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Abstract
Non-linear materials such as upconverting nanoparticles (UCNPs) are emerging technology with fast-growing applications in various fields. The power density dependence of the emission quantum yield (QY) of these non-linear materials makes them challenging to characterize using currently available commercial QY systems. We propose a multimodal system to measure QY over a wide dynamic range (1:104), which takes into account and compensates for various distorting parameters (scattering, beam profile, inner filter effect and bandwidth of emission lines). For this, a beam shaping approach enabling speckle free beam profiles of two different sizes (530 µm or 106 µm) was employed. This provides low noise high-resolution QY curves. In particular, at low power densities, a signal-to-noise ratio of >50 was found. A Tm-based core-shell UCNP with excitation at 976 nm and emission at 804 nm was investigated with the system.
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1. Introduction
The use of upconverting nanoparticles (UCNPs) has developed rapidly and today spans a wide range of research fields including, among others, molecular interactions in cell biology, specific staining of tissue histopathology slides, in vivo luminescence imaging, and photodynamic therapy [1–8]. Upconversion (UC) capable nanomaterials thus have demonstrated remarkable optical features, which have facilitated the generation of high impact findings in various fields of research in biomedicine. Each of these avenues of research stems from the discovery of the UC process by Bloembergen [9] and an early series of studies conducted by Auzel [10–12]. UC is the unique feature of certain materials to generate a high energy photon as a consequence of the sequential absorption of multiple low energy photons.
Due to the varying requirements in different biomedical applications, both material design and optimization of these particles are playing key roles in framing further research. To date, numerous properties of UCNPs have been investigated, including crystal composition and structure, surface functionality, and biocompatibility [13–18]. Recently, one of the important areas of UCNP-related research has focused on understanding the non-linear behaviour of UCNPs and their luminescence efficiency, in particularly for superresolution microscopy [19]. The low quantum yield (QY) nature of UCNPs has been a limiting factor in the extensive adoption of UCNPs in many other biomedical applications. The QY is typically in the range of a few percent at high excitation power densities and decreases dramatically to a fraction of a percent in the low power density regime [20]. The power dependence of the QY is critically important, especially for deep tissue applications - where the excitation light is heavily attenuated and the fluence rate varies spatially. Various factors influence the QY of UCNPs, including crystal properties (e.g. host matrix, size, phase, and dopant ratio), surface properties, and dispersion medium [21–26]. Hence, extensive research aimed at improving the QY of UCNPs to obtain a stronger signal is well motivated. Many different approaches have been applied to increase QY, including but not limited to optimization of particle composition [27], engineering of the excitation source [28–30], and implementation of core-shell structures [31,32].
The efforts in improving the luminescence efficiency of UCNPs make standardized QY measurements essential. More recently, the importance of the excitation beam profile in such measurements was reported [33–36]. Traditionally, integrating sphere instruments are used to measure the absolute QY, as these can collect emission in all directions. The alternative would be to use a detector measuring at a limited solid angle. This can then yield the QY by referring to measurements of a dye with a known QY in the same measurement geometry, assuming that the spatial distribution of the emission from the two samples are identical. The advantage of the latter relative QY technique is that the power density across the beam profile can be controlled to a greater degree, which is of significant importance as the QY depends on the power density of the excitation light. The efficient reflections within an integrating sphere, together with a complication in measuring the actual beam profile in an integrating sphere, make it difficult to control the exact beam profile of the excitation light in this type of setup. Also, the QY measurements using an integrating sphere provide a poor signal-to-noise ratio (SNR) owing to the low fraction of emitted light being detected, making it hard to measure at low power densities, which is critical for biomedical application.
Currently, there is no complete system that adequately characterizes the QY of UCNPs. Therefore, there is a high demand for a compact QY characterization system specifically designed to characterize non-linear materials. The ideal system should include:
- 1. a speckle free excitation beam profile for power density compensation,
- 2. a multi-variable characterisation namely absorption, scattering, emission spectra for compensation of their influence on QY curves,
- 3. a high SNR in low power density region to enable low noise, repeatable and reproducible characterisation in this important regime of operation,
- 4. a high-resolution step size of laser excitation power to resolve subtle features that would enhance the understanding of the QY curve,
- 5. a wide dynamic range of QY curves,
- 6. a broad spectral range of excitation and detection to allow for measurements of all emission lines of interest,
- 7. a systematic QY characterization protocol that would allow for direct comparisons between different materials that are developed by various research groups,
- 8. and a fully automated and well-controlled experimental setup with a subroutine to excite and acquire absorption and emission under the same experimental conditions across various power densities.
2. Material and methods
2.1 System setup
Figure 1 shows the schematic layout of the multi-modal QY system. The system consists of 6 arms designed to shape the beam (arms 1 and 2) and acquire the beam profile (arm 3) and characterize various optical parameters (absorption and scattering, emission spectra, and luminescence signal) of the UCNP sample (arms 4, 5, 6, respectively). Arm 1 (UCNP excitation arm) consists of temperature stabilized single-mode fibre-coupled diode laser (Thorlabs, BL976-PAG500) at 976 nm and an optical arrangement to achieve speckle free beam profile of adjustable size. Two sets of optics in arm 1 (L1 f = 30 mm; L2 f = 30 mm or nil; L3 f = 6 mm or nil; and L4 f = 200 mm) were used to achieve two different spot sizes: 530 µm or 106 µm in beam diameter (full width at half maximum-FWHM). The two spot sizes were chosen to achieve a high SNR in the low power density region and thereby a high dynamic range of power densities, while also having an optimal overlap of the two QY curves generated. A filter F1 (Thorlabs CM1-BP145B2) was employed to further increase the range of QY curves at low power densities for a given spot size. Lenses L1 and L3 on arm 1 were mounted on XY stages (Thorlabs CXY1) for precise alignment of the beam spot in the middle of the cuvette holder. A pair of irises (I1 and I2) were used to align the spot in the middle of the cuvette. A polarizer P1 (Thorlabs, LPNIRE200-B) was placed in between lenses L1 and L2 to vertically polarize the light impinging on the sample. A power meter (PM, Thorlabs, PM100D) was attached at the distal end of the cuvette holder to measure the power of the light passing through the sample. Arm 2 (reference dye excitation arm) is a replica of arm 1 with a single-mode fibre-coupled 785 nm laser (Thorlabs FPL785S-250 at 785 nm) used for excitation. The arm is enabled by flipping the flip mirror (FM2). The purpose of this arm is to acquire the luminescence signal from the dye whose emission line is similar to the emission of the UCNPs. The QY value of the dye (DY-781-01, Dyomics) used was well known from the literature (12.4%) [34,41]. In this reference, the dye has been validated using a well established nature protocol publication method [42], which enables the relative QY assessment by calibrating the experimentally obtained QY values from the UCNPs.
Fig. 1. Optical layout of multimodal QY system. Arm 1 and 2 are identical excitation arms used to shape the excitation beams: 976 nm and 785 nm for the excitation of the UCNPs and reference dye, respectively. Arm 3 collects a fraction of the laser light for beam profile measurements. Arm 4 includes a broadband white light to the main excitation path to perform scattering measurements, replacing the power meter, PM, with a spectrometer. Arm 5 is used for emission spectrum evaluation and arm 6 is used to collect the luminescence signal from UCNPs or Dye (once for calibration).
Arm 3 (beam profile arm) consists of a compact high-resolution CMOS camera (Thorlabs DCC3240M) and a set of neutral density filters to acquire the beam profile of the excitation light employed, identical to the beam profile in the cuvette. A pellicle beam splitter (BS, Thorlabs, CM1-BP108, 92:8) was used to split the rays and the camera was placed equidistant to the center of the cuvette holder thus acquiring a beam profile resembling the midpoint of the sample cuvette. Arm 4 (scattering spectra arm) provides a broadband light source (Ocean Optics HL 2000) to enable a wide range of absorbance spectra to estimate the scattering contribution of the sample. This arm is enabled by flip mirrors (FM1 and FM2) which guide the white light to the sample in the sample holder. For this measurement, the power meter (PM) was replaced with a fiber bundle (Thorlabs BFL200HS02) which was connected to the spectrometer (Ocean Optics QE Pro, 350-1100 nm). The intensity and spot size of this arm is controlled using an iris (I3). Arm 5 collects the luminesce light from the sample and sends it to the fiber-coupled (Thorlabs BFL200HS02) spectrometer (Ocean Optics QE Pro) to measure the emission spectra of UCNP samples. The arm consists of lenses (L9, L10 f = 30 mm), and a short pass filter SP1 (Thorlabs, FES0900) to remove excitation stray light at 976 nm. Arm 6 is the luminescence arm, which collects the luminescence light emitted by the sample using a set of lenses (L5, L6, L7, L8 = 30 mm) and focuses that light onto an avalanche photodiode (APD) detector (Thorlabs, APD410A). A slit (1 mm) is placed between L6 and L7 to limit the measured light to the emission generated in the center of the cuvette and also manage to avoid luminescence signals overfilling the APD. A polarizer P2 (Thorlabs, LPNIRE100-B) is kept at 54.7° with respect to the linear polarizer in excitation arms to remove distortion caused due to any anisotropic nature of the samples. A set of two filters, a bandpass BP (Thorlabs, FBH800-40) and a short pass SP2 (Thorlabs, FES0900) are placed in the path to filter excitation light and only allow the luminescence signal (804 nm) to pass and be focused on the APD active area. Similarly, bandpass BP (Thorlabs, FBH800-40) and long pass LP (Semrock - LP02-785RE-25) filters are adopted to avoid excitation light while measuring the reference dye signal. An XY stage (Thorlabs CXY1) was used to precisely center the APD detector and thereby enable accurate alignment. A data acquisition (DAQ) device (National Instruments, USB-6216) along with a preamplifier (Femto – DLPVA) was used to acquire the signal from the APD. All parameters varied in the system (e.g. laser current, power meter settings, beam profile, spectrometer settings) and the measurement routine are controlled and automated using a user-specified Python script interface.
2.2 Sample preparation
The samples used for this study were water-soluble NaYF4:Tm core-shell UCNP samples procured from Creative Diagnostics at a concentration of 10 mg/ml. These UCNPs were optimized during the synthesis for enhancement of the 804 nm emission transition. The reference dye used to calibrate the UCNPs signal was procured from Dyomics (Dy-781-01). The dye has been chosen to match the emission wavelength of the UCNPs (804 nm) and a QY of 12.4% (in ethanol) has been previously characterised [34] following standard protocols from certified laboratories for linear fluorescent materials [42]. The dye was diluted in ethanol solvent in a concentration that yielded luminescence 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. A volume of 1 ml was the minimum quantity needed to perform the measurement, and ml of each sample were placed in a quartz cuvette (Thorlabs, CV10Q3500FS).
2.3 Measurement protocol and data analysis
A systematic procedure was adopted for both the collection of data from the sample and the analysis algorithm to extract compensated QY values. The workflow for data collection and analysis is depicted in Fig. 2.
Fig. 2. Schematic flow chart depicting the measurement protocol and analysis algorithm. The left side shows the measurement protocol and the right side provides the outline of analysis methods.
2.3.1 Measurement protocol
At first, the UCNP and dye samples were prepared as mentioned in section 2.2. and sonicated for 15 minutes. The broadband (350-1100 nm) emission spectrum of the UCNPs was acquired using arm 5. This was followed by broadband transmission measurements of samples in the following order. Then the system was set up to create spot size S1 in both arms 1 and 2. The beam profile of S1 was captured using arm 3 with the beam profile camera, followed by luminescence signal measurements of UCNPs in arm 6 at various laser currents (976 nm, 0 - 800 mA, step size 12 mA). The same process was directly repeated thereafter for the dye sample at various laser currents (785 nm, 0 - 250 mA, step size 1.5 mA). The whole procedure of measuring the luminescence signal of UCNPs and dye at S1 was repeated with filter F1 & F1’ in the respective arms. Then, the whole sequence of measurements was repeated for beam profile S2. In total, eight luminescence measurement series were conducted in the following order: UCNP-S1, dye-S1, UCNP-S1-F1, dye-S1-F1’, UCNP-S2, dye-S2, UCNP-S2-F1, dye-S2-F1’. This completes the measurement sequence of the multimodal system. The outputs of the measurement sequence are the emission spectrum of UCNPs, in total five transmission data of UCNP and dye samples, two beam profiles of S1 and S2, and eight luminescence signals of UCNPs and dye combined.
2.3.2 Analysis methods
The analysis algorithm takes all the outputs of the measurements as inputs and calculates various optical parameters (wide dynamic range QY, absorption, scattering, the processed emission spectra, beam profile) and subsequently uses the calculated data to compensate for possible distortions in the estimated high dynamic range QY values of UCNPs. The steps are briefly described in this section, more detailed equations and calculation steps could be found in Supplement 1. The first step is to calculate the absorption and scattering spectra of UCNPs from the transmission measurement data. Beer Lamberts’ and Mie scattering laws were used for the estimation of absorbance and scattering spectra of the samples as described in Supplement 1-sec.1. The compensated absorption of UCNPs at 976 nm was used as input to the QY calculations. The acquired beam profiles of the 976 nm laser (S1 and S2) were employed to create i) image-based intensity matrix (see Supplement 1-sec.2), utilized to calculate the image compensated QY value of UCNP. The experimental QY (not compensated with dye QY) is obtained by taking the ratio of luminescence signal of UCNP to the absorbed power per unit length in the centre of the cuvette (see Supplement 1-sec.3). The relative QY is estimated by calibrating the UCNP experimental QY values with dye QY values (see Supplement 1-sec.3). The above process to find the relative QY was repeated for all spot sizes (S1, S2) and filter (nil, F1) configurations which provides in total four QY curves. The relative QY curves were stitched without any fitting parameters to provide a high dynamic range relative QY curve. The relative QY curves were compensated for the beam profile to avoid underestimating the QY values. This compensation was implemented by solving the QY for each pixel in the beam profile using a 2-level rate equation for the 804 nm line [34]. In this way the QY is obtained as a function of excitation power density. The output of the compensation method is balancing power density (ρb) and balancing QY (фb) for the overall high dynamic range image compensated QY curve. The ${\rho _b}$ determines the transition point from the linear regime to the non-linear regime of the UCNPs emission. At this point the QY is exactly ${\phi _b}$, which is one-half of the QY saturation value reached at high power densities [43]. The details of the rate equation and compensation methods are discussed in Supplement 1-sec.4.
3. Results and discussion
3.1 Temporal and spatial beam profile characterization
One of the key features of the system is the ability to obtain a speckle free uniform beam profile for a given spot size. This is achieved by using a single-mode fiber followed by an optical beam shaping lens set up to obtain spot size while preserving the speckle free beam profile. It is critical to have the power density (light intensity) across the beam profile as uniform and stable as possible. Such a profile will lead to fewer compensations and thereby accurate estimations of relative QY values of non-linear material. Figure 3(a-b) shows the beam profile acquired by the camera equidistant to the midpoint of the sample cuvette. The smooth speckle-free profile enables the algorithm to easily compensate for the beam-profile-based distortion to the evaluated QY curves. However, it is also important to ensure that the laser is temporally stable over the entire measurement period. To test this, the power meter at the distal end of the sample holder was temporarily replaced by a single-mode fiber (6 µm core diameter) with the other end of the fiber connected to an APD detector. The small fraction of light collected by the APD detector was acquired for 60 sec by using a DAQ card at a collection rate of 1 Mega sample/sec. Figure 3(c) shows the raw data acquired by the APD detector. The variation in the amplitude of the laser during one minute is less than 0.5%. To understand the key variation frequencies we performed a Fast Fourier transform (FFT) of the acquired signal. The results are shown in Fig. 3(d) where 1/f noise is evident and signal at 50 Hz is observed which might be corresponding to the power supply frequency (50 Hz). The generation of such a speckle free beam profile to avoid beam profile speckle related distortion, along with shaping beam width to increase SNR at various power density ranges were not found in the QY system published in the literature to date [34]. This novel design proposed in this work can achieve high dynamic range, speckle distortion-free QY values have the potential to bring insights to various works explored in literature, in particular, standardization efforts of UCNP emission across the community [35].
Fig. 3. Beam profile characterization a) Beam profile image b) Beam profile in 3D plot c) Raw temporal signal from one pixel of the beam profile shows no temporal fluctuations in laser intensity d) Fourier transform of the temporal signal show low frequency 1/f noise and harmonics of power suppy.
3.2 Multi-modal measurements and compensation
The accuracy of the estimated QY values of UCNPs depends on the accurate evaluation of various optical properties of UCNPs and optimal compensation for all contributing factors. It is ideal to measure all parameters of UCNPs simultaneously using a well-calibrated system to avoid possible changes in sample properties over time. The developed measurement protocol (section 2.3) allows for the efficient collection of data while minimizing delay, thereby maximizing accuracy when compensating for various contributing factors.
3.2.1 Absorption and scattering compensation
The UCNP absorbance spectrum measured by arm 5 contains both the absorption and scattering contribution. The measured transmission spectrum was fit to the wavelength dependence of Mie scattering. Figure 4(a) (red, black line) shows the Mie-scattering fit to the transmitted spectrum. The difference between the fitted scattering and the measured transmission spectra yields the actual absorption of the UCNP sample. To understand the importance of simultaneous measurement of scattering, we have performed extensive measurements on the sample by changing variables like sonication and repeated intra-day measurements. Under various conditions, the absorption of our sample is estimated to be around 10% percent of the sample’s attenuation. In the absence of scattering compensation, the UCNP QY would be underestimated by 90% at low power densities. The uncompensated QY curves were described in Supplement 1-sec.5. The literature work on the QY system has considered to some extent the effect of scattering compensation. However, this compensation was limited to non-simultaneous measurements. In this work, we quantify the error of uncompensated scattering under various experimental conditions (see Supplement 1-sec.5). The errors for these tests on QY were found to be fluctuating by 100% under certain conditions, these results emphasise the necessity to perform simultaneous scattering measurements using a multimodal system. The sample considered in this work is highly turbid, containing aggregated UCNPs or residuals from the synthesis, resulting in high scattering compensation values. For low turbid or transparent samples, we expect the compensation values to be lower. This compensation is in particular useful to assess QY during various synthesis steps before the final washing process [44]. Figure 4(b), shows an example curve of the change in luminescence signal for the absorbed power per unit length by the UCNPs for a given spot size S2. The UCNPs emission line (804 nm) explored in this paper follows two-photon process, the emission is known to be proportional to ρn where n is the number of photons involved in the upconversion process [34], the red line Fig. 4(b) shows the fitted curve.
Fig. 4. Multimodal features of QY system a) Absorbance (green dots) and fitted scattering (red line indicating fitting range) spectra b) Raw Luminescence signal (black dots), second-order polynomial fitted curve (red line) of UCNP sample c) Emission spectrum of UCNP d) Beam profile of 976 nm laser.
3.2.2 Reference dye calibration, inner filter effect and emission filter compensation
A calibration reference dye was used to convert the experimentally measured QY values to corrected relative QY values. To make this accurate and independent of dye concentration, the inner filter effect has been accounted as described in (see Supplement 1-sec.1). In addition, the spatial and spectral distribution of the luminescence emission needs to be the same for both samples. Both samples should emit isotropically, so the spatial assumption is believed to be fulfilled. In addition, the detection filters have to be compensated for, as the emission of the dye, in particular, is broader than the optical detection filter. For both samples, the percentage of the incident light was corrected by calculating the fraction of the emission profile being transmitted through the filters. It is also important to use a dye with a similar emission profile to the UCNPs as possible so that this correction factor can be minimized (yielding as small an error as possible). The emission spectrum of the UCNPs (acquired at 1.8 W/cm2), shown in Fig. 4(c), is processed with filters transmission spectra to account for errors related to the limited spectral bandwidth of the emission filters. This correction procedure is described in detail in Supplement 1-sec.3.
3.2.3 Beam profile compensation
The luminescence signal (e.g. Figure 4(b)) at all spot sizes provide raw data for QY values calculations (see Supplement 1-sec.3). The shape of the beam profile and characteristics of the laser affects the estimated QY values. The absence of speckle and a smooth close to Gaussian beam profile is evident from Fig. 4(d). Importantly, any presence of speckles results in a random distribution of a light speckle pattern with different power densities across the beam profile. This makes it difficult to accomplish accurate compensation for the beam profile. The system in this work is effectively free of both temporal and spatial speckles. The beam profile compensation algorithm (see Supplement 1-sec.4) can thereby accurately compensate for the different power densities across the beam profile. In the absence of compensation, the beam profile related error can be as much as 50% at low power density values. A similar approach for beam profile compensation can be found in literature and it was limited to single spot size low dynamic range measurements [34]. In this work, we have extended the compensation to multi-spot size which enabled accurate estimation of QY values over a high dynamic range of power densities.
3.2.4 High dynamic range and SNR of QY curves
Figures 5(a-d) show the relative QY values estimated for different spot sizes and filter combinations. Unlike dyes, the QY of UCNPs is dependent on the excitation power density. Therefore, determining the QY of UCNPs for a broad range of power densities is of great value. For example, Fig. 5 (a-b) have valuable inputs for in vivo studies of deep tissue, as it needs QY values for low power densities limited by light attenuation in tissue and safe human exposure levels whereas the microscope studies need to understand QY values at high power density. Depending on the microscope objective and sample, Fig. 5(c-d) provides insights into the emitted light from the sample. The system described in this paper has been designed to perform QY measurements over a broad range of power densities (1:104). This is achieved by using two different spot sizes (S1, S2) in combination with/without the use of an ND filter. In total 4 measurement series were conducted for each sample to extract QY values of UCNPs at a range of power densities and the results were stitched without any fitting parameter to obtain high dynamic range QY curves. Figure 5(e) shows the stitched relative QY curve along with the image beam profile compensated QY curve. The balance point of the image compensated curve is estimated to be 38.7 W cm-2 balancing power density (ρb) and 1.9% balancing QY (фb). The dynamic range can be further extended by adding new beam width to the setup by proper choice of lenses that can cover expand higher or lower dynamic range boundary.
Fig. 5. High dynamic range QY curve of UCNP a) Spot size S2 with filter F1, b) Spot size S2 without filter F1 c) Spot size S1 with filter F1 d) Spot size S1 without filter F1 e) Stitched relative (discrete markers), image compensated (red line).
The novel beam shaping methods in this work has addressed some challenges found in the literature. This includes a high dynamic range, high SNR, speckle. The QY systems in the literature reported around 2-3 orders of dynamic range [34,35]. In addition, all of them suffered from high noise, leading to large error bars in measured data, in particular, at low power density region, which is critical for light-sensitive applications like deep tissue imaging for biomedical applications. The proposed system effectively overcomes this challenge by employing a beam profile of large width (530 µm) to excite and collect a large number of photons at low power density. This effectively increases the signal level (25 times in area) of UCNP emitted light as compared to the small beam width (106 µm). The SNR of the system at the low power density region was found to be SNR (>50). To the best of our knowledge, this is the first demonstration of a high dynamic range QY with superior SNR at low power densities.
4. Conclusion and future outlook
In this work, we have presented for the first time high resolution and high SNR QY curves of UCNPs by carefully compensating for various distorting effects (scattering, beam profile, inner filter effect, bandwidth of filters) which influence the accuracy of estimated QY values. The study revealed that obtained QYs of UCNPs could vary by 90% in the absence of the multi-modal corrections. A systematic and standardized measurement and analysis protocol is proposed and tested on the commercially available core-shell UCNPs sample. The key results obtained in this work point to the importance of multi-modal measurements and compensation to achieve accurate and consistent QY values for UCNPs. With the increasing innovation rate in the field of UCNPs, we believe our work can act as a precursor for standardizing the measurements of QY values.
This work considers only the 804 nm emission line of the NaYF4:Tm UCNPs. An extension to other emission lines could be achieved easily, by changing emission filters in the system and by selecting a proper dye matching the emission wavelength of UCNPs. The UCNP laser remains the same, while the excitation laser of the reference dye needs to be chosen based on the measurement protocol used for the tabulated QY of the selected dye. Importantly, the rate equations developed for the 804 nm emission line will no longer be sufficient for processes involving 3, 4 or 5 photons and therefore will need to be extended to account for higher order transitions. Hence, the image-based beam compensation algorithms used in this paper no longer be valid for those transitions. Therefore there is an obvious demand for building compensation methods for higher order transitions. In the current configuration of the system, the sample and solvent are measured in sequence. Changes in the intensity of the light source may be present, leading to errors in the assessed optical properties and the obtained QY. This could be avoided by adding an extra arm with a cuvette holder for the blank sample containing solvent only. This would permit the transmittance spectral measurements to be performed simultaneously, thus avoiding possible errors. Also, a careful look into state-of-the-art detection technology might make it possible to further increase the dynamic range of the measured QY curves. To thoroughly validate the system we were using two different arms for UCNP laser 976 nm and dye reference laser 785 nm. However, the purpose of the dye arm is purely related to the calibration of the QY of UCNPs. This calibration factor will remain constant, provided, the system is robustly built and well tested under different experimental conditions. Therefore, the need for a reference arm could be eliminated once the calibration factor at each emission line is robustly estimated. Another possibility is to make transparent long-lasting solid phantoms with UCNP which can be used as a calibration tool [45,46]. Another important parameter influencing the optical properties of UCNPs and solvents is temperature. The future system could potentially include temperature stabilization to avoid errors when performing measurements in different laboratories under various conditions
Funding
Science Foundation Ireland (SFI/15/RP/2828).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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