Method for determining quantum yield from a sample by using combined interferometric scattering and fluorescence microscopy

Abstract

The invention relates to a method for determining the quantum yield from a sample (22) by using a combined interferometric scattering and fluorescence microscope (10), comprising the following steps: a) calibrating each of the illumination and detection channels (16, 18) for scattering measurements, the excitation unit (32) and the detection channel (34) for fluorescence measurements; b) performing an interferometric scattering measurement on the sample (22), thereby obtaining an interferometric scattering signal from the sample (22); c) illuminating the sample or applying an electrical bias to the sample, thereby obtaining a fluorescence signal from the sample (22); and d) calculating the quantum yield of the sample (22) from the interferometric scattering and fluorescence signals. Furthermore, a microscope (10) is proposed which is suitable for performing the above-mentioned method.

Description

Technical Field

[0001] The present invention relates to a method for determining quantum yield from a sample using a combination of interference scattering and fluorescence microscopy. Background Technology

[0002] Quantum yield (QY), also referred to herein as quantum efficiency (QE), is a key parameter in a wide range of scientific and technological fields. For example, quantum yield measurements are performed in photonics, biosciences, and materials science, particularly in important growth areas such as nanotechnology involving quantum dots and nanoparticles, and photovoltaics. In short, quantum efficiency defines the efficiency with which a system can convert absorbed photons or electrons into usable energy (e.g., emitted photons or electrons in the case of luminescent materials). Therefore, this parameter is crucial for determining the properties of materials capable of absorbing and converting photons.

[0003] Measuring the quantum efficiency of macroscopic or bulk samples is a standard procedure in the prior art. For example, for quantum dots or nanoparticles, the quantum yield of a colloidal solution is routinely measured to obtain information about the quality of its luminescent properties. In photovoltaics, quantum efficiency is routinely measured and provides information about the conversion rate of sunlight to electricity. Moreover, in the field of light-emitting diodes (LEDs), determining quantum efficiency is a standard measure; quantum efficiency gives information about how much light is obtained as output for a given electrical input.

[0004] Current methods for measuring quantum efficiency require extensive sample preparation and / or only provide information on the average quantum efficiency of bulk samples. In particular, methods used to determine quantum efficiency cannot distinguish or characterize the quantum efficiency of smaller portions of the sample (e.g., individual single particles or specific regions within the sample).

[0005] However, quantum yield measurements at such smaller fractions become important for luminescent samples. Photoluminescence quantum yield (PLQY) provides information about the efficiency with which a luminescent material converts absorbed photons into emitted photons. PLQY values ​​are a detrimental parameter for colloidal nanoparticle samples such as perovskite quantum dots. Here, PLQY values ​​are strongly dependent on size, shape, ligand interactions, defect density, and material composition, as well as inhomogeneities at the individual particle level. Until now, known PLQY measurements in the prior art have failed to capture these characteristics because they can only measure bulk solutions of millions to billions of particles and give their average values.

[0006] To measure PLQY or general QY from a smaller portion of a sample, fluorescence measurements that determine the incident and emitted photons from the sample are combined with auxiliary methods that determine the size of the smaller portion of the sample being tested. For example, scanning electron microscopy is used in conjunction with photoluminescence microscopy to determine the PLQY of individual particles.

[0007] Such combined measurements are known in the prior art and have been published in several scientific journals. However, these methods are impractical because they typically require complex sample preparation, which ultimately alters the properties of the measured sample. Furthermore, these techniques do not allow for operational measurements or the acquisition of statistically relevant data from the measured sample, hindering their effective commercialization.

[0008] Therefore, it is not yet possible to routinely measure the quantum yield of a small fraction of a sample, such as the QE of a single particle or a small region within the sample, in a fast and convenient manner. Summary of the Invention

[0009] Therefore, the object of the present invention is to provide a method for solving the above-mentioned problems.

[0010] In a first aspect, the present invention relates to a method for determining the quantum yield of a sample using a combined interferometric scattering and fluorescence microscope. The fluorescence microscope includes an excitation unit for illuminating the sample or applying an electrical bias voltage to the sample, a detection channel for fluorescence measurement, and an illumination and detection channel for scattering measurement. The method includes the following steps:

[0011] a) Calibrate each of the irradiation and detection channels used for scattering measurements, and the excitation and detection channels used for fluorescence measurements;

[0012] b) Performing an interference scattering measurement on the sample to obtain an interference scattering signal from the sample; c) Irradiating the sample or applying an electrical bias voltage to the sample to obtain a fluorescence signal from the sample; and

[0013] d) Calculate the quantum yield of the sample based on the interference scattering and fluorescence signals.

[0014] The basic idea of ​​this invention is to first perform a calibration step, which calibrates the irradiation and detection channels used for scattering measurements, the excitation unit used for fluorescence measurements, and the detection channel, so that the calibrated scattering and fluorescence signals obtained from the aforementioned channels in steps b) and c) can be directly used to calculate QY in step d). Here, the inventors surprisingly discovered that interferometric scattering microscopy (hereinafter referred to as iSCAT) can be combined with fluorescence microscopy to determine the QY of a sample.

[0015] In principle, interferometric scattering microscopy is a technique that utilizes the interference between scattered light from a sample and directly reflected light from nearby interfaces to provide high-contrast, label-free images. In particular, iSCAT allows for real-time observation and can be operated under ambient conditions. iSCAT's primary focus is on analyzing single-particle tracking down to the molecular level.

[0016] The conceptual principles of iSCAT have been published in several scientific journals, such as “Detection and spectroscopy of gold nanoparticles using supercontinuum white light confocal microscopy”, Biochemistry, 2004, Vol. 93, No. 3, and “Direct optical sensing of single, unlabeled proteins and superresolution imaging of their binding sites”, Nature Communications, 5, 4495 (2014). However, it has not yet been possible to use iSCAT to measure the quantum yield of single particles.

[0017] In the context of this invention, iSCAT offers the advantage of being able to study samples in their natural state without altering their properties. Furthermore, since the combined interference scattering and fluorescence microscope has already been calibrated due to step a), subsequent measurements can be performed without using auxiliary measurement techniques for each individual measurement. In other words, once the microscope has been calibrated for the type of sample to be measured, subsequent measurement and calculation steps can be applied directly. Therefore, the method according to the invention eliminates the need for complex sample preparation and overcomes the problem that prior art methods cannot capture the heterogeneity between different regions and / or particles of the sample.

[0018] Fluorescence signals can be obtained by illuminating the sample with light of suitable intensity and wavelength to excite the sample, i.e., generating a fluorescence signal, or by applying an electrical bias voltage to the sample. The second case is also called electroluminescence. Therefore, the excitation unit may include a power supply suitable for applying an electrical bias voltage, or an irradiation source suitable for illuminating the sample with light. In the case of illuminating the sample, photoluminescence quantum yield (PLQY) is obtained, while in the case of applying an electrical bias voltage, electroluminescence quantum yield (ELQY) is obtained in step d).

[0019] Preferably, the excitation unit includes an irradiation source suitable for irradiating the sample.

[0020] Preferably, the measured scattering signal is a contrast value, which is defined by the following equation:

[0021]

[0022] Where C is the contrast as a percentage, I(b) is the intensity of the background, and I(s) is the intensity of the sample.

[0023] In the context of this invention, the QY obtained from step d) does not reflect the QY of the larger (i.e., macroscopic or bulk portion) of the sample. Specifically, the method described above allows for the measurement of the QY of portions with dimensions of 8-64,000 nm³, preferably 125-8000 nm³. Therefore, calculating the QY of such larger sample portions is excluded from this invention.

[0024] According to a first embodiment of the present invention, step a) further includes the step of determining the conversion relationship between size-related parameters and the measured interference scattering signal.

[0025] By doing so, the measured scattering signal from the sample can be set to be related to the sample's physical dimensions (size-dependent parameters), i.e., converted into the sample's physical dimensions (size-dependent parameters). In this respect, the term "conversion relation" refers to a module, table, or mathematical equation that converts the measured interference scattering signal into the sample's size-dependent parameters. The use of conversion relations allows for the rapid and simple calibration of the detection and illumination channels used for scattering measurements. Therefore, determining the conversion relation "calibrates" the illumination and detection channels used for scattering measurements because once the conversion relation is established for the sample, the scattering signal measured from that sample is automatically calibrated and can therefore be directly converted into physical dimensions.

[0026] According to another embodiment of the invention, the conversion relationship is determined by a calibration curve obtained by measuring the scattering signal of a sample with known size-related parameters.

[0027] In other words, a calibration curve is obtained by measuring the scattering signal of a sample with known size-related parameters on a combined interference scattering and fluorescence microscope, which is then used for the actual measurement in step b). This allows a single microscope to perform calibration and subsequent measurements without the need for additional equipment and extensive sample preparation, e.g., handling samples between different microscopes and settings. For example, the calibration curve can be linear. It should be noted that the above steps only require samples of the same type, but not necessarily the exact same sample used in step b). For example, materials of the same category can be used. Another advantage of the above embodiment is that the sample properties do not change during the scattering measurement. In fact, the sample used for calibration can also be used for later measurements.

[0028] Alternatively, the conversion relationship can be determined from a simulation based on the sample's refractive index. The simulation requires no microscope and is therefore unaffected by environmental factors. Based on the sample's refractive index, the simulation allows for the rapid and simple acquisition of the desired conversion relationship. This embodiment is advantageous for valuable samples, which can be used solely for the measurements in step b) to avoid damage or contamination of the sample during step a).

[0029] The conversion relationship can also be obtained from a calibration curve, which is obtained by measuring the sample's size-related parameters using electron microscopy and by measuring the sample's scattering signal using interferometric scattering measurements. This method uses electron microscopy and interferometric scattering measurements to obtain a calibration curve from which the conversion relationship can be derived. Although the above method requires more sample preparation compared to the previous two alternatives, the combination of electron microscopy and interferometric scattering microscopy produces the most accurate conversion relationship because the results of the two techniques can be correlated.

[0030] Preferably, the conversion relationship is determined based on a calibration curve obtained by measuring the scattering signal of a sample with known size-related parameters.

[0031] According to another embodiment of the invention, the size-related parameter is selected from sample weight, sample volume, and sample density. Preferably, the size-related parameter is sample volume. For example, the sample volume can be provided in nm³.

[0032] According to another embodiment of the invention, between or during steps b) and d), the method further includes a step of using a transformation relation to transform the scattering signal from the detection channel used for scattering measurement to obtain a dataset of size-related parameters, in particular, the scattering signal being a contrast value.

[0033] In other words, the measured scattering signal obtained from step b) can be directly transformed using a transformation relation, such as the contrast value, to generate a dataset of size-related parameters. This dataset can then be used to calculate QY in step d). Therefore, the use of the transformation relation allows for easy and rapid transformation of the measured signal without requiring extensive reprocessing of the scattering signal.

[0034] In another embodiment of the invention, step a) further includes determining a correction factor that takes into account the loss of photons emitted by the sample in the detection channel used for fluorescence measurement.

[0035] The idea behind the above embodiments is to consider all photons emitted by the sample that do not reach the detector in the QY calculation of step d). In principle, there are several losses in the detection channel used for fluorescence measurements caused by correction factors, such as the objective's collection efficiency, transmission loss, and losses caused by beam splitters, lenses, and other filters. Furthermore, the camera's quantum efficiency can also be considered. While the losses of most components are provided by the manufacturer, the objective's collection efficiency must be determined. A preferred method for measuring the objective's collection efficiency is to measure the emission of the samples in question, both with and without oil immersion, under the same excitation conditions.

[0036] According to another embodiment, between steps b) and d), or during step d), the method further includes a step of converting the fluorescence signal from the detection channel used for fluorescence measurement using a correction factor to obtain a dataset of corrected fluorescence signals. Due to the use of the correction factor, the above method steps allow for the determination of a more accurate fluorescence signal. Therefore, the quantum yield can also be calculated more precisely.

[0037] In another embodiment of the invention, if the sample is irradiated in step a), the method further includes the step of determining the amount of incident photons on the sample in the irradiation channel for fluorescence measurement. Specifically, the amount of incident photons is obtained to measure the irradiation area and irradiation distribution in the absence of a sample at the sample location, taking into account a calibration factor that accounts for the laser power loss in the irradiation channel for fluorescence measurement.

[0038] The above-described method allows for precise determination of the amount of photons reaching the sample from the irradiation source. Losses typically occur in the irradiation path due to the optical devices used (such as beam splitters or lenses), stray light generation, and transmission losses caused by the objective lens.

[0039] These losses can be summarized into a single calibration factor, which allows for the correction of the amount of photons reaching the sample. The calibration factor can be obtained by measuring the initial laser power after the laser output before the optics and the reduced laser power reaching the sample. Preferably, the calibration factor is measured once before each measurement.

[0040] Determining the illumination distribution and the illumination area is crucial because they can be used to calculate the amount of incident photons on the detector. Typically, the detectors used are 2D detectors, such that the amount of photons incident on the sample is given in units of photons per pixel. The inventors have discovered that the illumination distribution, along with the illumination area, is not uniform. For example, the illumination distribution can follow a Gaussian distribution shape. In other words, this means that fewer photons strike the sample at the edges compared to the central portion. Therefore, by considering the illumination distribution together with the thus obtained illumination area, the amount of incident photons on the sample in the illumination channel used for fluorescence measurements can be obtained more accurately.

[0041] In another embodiment of the invention, in step d), the quantum yield of the sample is calculated by using a dataset of corrected fluorescence signals, a dataset of size-related parameters, and the amount of incident photons or current applied to the sample.

[0042] The combination of the above data and parameters results in the most accurate quantum yield, which can be obtained using combined interference scattering and fluorescence microscopy. Preferably, a dataset of size-related parameters is used to calculate the absorption cross-section of the sample. For example, the absorption cross-section ( This can be calculated using the following equation:

[0043]

[0044] in It is the light wave vector ( (is wavelength), and This is polarizability. For deep subwavelength (i.e., wavelengths below 1000 nm) particles in a homogeneous medium, the polarizability can be approximated as:

[0045]

[0046] Where V is the particle volume, and , These are the dielectric functions of the particles and the environment, respectively.

[0047] The obtained absorption cross section, together with the amount of incident photons on the sample, gives the amount of photons absorbed for the corresponding sample portion. Finally, the photoluminescence quantum yield of the sample portion can be obtained by simply dividing the emitted photons (i.e., the dataset of the corrected fluorescence signal) by the absorbed photons of the sample.

[0048] To apply an electrical bias voltage, the following principle, as described above, can be used. To obtain ELQY, the emitted photon is divided by the applied normalized current.

[0049] According to another embodiment of the invention, the sample is selected from the group consisting of: single particles, preferably nanoparticles, more preferably nanoparticles fixed on a substrate; nanoregions within a membrane, preferably membranes having a nanometer thickness; devices, preferably nanoregions within a solar cell or battery pack; nanoregions within a substrate; and biomolecules, preferably proteins.

[0050] In other words, the sample fractions described above are preferably selected from one of the samples listed above.

[0051] Preferably, the sample in question is a single particle, more preferably a nanoparticle. These individual particles can be attached to a substrate that can be held by a substrate holder. Upon attachment, the local refractive index change allows these individual particles to be detected by interference scattering microscopy. Simultaneously, photons of different wavelengths emitted by the individual particles can be detected by using an illumination and detection channel for fluorescence measurements.

[0052] According to another embodiment of the invention, if the sample is irradiated by the excitation unit, during step b), the sample is irradiated with both the irradiation light in the irradiation channel for fluorescence measurement and the irradiation light in the irradiation channel for scattering measurement, wherein the irradiation light in the irradiation channel for fluorescence measurement has a lower intensity than the irradiation light in the irradiation channel for scattering measurement. This is to increase the signal-to-noise ratio in the irradiation channel for scattering measurement to avoid sample damage due to absorption at the wavelength used for irradiation. In particular, the irradiation light in the irradiation channel for scattering measurement is linearly polarized or circularly polarized.

[0053] The above steps enable accurate fluorescence and scattering measurements. Preferably, fluorescence and scattering measurements are performed in parallel, i.e., within the same time period. Therefore, measurements can be performed in a short time.

[0054] If the electrical bias is applied to the sample by the excitation unit, then step b) should be performed before step c).

[0055] According to another embodiment of the invention, in steps b) and c), the fluorescence signal and the scattering signal are recorded as image frames, and / or between steps b) and d) or during step d), the method further includes the step of associating the position of each fluorescence signal and the position of each scattering signal to obtain a correlated image frame with superimposed fluorescence signal and scattering signal.

[0056] The superimposed fluorescence and scattering signals within a single image frame allow for precise determination of the positional differences between the fluorescence and scattering signals, as well as the positional differences between each fluorescence signal and each scattering signal. Therefore, an image frame with superimposed fluorescence and scattering signals provides detailed information about the aforementioned measurements. Specifically, this information can be used to correct the obtained measurement data, thereby eliminating measurement errors or taking into account other effects of the measurement.

[0057] According to another embodiment, the method further includes the step of selecting false positive signals by analyzing related image frames with superimposed fluorescence and scattering signals.

[0058] Preferably, the analysis includes the step of determining the distance between the superimposed fluorescence and scattering signals, wherein if the determined distance is found to be below or above a predetermined threshold, the superimposed fluorescence and scattering signals corresponding to the determined distance are discarded in step d).

[0059] In other words, the above measurement steps allow for the selection of false positive signals from the remaining acquired data. This increases the stability and accuracy of the measurement. Furthermore, by obtaining image frames with superimposed fluorescence and scattering signals, analysis can be performed directly using the images, making data processing and interpretation easier compared to processing the raw data. This also increases the visualization of the measurement, allowing the operator to directly see the distance differences between the superimposed scattering and fluorescence signals.

[0060] The threshold can be selected based on the sample being measured. A larger threshold is used for larger samples, while a lower threshold is used for smaller samples. The signal intensity can also affect the threshold. In particular, a larger threshold is used for many signals compared to a small number of particles.

[0061] According to another embodiment of the present invention, between steps b) and d), the method further includes the following steps:

[0062] e) Apply an external stimulus to the sample to reduce the fluorescence of a specific region of the sample.

[0063] In particular, the method also includes the step of repeating steps b), c) and e) at predetermined times or until a predetermined number of cycles are completed.

[0064] The reason for the photobleaching step e) described above is that after a certain measurement time, the substrate may be covered by fluorescent particles, which mask the signal of each newly attached particle. By implementing step e) of the above method, the fluorescence of the already attached sample can be weakened, that is, "destroyed" in the sense that they no longer fluoresce, which realizes the original environment ready for a new batch of particles to attach to the substrate.

[0065] In essence, this mechanism "resets" the measurement area and allows for the measurement of signals from newly attached particles in the same area, significantly increasing the number of particles that can be analyzed. This, in turn, improves the statistical accuracy of the measurements. Furthermore, the methodological steps described above provide a seamless transition between measurement cycles, eliminating the need for manual intervention.

[0066] Preferably, the sample is irradiated with a laser of sufficient intensity to photobleach a certain area of ​​the sample.

[0067] Alternatively, an electrical bias can be applied to the sample, causing the sample to separate from the substrate surface or the substrate to be photobleached.

[0068] In particular, the above cycle can be repeated indefinitely.

[0069] Preferably, the cycle is performed at least twice, more preferably at least ten times, and even more preferably at least twenty times.

[0070] Alternatively, the cycle may be repeated for at least 10 minutes, more preferably at least 20 minutes, or even more preferably at least 30 minutes.

[0071] According to a second aspect of the invention, the present invention relates to a combined interference scattering and fluorescence microscope, the microscope comprising an excitation unit including an irradiation channel or power supply for performing fluorescence measurements using a second irradiation source. The microscope also includes a detection channel for fluorescence measurement and an illumination and detection channel for scattering measurement, a sample holder for holding the sample in the sample position, a first illumination source allocated to the illumination channel for scattering measurement and configured to provide illumination light to the sample holder, a second illumination source allocated to the excitation unit and configured to provide illumination light to the sample holder, or a power supply allocated to the excitation unit and configured to apply an electrical bias voltage to the sample holder, a first detector allocated to the detection channel for scattering measurement and configured to receive output light from the sample holder, the output light including scattered light from the sample position and illumination light from the sample position, a second detector allocated to the detection channel for fluorescence measurement and configured to receive fluorescence from the sample, and a control and processing unit connected to the excitation unit and detection channel for fluorescence measurement and each illumination and detection channel for scattering measurement, wherein the control and processing unit is adapted to perform steps a) to c) according to the first aspect of the invention.

[0072] The microscope according to the second aspect of the invention allows for the measurement of the quantum yield of fluorescent particles in a short time without altering the properties of the sample. Regarding further advantages, the above description of the first aspect also applies to the second aspect. Attached Figure Description

[0073] The invention will now be described in detail with reference to the accompanying drawings, in which:

[0074] - Figure 1A This is a schematic diagram of a combined interference scattering and fluorescence microscope according to the present invention, wherein the excitation unit includes an irradiation source;

[0075] - Figure 1B This is a schematic diagram of a combined interference scattering and fluorescence microscope according to the present invention, wherein the excitation unit includes a power supply;

[0076] - Figure 2 It is a schematic flowchart that includes method steps for performing a method to determine the quantum yield of a sample by using a combined interference scattering and fluorescence microscope from Figure 1;

[0077] - Figure 3 This is a schematic diagram of the photobleaching mechanism, which can be achieved through... Figure 2 The method and steps are as follows; and

[0078] - Figure 4 It comes from Figure 1B A schematic side view of the sample substrate arrangement under a microscope. Detailed Implementation

[0079] Figure 1A A combined interference scattering and fluorescence microscope 10 according to the present invention is depicted.

[0080] The combined interference scattering and fluorescence microscope 10 includes an optical system 12 that provides an optical path 14.

[0081] In detail, the optical path 14 includes an illumination channel 16 for scattering measurement and a detection channel 18 for scattering measurement. The illumination channel 16 for scattering measurement optically connects a sample holder 20 for holding the sample 22 in the sample position to a first illumination source 24, which is configured to provide a first illumination light 26 (scattered light).

[0082] For example, the first irradiation light 26 can be coherent light or circularly polarized light. Specifically, the first irradiation source 24 can be a laser. Typically, the wavelength and intensity of the first irradiation light 26 are selected according to the sample 22 to be imaged. Preferably, the first irradiation light 26 can be ultraviolet light (which can be defined herein as having a wavelength in the range of 10 nm to 380 nm); visible light (which can be defined herein as having a wavelength in the range of 380 nm to 740 nm); or infrared light (which can be defined herein as having a wavelength in the range of 740 nm to 300 μm). The first irradiation light 26 can also be a mixture of the above wavelengths.

[0083] Furthermore, the detection channel 18 for scattering measurements optically connects the sample holder 20, which holds the sample 22 in its sample position, to a first detector 28 configured to receive output light 30 (alternating square scattered lines) from the sample 22, including light scattered from the sample position (square lines) and illumination light reflected from the sample position (scattered lines). Therefore, the principle of interference scattering microscopy is that scattered light from or near the surface of the sample 22 interacts constructively with reflected light, causing the detector 28 to capture an image of the object.

[0084] The illumination light reflected from the sample position is mainly reflected from the interface between the sample holder 20 and the sample 22.

[0085] For example, the interface could be a glass-water interface.

[0086] Specifically, the intensity of the reflected illumination light can be approximately 0.5% of the intensity of the incident light 26. The scattered light is scattered by the object to be imaged in sample 22.

[0087] Sample 22 may be a liquid sample containing the object to be imaged.

[0088] The objects that can be imaged by the combined interference scattering and fluorescence microscope 10 can be selected from the group consisting of: single particles, preferably nanoparticles, more preferably nanoparticles fixed on a substrate; nanoregions within a membrane, preferably membranes with nanometer thickness; nanoregions within a device, preferably within a solar cell or battery pack; and nanoregions within a substrate and protein.

[0089] Typically, the sample holder 20 holds the sample 22 on the surface that forms the interface between the sample holder 20 and the sample 22.

[0090] The first detector 28 is arranged to receive the output light 30 from the sample location. The first detector 28 may be a CMOS detector (complementary metal-oxide-semiconductor), an image sensor, or a CCD (charge-coupled device).

[0091] So far, the components that are formally part of the interference scattering section of the combined interference scattering and fluorescence microscope 10 have been described. Next, the components of the fluorescence section of the combined interference scattering and fluorescence microscope 10 will be described in more detail.

[0092] Specifically, the optical path 14 includes an excitation unit 32 and a detection channel 34 for fluorescence measurement. Figure 1A In the embodiment shown, the excitation unit 32 is an irradiation channel 33 for fluorescence measurement, which includes a second irradiation source 36.

[0093] The irradiation channel 33 for fluorescence measurement optically connects the second irradiation source 36 to the sample holder 20, which holds the sample 22 in the sample position.

[0094] Specifically, the second irradiation source 36 is configured to provide a second irradiation light 38 (dashed line). The second irradiation light 38 may be light having a wavelength suitable for exciting the sample 22. Preferably, the second irradiation source 36 may be a laser.

[0095] In detail, the second illumination source 36 is optically coupled to a single-mode fiber 40, which guides the second illumination light 38 from the second illumination source 36 into the illumination channel 16 for scattering measurement. Alternatively, instead of the single-mode fiber 40, multiple optical mirrors can be used to guide the second illumination light 38 into the illumination channel 16 (not shown here) for scattering measurement.

[0096] To change the intensity of the second illumination light 38, an ND filter 42 (neutral density filter) is arranged between the second mode fiber 40 and the second illumination source 36. The ND filter 42 is controlled by a motor unit (not shown here). The ND filter 42 can be controlled by the motor unit to increase or decrease the power intensity of the second illumination light 38.

[0097] In addition, the detection channel 34 for fluorescence measurement optically connects the sample holder 20 for holding the sample 22 in the sample position to the second detector 44, which is configured to receive fluorescence 46 (double line) from the sample 22.

[0098] Fluorescence 46 is the light emitted by sample 22 when sample 22 is excited by the second irradiation light 38.

[0099] Typically, the second detector 44 is a CMOS detector, an image sensor, or a CCD.

[0100] In order to guide the first and second illumination lights 26 and 38 from the first and second illumination sources 24 and 36 to the sample position and from the sample position to the first and second detectors 28 and 44, respectively, the optical system 12 also includes a plurality of adjustable dichroic mirrors 48, adjustable mirrors 50 and focusing lenses 52.

[0101] Specifically, the dichroic mirror 48 is adapted to separate different types of light from each other. For example, the dichroic mirror 48 is arranged along the optical path 14 between detectors 28 and 44 to separate the detection channel 18 for scattering measurements from the detection channel 34 for fluorescence measurements. Another dichroic mirror 48 can be used to combine the illumination channel 16 for scattering measurements with the illumination channel 33 for fluorescence measurements upstream of the sample location.

[0102] In addition, one of the focusing lenses 52 serves as a condenser lens, which focuses each of the first and second irradiation lights 26 and 38 from the first and second irradiation sources 24 and 36 onto the sample 22, respectively, and one of the focusing lenses 52 serves as a barrel lens, which focuses each of the output light 30 and fluorescence 46 from the sample 22 and the sample position onto the first and second detectors 28 and 44, respectively.

[0103] Additionally, the optical system 12 includes an objective lens 54, which is adapted to guide the first illumination light 26 and the second illumination light 38 to the sample position and collect fluorescence 46 and output light 30, and guide the fluorescence 46 and output light 30 to the first detector 28 and the second detector 44, respectively.

[0104] Furthermore, the optical system 12 includes a beam splitter 56 adapted to separate the first and second illumination beams 26, 38 from the output beam 30 and fluorescence 46, respectively, and to guide the output beam 30 and fluorescence 46 to the first and second detectors 28, 44, respectively, via a tunable mirror 50, while the first and second illumination beams 26, 38 are guided to the objective lens 54. Typically, the beam splitter 56 allows for partial reflection and partial transmission of the incident light thereon, thereby allowing the incident light and the back-propagating light to be split into two different optical paths.

[0105] For example, beam splitter 56 may be a plate typically provided with a film, which may be metallic or dielectric, arranged at a 45° angle relative to optical path 14. Alternatively, the beam splitter may be a cubic beam splitter formed by multiple prisms, each prism having a partially reflective film at the interface between the prisms.

[0106] In addition, the combined interference and fluorescence microscope 10 includes a stage unit 58 adapted to adjust the focus of the objective lens 54.

[0107] Specifically, the stage unit 58 is adapted to change the distance between the objective lens 54 and the sample 22, thereby adjusting the focus.

[0108] The combined interference scattering and fluorescence microscope 10 is connected to the control and processing unit 60 via a connection 62. In particular, the connection 62 can be wired; however, a wireless connection is also possible.

[0109] The control and processing unit 60 is connected to each of the irradiation and detection channels 16, 18, 33, 34 and is adapted to perform a method for determining the quantum yield from the sample 22 by using the combined interference scattering and fluorescence microscope 10.

[0110] Figure 1B An embodiment of the combined interference scattering and fluorescence microscope 10 is shown, wherein the excitation unit 32 is not an illumination channel for fluorescence measurement 33, but a power supply 78. The remaining features remain unchanged and are incorporated herein by reference to the above description.

[0111] Power supply 78 is connected to sample holder 20 via electrical connection 76 (e.g., wire, etc.). In particular, power supply 78 is adapted to apply an electrical bias voltage, such as current, to sample 22. The electrical bias voltage should be sufficient to excite sample 22 and generate an electrofluorescence signal from sample 22.

[0112] A suitable power supply 78 can be a potentiostat.

[0113] Power supply 78 is connected to control and processing unit 60, which is further connected to each of irradiation and detection channels 16, 18, 34, and is adapted to perform a method for determining the quantum yield of sample 22 by using combined interference scattering and fluorescence microscope 10.

[0114] Next, we will combine Figure 1A The microscope examples shown describe in more detail the method for determining the quantum yield of a sample.

[0115] Figure 2 The document provides a schematic overview of the steps involved in determining quantum yield from a sample, which will be described below.

[0116] In the first step S1, each of the irradiation and detection channels 16, 18, 33, and 34 is calibrated.

[0117] In the next step S2, fluorescence and interference scattering measurements are performed on the sample to obtain interference scattering and fluorescence signals.

[0118] Subsequently, in step S3, the quantum yield of the sample is calculated based on interference scattering and fluorescence signals.

[0119] Each of steps S1 through S3 described above is described in more detail below.

[0120] First, step S1 will be described in more detail.

[0121] Step S1 may also include at least one of steps S4 to S6.

[0122] In step S4, a conversion relationship between size-related parameters and the measured interference scattering signal is determined. For example, a conversion relationship can be used to convert the measured interference scattering signal (e.g., contrast value) from the sample into a size-related parameter (e.g., sample volume). The conversion relationship can be a table, mathematical equation, or theoretical module that associates the size-related parameter with the interference scattering signal. In other words, the interference scattering signal measured during the calibration steps in interference scattering and fluorescence microscopy is calibrated to a specific size-related parameter, such as the cubic nanometers of the sample.

[0123] Specifically, the conversion relationship can be determined from a calibration curve obtained by measuring the scattering signal of a sample with known size-related parameters or based on a simulation of the sample's refractive index, or from a calibration curve obtained by measuring the size-related parameters of the sample using an electron microscope and measuring the scattering signal of the sample using an interferometric scattering measurement.

[0124] Preferably, the size-related parameters are selected from the group consisting of sample weight, sample volume, and sample density.

[0125] In addition, in step S5, a correction factor is determined that takes into account the loss of emitted photons from the sample in the directional channel used for fluorescence measurement.

[0126] The correction factor may include at least one of the following sub-factors, such as the collection efficiency of the objective lens, the transmission loss objective lens, the quantum efficiency of optics in the detection path, lenses, filters and cameras.

[0127] For example, the collection efficiency of an objective lens can be determined by measuring the emission of the sample in question with and without top oil immersion under the same excitation conditions, in order to calculate the collection efficiency of the used oil-immersion objective lens. The collection efficiency and the aforementioned sub-factors are combined into a calibration factor.

[0128] In a further step S6, the amount of incident photons on the sample in the irradiation channel used for fluorescence measurement is determined.

[0129] Step S6 may include the following steps: determining the amount of incident photons by measuring the irradiation area and irradiation distribution at the sample location in the absence of a sample, and taking into account a calibration factor that accounts for laser power loss in the irradiation channel used for fluorescence measurement.

[0130] It should be noted that steps S4 to S6 of the above method do not have to be performed one after another, but can be combined, so the order is arbitrary.

[0131] In the following text, we will examine method step S2 in more detail.

[0132] During step S2, the sample can be irradiated using both the irradiation light in the irradiation channel for fluorescence measurement and the irradiation light in the irradiation channel for scattering measurement, with the irradiation light in the irradiation channel for fluorescence measurement having a lower intensity compared to the irradiation light in the irradiation channel for scattering measurement.

[0133] In particular, the illumination light in the illumination channel used for scattering measurements is linearly polarized or circularly polarized.

[0134] In principle, the sample can be irradiated simultaneously through two irradiation channels.

[0135] Step S3 is discussed in more detail below. Step S3 may include at least one of steps S7 to S10.

[0136] In S7, the scattered signal from the detection channel used for scattering measurement is transformed using a transformation relation to obtain a dataset of size-related parameters. Specifically, the scattered signal is a contrast value. However, this particular step can also be performed between steps S2 and S3.

[0137] Step S8 involves converting the fluorescence signal from the detection channel used for fluorescence measurement using a correction factor to obtain a dataset of corrected fluorescence signals. This step can also be performed between steps S2 and S5.

[0138] In step S8, the fluorescence and scattering signals can be transformed or recorded as image frames. This step is performed during step S2.

[0139] Furthermore, between or during steps S2 and S3, the method further includes step S9: correlating the position of each fluorescence signal with the position of each scattering signal to obtain a correlated image frame with superimposed fluorescence and scattering signals.

[0140] In addition, step S9 may include at least one of the following steps:

[0141] -Perform background correction, preferably ratio background correction;

[0142] - Locate smaller portions of the sample based on the measured scattering signals, particularly the location of particles or regions within the sample;

[0143] - Process the scattering signal to extract the measurement amplitude and spatial and temporal location of each smaller portion of the sample;

[0144] - The obtained superimposed fluorescence and scattering image frames are rescaled to the same pixel size and interpolated to the same image amount, optionally taking into account different exposure times and different image amounts;

[0145] - Exclude all separated particles and particles that are extremely close in time and location from the interferometric scattering data;

[0146] -Consider the possible offset between the scattered signal and the fluorescence signal;

[0147] - Subtract the background of the fluorescence image from the illumination distribution;

[0148] - Convert the grayscale level into detected photons;

[0149] - Use right-angled or circular regions to calculate the number of photons from a pixel.

[0150] Then, an additional step S10 can be performed, which includes selecting false positive signals by determining the distance between the superimposed fluorescence and scattering signals, wherein if the determined distance is found to be lower or higher than a predetermined threshold, the superimposed fluorescence and scattering signals corresponding to the determined distance are discarded in step S3.

[0151] In addition, step S10 may include at least one of the following steps:

[0152] - If the offset is too large, temporarily discard the same frame;

[0153] - If the offset is too large, discard the same position in the image space;

[0154] - The signal is linearly averaged by rolling average of spatial and temporal regions, where the angle coefficient is displayed for the attached particles at spatial and temporal points;

[0155] - Discard smaller portions of the sample (especially particles) that overlap with another smaller portion (especially particles) of the sample, for example if the distance between them is too close;

[0156] -Average the signal, where the average signal must exceed a certain threshold, and if the signal is below the threshold, the specific signal is discarded;

[0157] - Discard the smaller, free portions of the sample from the outset, especially particles;

[0158] - Consider a minimum threshold number of images that discards smaller portions of the sample, particularly particles, that remain attached at the end of the measurement;

[0159] It should be noted that steps S7 to S10 of the above method do not have to be performed one after another, but can be combined, so the order is arbitrary.

[0160] In detail, step S3 may also include at least one of the following steps:

[0161] - Use the following transformation relationship to convert the measured scattering signal into size-related parameters:

[0162] - The size-related parameters are converted into an absorption cross section by using the material's refractive index and the classical formula described above.

[0163] - The absorbed photons of the sample are calculated by using the absorption cross section and calibrated incident photons per unit area;

[0164] - The quantum yield of a sample is calculated using a dataset of absorbed photons and corrected fluorescence signals.

[0165] Preferably, the quantum yield of the sample in step S3 is calculated using a dataset of corrected fluorescence signals, a dataset of size-related parameters, and the amount of incident photons on the sample.

[0166] More preferably, the sample is selected from single particles, preferably nanoparticles, more preferably nanoparticles fixed on a substrate; nanoregions within a membrane, preferably membranes with nanometer thickness; nanoregions within a device (preferably solar cells or battery packs), nanoregions within a substrate, and proteins.

[0167] Additionally, a further step S11 can be performed, in which an external stimulus is provided to attenuate the fluorescence of the sample. In a preferred embodiment, the sample is irradiated with a laser of sufficient intensity to photobleach a region of the sample. However, an electrical bias voltage that causes the sample to separate from the substrate surface can also be used. This step can be performed after step S2.

[0168] Specifically, the method also includes the step of repeating steps S2 and S11 for a predetermined time or until a predetermined number of cycles are completed.

[0169] Specifically, step S11 allows photobleaching of a certain area of ​​the sample, which produces a transparent substrate free of fluorescent sample. Therefore, a new sample can be attached to the surface and measured.

[0170] The above steps S1 to S11 can also be performed by Figure 1B The microscope 10 shown performs this operation, except that instead of irradiating the sample, an electrical bias voltage is applied to it. Furthermore, the calibration step regarding the irradiation channel used for fluorescence measurements is omitted, since the amount of charge applied to the sample is known through the power supply used. Preferably, the power supply is coupled to a computer, and their combination allows for precise determination of the amount of charge, i.e., the current induced in the sample.

[0171] In contrast to the method described in steps S1 to S11 above, it is preferable to apply an electrical bias voltage after the scattering measurement is performed.

[0172] To be described later Figure 4 The details of measuring electroluminescence are provided.

[0173] exist Figure 3 The principle of step S11 is explained in detail.

[0174] At the start of the photobleaching process S11, the region of sample 64 located on substrate 66 does not have sample material attached thereto. After a certain amount of time, a certain amount of sample attaches to substrate 66, which results in a sample signal 68 from sample 64.

[0175] However, after an additional time period, more samples attach to base 66, resulting in additional sample signals 70 from the newly attached samples. Now, signals 68 and 70 from different samples interfere with each other. In the worst case, signals 68 and 70 partially or completely overlap with each other. To avoid this overlap, a photobleaching step S11 can be performed after a certain period of time.

[0176] By doing so, a certain area of ​​sample 72 can be photobleached, meaning that the fluorescence of the sample attached to substrate 66 so far is destroyed. In other words, the process reduces the fluorescence signal from the sample attached to the surface of substrate 66.

[0177] After a certain period of time, due to the adhesion of the new sample, the signal of the new fluorescent sample 74 appears on the substrate 66, and further fluorescence measurements can be performed.

[0178] In principle, the order of steps S11 and S2 can be repeated indefinitely.

[0179] Figure 4 Showing from Figure 1B The side view of the microscope sample 22 and substrate 66 is given in the figure.

[0180] Sample 22 is reversibly or irreversibly attached to base 66, which is conductive or coated with a conductive top layer facing sample 22.

[0181] Furthermore, base 66 is electrically connected to power supply 78 via electrical connection 76. By applying an electrical bias voltage to base 66, sample 22 attached thereto is excited and emits fluorescence 46, which is coupled into detection channel 34 for fluorescence measurement.

Claims

1. A method for determining the quantum yield of a sample (22) using a combined interference scattering and fluorescence microscope (10), the microscope comprising an excitation unit (32) for irradiating the sample or applying an electrical bias voltage to the sample, a detection channel (34) for fluorescence measurement, and irradiation and detection channels (16, 18) for scattering measurement, wherein the method comprises the following steps: a) Calibrate each of the irradiation and detection channels (16, 18) for scattering measurements, the excitation unit (32) for fluorescence measurements, and the detection channel (34); b) Perform an interference scattering measurement on the sample (22) to obtain an interference scattering signal from the sample (22); c) Irradiate the sample or apply an electrical bias voltage to the sample to obtain a fluorescence signal from the sample (22); and d) Calculate the quantum yield of sample (22) based on interference scattering and fluorescence signals.

2. The method for determining quantum yield according to claim 1, wherein, Step a) also includes determining the conversion relationship between size-related parameters and the measured interference scattering signal.

3. The method for determining quantum yield according to claim 2, wherein the conversion relationship is determined by a calibration curve obtained by measuring the scattering signal of a sample having known size-related parameters, or by a simulation based on the refractive index of the sample, or by a calibration curve obtained by measuring the size-related parameters of the sample using an electron microscope and measuring the scattering signal of the sample using interferometric scattering measurement.

4. The method for determining quantum yield according to claim 2 or 3, wherein, The size-related parameters are selected from a group consisting of sample weight, sample volume, and sample density.

5. The method for determining quantum yield according to any one of claims 2 to 4, wherein, Between or during steps b) and d), the method further includes a step of using a transformation relation to transform the scattering signal from the detection channel used for scattering measurements to obtain a dataset of size-related parameters, in particular, the scattering signal being a contrast value.

6. The method for determining quantum yield according to any one of the preceding claims, wherein, Step a) also includes determining a correction factor that takes into account the loss of photons emitted from the sample (22) in the detection channel (34) used for fluorescence measurement.

7. The method for determining quantum yield according to claim 6, wherein, Between or during steps b) and d), the method further includes a step of using a correction factor to convert the fluorescence signal from the detection channel used for fluorescence measurement to obtain a dataset of corrected fluorescence signals.

8. The method for determining quantum yield according to any one of the preceding claims, wherein, If the sample (22) is irradiated in step a), the method further includes the step of determining the amount of incident photons on the sample (22) in the irradiation channel (32) for fluorescence measurement. Specifically, the amount of incident photons is obtained by measuring the irradiation area and irradiation distribution in the absence of a sample at the sample location and taking into account a calibration factor that accounts for the loss of laser power in the irradiation channel used for fluorescence measurement (32).

9. The method for determining quantum yield according to claims 5, 7, and 8, wherein, In step d), the quantum yield of the sample is calculated by using a dataset of corrected fluorescence signals, a dataset of size-related parameters, and the amount of incident photons or current applied to the sample (22).

10. The method for determining quantum yield according to any one of the preceding claims, wherein, The sample is selected from the group consisting of: single particles, preferably nanoparticles, more preferably nanoparticles fixed on a substrate; nanoregions within a membrane, preferably nanoregions within a membrane having a thickness of 30 nanometers; nanoregions within a device, preferably a solar cell or battery pack; nanoregions within a substrate; and biomolecules, preferably proteins.

11. The method for determining quantum yield according to any one of the preceding claims, wherein, If the sample (22) is irradiated by the excitation unit (32), then during step b), the sample (22) is irradiated by the irradiated light (38) in the irradiation channel (32) for fluorescence measurement and the irradiated light (26) in the irradiation channel (16) for scattering measurement, wherein the irradiated light (38) in the irradiation channel (32) for fluorescence measurement has a lower intensity than the irradiated light (26) in the irradiation channel (16) for scattering measurement. Specifically, the illumination light (26) in the illumination channel (16) used for scattering measurements is linearly polarized or circularly polarized, and / or If the electrical bias is applied to the sample (22) by the excitation unit (32), then step b) is performed before step c).

12. The method for determining quantum yield according to any one of the preceding claims, wherein in steps b) and c), the fluorescence and scattering signals are recorded as image frames, and / or in, Between or during steps b) and d), the method further includes the step of associating the location of each fluorescence signal with the location of each scattering signal to obtain a correlated image frame with superimposed fluorescence and scattering signals.

13. The method for determining quantum yield according to claim 12, wherein, The method further includes the following steps: selecting false positive signals by analyzing relevant image frames with superimposed fluorescence signals and scattering signals, preferably by determining the distance between the superimposed fluorescence signals and scattering signals, wherein if the determined distance is found to be lower than or higher than a predetermined threshold, the superimposed fluorescence signals and scattering signals corresponding to the determined distance are discarded in step d).

14. The method for determining quantum yield according to any one of the preceding claims, wherein, Between steps b) and d), the method further includes the following steps: e) Apply an external stimulus to the sample (22) to reduce the fluorescence of a certain region of the sample (64), In particular, the method also includes the step of repeating steps b), c) and e) at predetermined times or until a predetermined number of cycles are completed.

15. A combined interference scattering and fluorescence microscope (10), comprising: Excitation unit (32), the excitation unit (32) includes an irradiation channel (33) or power supply (78) for fluorescence measurement having a second irradiation source (36). The detection channel (34) is used for fluorescence measurement, and the irradiation and detection channels (16, 18) are used for scattering measurement. A sample holder (20) is used to hold the sample (22) in the sample position. A first irradiation source (24), which is assigned to an irradiation channel (16) for scattering measurements and configured to provide irradiation light (26) to the sample holder (20), A second irradiation source (36) or a power supply (78) is provided to the excitation unit (32) and configured to provide irradiation light (38) to the sample holder (20), and the power supply (78) is provided to the excitation unit (32) and configured to apply an electrical bias voltage to the sample holder (20). A first detector (28) is assigned to the detection channel (18) for scattering measurement and is configured to receive output light (30) from the sample holder (20), the output light (30) comprising light scattered from the sample location and illumination light reflected from the sample location; and The second detector (44) is assigned to the detection channel (34) for fluorescence measurement and is configured to receive fluorescence (46) from the sample (22). and a control and processing unit (60), which is connected to each of the excitation unit (32) and the detection channel (34) for fluorescence measurement and the irradiation and detection channels (16, 18) for scattering measurement. The control and processing unit (60) is adapted to perform steps a)-d) according to claim 1.

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