled water at 5 mg/ml, 2.5 mg/ml for concentration repeatability measurements. These UCNPs have an emission wavelength around 800 nm. A reference sample containing a reference dye was used to calibrate the UCNPs QY value. The reference dye was sourced from Dyomics (Dy- 781). The reference dye has an emission wavelength centred similar to UCNP (800 nm) and a factory tabulated quantum yield of 11.9 % when dissolved in ethanol. The reference dye was diluted in ethanol solvent to have absorption values similar to 10 mg/ml UCNP samples used for this study. Two dedicated cuvettes with water and ethanol solvents were prepared as blank references to obtain absorption values of pure UCNPs and dye. 2 ml of each sample were placed in a quartz cuvette (Thorlabs, CV10Q3500FS) and the sealed to avoid any evaporation of the ethanol and water solvents.

Specific details of the apparatus 100 used in the experimental tests are as follows. Light sources: 10A = temperature stabilised diode laser at λ_pumpi = 976 nm coupled to a single mode fiber (Thorlabs, BL976-PAG500); 10B = temperature stabilised diode laser at _pump2 = 785 nm coupled to a single mode fiber (Thorlabs, FPL785S-250); 80 = Ocean Optics HL-2000. Optical wavelength filters: BP1= band pass (Edmund Optics, FBH800) and short pass (Thorlabs, FES0900) for samples 1 and 2, and band pass (Edmund Optics, FBH800) and long pass (Thorlabs, LP02785RU) for reference sample; BP2 = short pass for samples 1 and 2 (Thorlabs, FES0900). Detectors: 70E = APD (Thorlabs, APD120A), 70E1 = spectrometer (Ocean Optics QE Pro). Optical density filter: ND= optical density 1 (10% transmission). Lenses: L1 (f 1=30 mm); L2 (f2 =30 mm); L3 (f3=6mm); L4 (f4=200mm); L5-L11 (f=30mm). This selection of lenses produces two different widths at the detection region 65 of Wl=700μm when the lens arrangement 10, 10’ is in the first and second illumination optical paths 101, 101’and W2=150 pm when the lens arrangement 10, 10’ is moved out of the first and second illumination optical paths 101, 101'. The first light source 10A is used to excite the UCNPs in samples

1 and 2, and the second light source 10B is used to excite the dye in the reference.

IIA Measurement method

Broadband (350-1100 nm) transmission spectrum of samples 1 and 2, a cuvette filled with reference solvent (water), and an empty cuvette was measured to determine the net absorption spectrum of the UCNPs, as described in sections IA and IB. In this case, detector 70T was replaced with a fiber bundle (Thorlabs, BFL200HS02) which collected and sent the transmitted light to detector/spectrometer 70E1. The above transmission measurements were repeated for the reference sample, and a cuvette filled with reference solvent (ethanol).

The pump-induced luminescence emission signal and spectrum of the UCNPs in samples 1 and

2 is measured over a wide dynamic range of excitation power density. The luminescence measurements are taken at various laser currents (20mA-800mA, with a 4mA steps) with and without the optical density filter ND. Measurements with the first beam width W1 (700 μm) correspond to a low density regime, while measurements with the second beam width W1 (150 μm) correspond to a high density regime. The beam profile for each beam width and beam power was imaged in imaging detector 40D and recorded. The pump beam power at each beam width and beam power combination was measured using a power meter at the position of detector 70T. These measurements were repeated for the reference sample using the second pump beam 120 at _pump2 = 785 nm (20mA-850mA, with a 4mA steps).

The absolute absorbance (removing scattering contributions) for samples 1 and 2 and the reference dye was determined as described above. Transmission

correction factors, T_{UCNP 1}, T_UCNP2, T_Dye for the samples and the reference sample were determined using the measured emission spectra and transmission of the filter BP1.

The relative QY for the samples was calculated using equation 5 for each beam width, optical density filter and beam power combination to obtain four sets of QY values, one for each beam width and optical density filter combination (in this case present or not present) which were combined to provide a high dynamic range QY curve. Figure 11a- 11d show the relative QY versus average power density (calculated using the full-width at half maximum (FWHM) of the beam profile imaged in step S3) for the QY values obtained, respectively, with the first beam width W1 and without the filter ND, the first beam width W1 with the filter ND, the second beam width W2 without the filter ND, and the second beam width W2 without the filter ND. Figure lie shows the resulting high dynamic QY curve combining all the data, in this case spanning a dynamic range of 10⁴.

Next, the relative QY values were compensated for the non-uniform beam profile, using the procedure described in section IB. The resulting beam profile compensated QY curve is shown in figure lie by the open circles (open squares show the results for Gaussian-based compensation). As is evident, the assumption of a uniform beam profile, which is the case when only the beam width is used to estimate the power density, leads to an underestimation of the QY. The approach described above properly takes the non-uniform beam profile into account leading to more accurate QY measurements.

The apparatus 100 was also tested under various experimental conditions to ensure its robustness. The following four variables were considered a) sonication of the samples b) intraday stability c) inter-day reproducibility d) different sample concentrations. The results of these measurements are summarized in figure 12. The sonication time was tested to understand optimal sonication time for the samples and it’s clear from figure 12a that sonication time between 5-15 minutes does not influence the measured QY of UCNPs significantly. Figure 12b indicates that the intra-day variations were found to be within 4% variation at high QY values. Inter-day reproducibility was found to have similar variations (see figure 12c). It is worth noting that in the absence of all measurements and compensations, we found the assessed QY values to vary by up to 100%. The different sample concentrations were attributed to the changes in the turbidity of the UCNP sample. Correct compensation for scattering in the transmission spectral measurements resulted in a remaining negligible variation in QY values as illustrated in figure 12d. In addition to the inter-sample variations, the absence of this compensation yielded on average a 60% underestimation in QY values. These tests emphasize the need for multimodal measurements and compensation for the optimal characterization of QY of UCNPs.

Although only the 800 nm emission line of the NaYF4:Tm UCNPs was consider in the above experimental results, it will be appreciated that detection of other emission lines in other samples can be achieved easily, by changing the optical wavelength filters BP1 in the detection optical paths 102, 102’ and by selecting a suitable dye matching the emission wavelength of UCNPs. In this case, the first light source 10 A used for exciting the UCNPs remains the same, while the second light source 10B used for exciting the reference dye should be chosen based on the measurement protocol used for the tabulated QY of the selected dye.

Notably, the equation 6 (derived from rate equations for a 2 photon transition) developed for the 800 nm emission line may no longer be valid for other emission lines, as these lines might involve a greater number of photon transitions. As such, in generally, a model describing an n-photon transmission suitable for the sample and measurement should be used.

The above validation example, transmission measurements on different samples and solvents were performed in sequence placed in the same position. In an alternatively layout, the third illumination optical path 103 may not join the first or second illumination optical paths 101, 101' but may instead comprise a separate sample holder for measuring the transmission spectrum of samples or reference sample (e.g. containing reference solvent) in a separate beam line, optionally concurrently with luminescence measurements. This may speed up the measurement sequence, thus reducing errors in the assessed optical properties and in the obtained QY associated with changes in experimental conditions with time.

Although the second illumination optical path 101' was used to measure a reference dye to calibrate the quantum yield of UCNPs, this calibration factor will remain constant, provided, the apparatus 100 is robustly built and well tested under different experimental conditions. Therefore, the need for a reference arm is not essential once the set-up is calibrated at each emission line.

The above results demonstrate that the apparatus 100 is well suited for stability and reproducibility with a variation of less than 4% under different experimental conditions. Further, the results show that the obtained quantum yields of UCNPs can vary by up to 100% and have an offset of 60% in the absence of the multi-modal corrections included in this proposed method. The system and methods described herein results can therefore act as a precursor for standardizing the measurements of quantum yield values.

III. Further applications

As described above, the apparatus 100 may be used to characterise various optical parameters of particles within a sample including absorption and scattering, luminescence emission spectra and luminescence signal and QY. Although the above description has focused on measurements of QY, the principle of exciting non-linear luminescent particles such as UCNPs with a substantially speckle-free uniform pump beam profile to improve the accuracy and reliability of the sample characteristics derived from the luminescence can be applied to a number of other applications. It is especially important for accurate quantification of luminescence of non-linear materials in the low power regime where the non-linearity is strongest, as applies to biomedical applications (e.g. where the sample comprises biological tissue) where high light levels can damage/denature the protein or other components of the biological media.

By way of example only, speckle-free uniform pump beam profile is important to accurately determine the radiative lifetime of electronic states in the particles. Figure 14 shows an example electronic transitions in Tm³⁺ ions resulting from multiphoton 975 nm absorption in Yb³⁺ ions in a rare earth NaYF4:Yb,Tm UCNP system. The upconversion emission shown by the 800nm and 474nm lines occurs due to the existence of long lifetimes present on intermediate excited energy levels. Measuring the radiative lifetime of individual levels (especially the ones involved on the multi electronic transitions steps) is important for characterizing the material as well as for modelling and understanding the optical processes. As well as the radiative lifetime, the time to populate and depopulate the energy levels (referred to here as raising and decay time, respectively) is also important to determine. Because the whole process is driven by the number of photons that the material is exposed to (i.e. the power density of the excitation line), it is also expected that the raising and decay time also exhibit a power density dependence. Therefore, having a proper beam illumination profile is important to properly evaluate the intrinsic properties of nonlinear optical materials. As such, the apparatus 100 and method of the invention provides an important step forward in developing novel materials with optimal features (e.g. UCNPs with high emission quantum yield). As an example, consider the energy levels a and b in the Yb3+ ions shown on figure 14. The population decay from the excited state to ground state depends on the b energy level population (N_b), spontaneous radiative lifetime ( _b), energy migration to the environment (W_dN_d), and the energy transfer processes to the Tm3+ ions i energy level (i.e. the population density of the i level, being i equals to 1 or 2). Thus, the decay time is represented by t = 1 /R_b , with R_b given by the following equation:

The population of the i levels are power density dependent and, thus, so are the raising and decay time. The same is valid to the energy levels in Tm3+ which are involved in the upconversion emission process. Figures 15a and 15b show example measurements of raising and decay time, respectively, for the energy level 2 responsible for the 800nm emission in NaYF4:Yb,Tm UCNPs under 975nm laser excitation with a speckle-free uniform beam profile at different power densities (indicated by the laser currents) using the apparatus 100 arrangement shown in figure 2. As is evident, the τ values for the population raising and decay are different since during the decay measurement the pump light is absent and mechanisms involved in the process lack the pumping up electrons due to energy transfer initially driven by laser light absorption in the Yb3+ ions.

As a further example, in general, non-linear luminescent particles such as UCNPs can be used as a substitute for traditional fluorescence probes where a narrow bandwidth emission, auto- fluorescence background free emission (providing high dynamic range detection) is important. In these applications, use of a speckle-free beam profile plays critical role in the accurate quantification of UNCP luminescence, in particular at low power density where the UNCP luminescence exhibits strong non-linear behaviour. Such luminescent probe-based applications include, but are not limited to optogenetics, real time polymerase chain reaction (PCR), and flow cytometer.

Optogenetics : When optogenetics is performed on superficial tissue layers or on the transparent deep tissue layers, the use of the apparatus 100 with a speckle-free uniform beam profile provides uniform stimulation across the region of interest, and is a key enabler to excite non-linear particles such as UCNPs in quantifiable way. In particular, there is a limitation on the power levels for biological tissues to avoid tissue damage.

Deep tissue probing applications using non-linear luminescent particles as biomarkers:. Deep tissue imaging systems measure luminescence particles deep within the tissue. The number and concentration of particles can be quantified from the measured luminescence if their emission properties are well characterised. However, the intensity of light is greatly attenuated while propagating in highly scattering media such as human tissue. As such, the use of the apparatus 100 and methods of the invention to accurately characterise the QY of such particles over a wide dynamic power density range (covering that which the particles will experience at depth) will enable better understanding of luminescence signals from biomarkers present deep inside tissue and deep tissue images.

Real time PCR monitoring. State of the art real time PCR apparatuses use fluorescence probes to monitor the amplification of DNA by the polymerase process. The fluorescence probes can be substituted with UCNPs. UCNPs exhibit narrower emission than fluorescence probes and also no background emission in the detection band. The reduced background signal enables better identification of the cycle quantification (Cq) value which is critical to assess amount of target nuclei acid present in the sample. Meanwhile, the narrower emission width of UCNPs means that multiple emission lines can be distinguished and detected simultaneously (multiplexed). For example, different UCNPs (that emit at different wavelengths) can be used to bind to different DNA strands, allowing for real time monitoring of multiple genes in the sample. In this case the assessment of DNA amplification requires the determination of UCNP concentration bound to DNA, which can be derived from the luminescence with improved accuracy using the apparatus 100 with a speckle-free uniform beam profile. The apparatus 100 can be also used to more accurately determine the Cq value.

Flow cytometry. In a flow cytometer, UCNPs can be used as substitute for traditional fluorophores. As above described above, the narrow bandwidth of emission lines and low background emission can be used for multiplexing multiple signatures of cells simultaneously. The use of the apparatus 100 with a speckle-free uniform beam profile provides more accurate quantification of UCNP bound to the target cells.

It will be appreciated that different detection geometries may be required for different measurements, applications and/or samples. Figure 16 shows another example arrangement of the apparatus 100 where light emitted from the sample is detected in reflection geometry. This may be suitable for application where the sample comprises substantially solid scattering media, e.g. biological tissue. Here, a dichroic mirror BS-d reflects and directs the pump beam 110 (at the first centre wavelength) toward the detection region 65 of the sample. Pump-induced light emitted from UCNPs in the sample (at a shorter wavelength) along the detection optical path 102 is collected by lens L5 and transmitted through the dichroic mirror BS-d onto the detector 70E via a band pass filter BP1 and polariser P2 as described previously. Pump light reflected from the sample is filtered by the dichroic mirror BS-d.

Figure 17 shows another example arrangement of the apparatus 100 where light emitted from the sample is detected in transmission geometry. For example, this arrangement may be suitable for PCR monitoring and/or flow cytometry. Here a beam splitter BS reflects and directs a portion of the direct the pump beam 110 (at the first centre wavelength) toward the detection region 65 of the sample, and pump-induced light emitted from UCNPs in the sample (at a shorter wavelength) along the detection optical path 102 in transmission geometry is collected by lens L5 and focused onto the detector 70E by lens L6 via a band pass filter BP1 and polariser P2 as described previously. The apparatus 100 of figures 16 and 17 may include any of the features described above with reference to figures 2-5 and 13.

In addition, the apparatus 100 can be used to perform dynamic light scattering (DLS) measurements to derive properties such as the particle size and distribution of the sample. In this case, the optical wavelength filter(s) BP1 in the first detection optical path 102 is removed to allow scattered pump light to reach the detector 70E (fast APD). The DLS measurement principle is well known. Brownian (random direction) motion of dispersed particles in the sample causes the pump light to scatter in all directions to a varying extent depending on the particle size and temperature. The scattered light thus varies over time and the autocorrelation function of the detector signal recorded over a period of time contains information on the particle size distribution in the sample which can be determined through the Stokes-Einstein equation using known methods. As such, the apparatus 100 may readily be configured to perform a DLS measurement based on the detector output.

From reading the present disclosure, other variations and modifications will be apparent to the skilled person. Such variations and modifications may involve equivalent and other features which are already known in the art, and which may be used instead of, or in addition to, features already described herein.

Although the appended claims are directed to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel feature or any novel combination of features disclosed herein either explicitly or implicitly or any generalisation thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention.

Features which are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination.

For the sake of completeness it is also stated that the term "comprising" does not exclude other elements or steps, the term "a" or "an" does not exclude a plurality, and any reference signs in the claims shall not be construed as limiting the scope of the claims.

Claims

1. A method of optical characterisation of a sample, comprising: illuminating a sample with a pump beam having a first centre wavelength, and a first beam width, a first excitation power density and a substantially speckle-free beam profile at a detection region within the sample; and detecting light emitted from the detection region of the sample produced by the interaction of the pump beam with the sample.

2. The method of claim 1, further comprising shaping the pump beam to have the substantially speckle- free beam profile and/or a substantially smooth continuous intensity distribution at the detection region.

3. The method of claim 2, wherein shaping the pump beam comprises: passing the pump beam through a single mode optical fiber; using a single mode laser as a light source producing the pump beam; and/or spatially filtering the pump beam.

4. The method of any preceding claim, wherein the pump beam further has a substantially uniform intensity distribution at the detection region; and/or the method further comprises: shaping the pump beam to have a substantially uniform intensity distribution at the detection region using one or more lenses in an illumination optical path between a light source producing the pump beam and the sample.

5. The method of any preceding claim, further comprising illuminating the sample with a pump beam having the first centre wavelength, and a second beam width, a second excitation power density and a substantially speckle-free beam profile at the detection region.

6. The method of claim 5, wherein the pump beam having the respective first and second beam width is produced by the same light source, and the step of illuminating the sample with a pump beam having the first centre wavelength, and a second beam width, a second excitation power density and a substantially speckle-free beam profile at the detection region comprises: adjusting the pump beam to have the second beam width and second excitation power density at the detection region.

7. The method of claim 6, wherein adjusting the pump beam to have the second beam width at the detection region comprises adjusting and/or changing a lens arrangement in an illumination optical path between a light source producing the pump beam and the sample; and/or optionally or preferably, wherein the lens arrangement comprises one or more lenses configured to provide a collimated beam of adjustable width; and adjusting and/or changing the lens arrangement comprises: moving at least one of the one or more lenses into or out of the illumination optical path; and/or directing the pump beam so as to pass through or bypass at least one of the one or more lenses.

8. The method of any of claims 5 to 7, further comprising shaping the pump beam having the second beam width at the detection region to have the substantially speckle-free beam and/or a substantially smooth continuous intensity distribution at the detection region.

9. The method of claim 8, wherein shaping the pump beam comprises: passing the pump beam through a single mode optical fiber; using a single mode laser as a light source producing the pump beam; and/or spatially filtering the pump beam.

10. The method of any of claims 5 to 9, wherein the pump beam having the second beam width at the detection region further has a uniform intensity distribution at the detection region; and/or the method further comprises: shaping the pump beam having the second beam width at the detection region to have a substantially uniform intensity distribution at the detection region using one or more lenses in an illumination optical path between a light source producing the respective pump beam and the sample.

11. The method of any preceding claim, further comprising: varying the excitation power density of the pump beam having the first beam width at the detection region, and illuminating the sample with said pump beam at a plurality of excitation power densities; and detecting, at a plurality of excitation power densities, light emitted from the detection region of the sample produced by the interaction of the pump beam with the sample.

12. The method of any of claims 5 to 11, further comprising; varying the excitation power density of the pump beam having the second beam width at the detection region, and illuminating the sample with said pump beam at a plurality of excitation power densities; and detecting, at a plurality of excitation power densities, light emitted from the detection region of the sample produced by the interaction of the pump beam with the sample.

13. The method of claim 11 or 12, wherein varying the excitation power density of the respective pump beam comprises: adjusting an optical power output of a light source producing the respective pump beam; and/or attenuating the respective pump beam.

14. The method of any preceding claim, further comprising: measuring an emission spectrum of the detected pump-induced emitted light; and compensating the detected pump-induced emitted light for a limited transmission bandwidth of one or more optical elements in a detection optical path based on the measured emission spectrum.

15. The method of any preceding claim, further comprising deriving one or more optical properties of particles within the sample based, at least in part, on the detected pump-induced emitted light.

16. The method of any preceding claim, further comprising deriving a quantum yield of particles within the sample based, at least in part, on the detected pump-induced emitted light by performing quantum yield analysis.

17. The method of claim 16, wherein the method further comprises: illuminating the sample with a broadband light beam having a broad spectral content; and detecting the light transmitted through the sample to derive an absorption spectrum of the sample, and wherein the quantum yield is derived based on the detected pump-induced emitted light and the derived absorption spectrum, optionally or preferably, the component of the derived absorption spectrum at the centre wavelength of the pump beam.

18. The method of claim 17, wherein the quantum yield analysis comprises compensating for or substantially removing scattering contributions in/from the derived absorption spectrum.

19. The method of any of claims 16 to 18, further comprising imaging an intensity profile of the pump beam to determine the excitation power density and/or beam profile or intensity distribution at the detection region; and, optionally or preferably, wherein the quantum yield analysis comprises compensating for a non-uniform intensity distribution of the respective pump beam at the detection region based on the measured or imaged beam profile and a model describing a power density dependence of quantum yield, optionally or preferably, wherein the model is derived from a rate equation describing the emission process in the sample.

20. The method of any of claims 16 to 19, further comprising: replacing the sample with a reference sample having a predetermined quantum yield characteristic; repeating the method steps of any of claims 16 to 19 to derive, at each beam width and excitation power density, a quantum yield of particles within the reference sample; and calibrating the derived quantum yield of particles within the sample using the derived quantum yield of particles in the reference sample and the predetermined quantum yield characteristics of the reference sample; and optionally or preferably, wherein the reference sample is illuminated using a pump beam having a different centre wavelength.

21. A particle characterisation apparatus, comprising: a first light source configured to produce a first pump beam having a first centre wavelength and a substantially speckle-free beam profile for illuminating, along a first illumination optical path, a sample to thereby produce emitted light by the interaction of the first pump beam with the sample; a first detector for detecting pump-induced emitted light from a detection region within the sample along a first detection optical path; and a first optical filter element in the first detection optical path for attenuating light at the first centre wavelength.

22. The apparatus of claim 21, wherein the first light source comprises a single mode fiber coupled to the output of a laser, and/or wherein the first light source comprises a single mode laser, so as to produce a first pump beam with a substantially speckle-free beam profile and/or a substantially smooth continuous intensity distribution at the detection region.

23. The apparatus of claim 21 or 22, further comprising a first beam shaping arrangement in the illumination optical path configured to transform the first pump beam to have a substantially uniform spatial intensity distribution at the detection region; and optionally or preferably, wherein the first beam shaping arrangement comprises one or more lenses, and optionally a diffraction element, to transform the beam profile using truncation, refraction and/or diffraction.

24. The apparatus of any of claims 21 to 23, comprising a first lens arrangement in the first illumination optical path configured to adjust the first pump beam to have a first beam width or a second beam width at the detection region.

25. The apparatus of claim 24, wherein the first lens arrangement comprises one or more lenses configured to provide a collimated beam of adjustable width; and, optionally or preferably: wherein at least one of the one or more lenses is moveable and/or removable to provide the collimated beam of adjustable width; and/or wherein the first lens arrangement comprises a plurality of mirrors arranged to selectively direct the first pump beam so as to pass through or bypass at least one of the one or more lenses to provide the collimated beam of adjustable width.

26. The apparatus of claim 25, wherein the first lens arrangement comprises a focusing lens and collimating lens, and wherein: the focussing lens and the collimating lens are moveable into and out of the first illumination optical path to provide the collimated beam of adjustable width; and/or the first lens arrangement comprises a plurality of mirrors arranged to selectively direct the first pump beam so as to pass through or bypass the focussing lens and the collimating lens to provide the collimated beam of adjustable width.

27. The apparatus of any of claims 21 to 26, further comprising a means for varying the excitation power of the pump beam at the detection region; and, optionally or preferably, wherein the means for varying the excitation power of the first pump beam at the detection regions comprises: an adjustable power controller of the first light source; and/or one or more optical filter elements in the first illumination optical path.

28. The apparatus of claim 27 when dependent from any of claims 24 to 26, wherein the means for varying the excitation power density of the first pump beam at the detection region and the first lens arrangement are configured to provide a dynamic range of excitation power densities at the detection region of up to 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸.

29. The apparatus of any of claims 21 to 28, further comprising a beam profiler in the first illumination optical path for measuring a two-dimensional spatial intensity distribution of the first pump beam at a location equivalent to the detection region; and, optionally or preferably, wherein the beam profiler comprises a beam-splitter that directs a percentage of the first pump beam towards a two-dimensional imaging detector.

30. The apparatus of any of claims 21 to 29, further comprising a second light source for producing a second pump beam having a second centre wavelength and a substantially speckle-free beam profile for illuminating, along a second illumination optical path, a reference sample to thereby produce emitted light by the interaction of the second pump beam with the reference sample; and, optionally or preferably, wherein the second illumination optical path joins the first illumination optical path.

31. The apparatus of claim 30, wherein the apparatus further comprises: a second lens arrangement in the second illumination optical path substantially identical to the first lens arrangement for adjusting the second pump beam to have a first beam width or a second beam width at the detection region; and optionally or preferably, wherein the second light source comprises a single mode fiber coupled to the output of a laser, and/or wherein the second light source is a single mode laser, so as to produce a second pump beam with a substantially speckle-free beam profile and/or a substantially smooth continuous intensity distribution at the detection region.

32. The apparatus of any of claims 21 to 31, wherein the first light source comprises a laser, and the apparatus further comprises a broadband light source for producing a broadband light beam having a broad spectral content for illuminating, along a third illumination optical path, the sample for measuring an absorption spectrum of the sample; and optionally or preferably, wherein: the third illumination optical path joins the first illumination optical path; the first detector is configurable to measure the absorption spectrum; and/or the apparatus comprises a second detector for detecting light transmitted through the sample for measuring the absorption spectrum.

33. The apparatus of any of claims 21 to 32, wherein the apparatus comprises an integrating sphere for holding the sample, the integrating sphere comprising an excitation port for receiving the first pump beam and one or more detection ports for coupling to the first detector.

34. The apparatus of any of claims 21 to 32, wherein the apparatus is operable to perform a quantum yield measurement of particles within the sample based, at least in part, on an output of the first detector.

35. The apparatus of claims 34, further comprising a processing device for deriving, at each excitation power density and beam width, a quantum yield measurement of particles within the sample based, at least in part, on an output from the first detector

36. The apparatus of any of claims 21 to 35, wherein the apparatus is operable to perform a dynamic light scattering measurement of particles within the sample based on an output of the first detector.

37. The apparatus of any of claims 21 to 36, wherein the apparatus is operable to perform flow cytometry measurements of particles within the sample based on an output of the first detector.

38. The apparatus of any of claims 21 to 37, wherein the apparatus is operable for use in monitoring a polymerase chain reaction in the sample based on an output of the first detector.

39. The apparatus of any of claims 21 to 38, wherein the apparatus is operable for detecting light emitted from particles within a liquid or solid sample.