Single molecule fluorescence detection system and method

Abstract

A solution-based single molecule fluorescence spectroscopy system (100) comprising: an excitation source (102) for irradiating an excitation volume of a fluorescently labeled sample solution (104) with excitation radiation; a detector (106) for detecting fluorescence emission radiation emitted from a detection volume (128) of the fluorescently labeled sample solution; one or more processors (108) configured to: receive a detection signal from the detector; calculating a sample signal occupancy, the sample signal occupancy representing a proportion of time at which the detection signal includes a sample emission signal from the fluorescently labeled sample solution; and outputting a sample quality signal based on the sample signal occupancy.

Description

Technical Field

[0001] This disclosure relates to a solution-based single-molecule fluorescence spectroscopy system and a method for analyzing sample quality in the system. Background Technology

[0002] Single-molecule Foster resonance energy transfer (smFRET) is a fluorescence technique for studying single molecules. smFRET enables the determination of different biomolecular conformations and their corresponding conformational transition rates at timescales associated with key cellular processes such as protein (un)folding, transcription, and DNA replication and repair. Due to its ease of sample preparation, rapid data acquisition, and high temporal resolution, confocal microscopy is also the preferred method for generating multiple smFRET constraints for integrated structural modeling. Summary of the Invention

[0003] According to a first aspect of this disclosure, a solution-based single-molecule fluorescence spectroscopy system is provided, comprising: An excitation source, which is used to irradiate the excitation volume of a fluorescently labeled sample solution with excitation radiation; A detector used to detect fluorescent emission radiation emitted from the detection volume of the fluorescently labeled sample solution; One or more processors, which are configured to: Receive detection signals from the detector; Calculate the sample signal occupancy rate, which represents the proportion of time the detection signal includes the sample emission signal from the fluorescently labeled sample solution; and The sample quality signal is output based on the sample signal occupancy rate.

[0004] The sample quality signal can include the sample signal occupancy rate.

[0005] One or more processors can be configured to compare the sample signal occupancy rate with an upper occupancy rate threshold. If the sample signal occupancy rate exceeds the upper occupancy rate threshold, the sample quality signal may include an alarm signal.

[0006] Alarm signals may include indications of excessively high sample signal occupancy and / or recommendations to dilute fluorescently labeled sample solutions.

[0007] One or more processors can be configured to calculate a recommended dilution value based on the sample signal occupancy, and wherein an alarm signal indicates the recommended dilution value.

[0008] The system can be configured to reduce the concentration of the fluorescently labeled sample solution if the sample signal occupancy exceeds an upper occupancy threshold.

[0009] The system may include a microfluidic sample holder. The system can be configured to reduce the concentration of the fluorescently labeled sample solution by increasing the flow rate of a first microfluidic supply line supplying a buffer solution and / or decreasing the flow rate of a second microfluidic supply line supplying the fluorescently labeled molecules.

[0010] This system is applicable to porous samples, where different pores contain fluorescently labeled sample solutions of varying concentrations. The system can be configured to reduce the concentration of the fluorescently labeled sample solution by positioning pores with relatively low concentrations of sample solution within the excitation volume.

[0011] One or more processors can be configured to compare the sample signal occupancy rate with a lower occupancy rate threshold. If the sample signal occupancy rate is less than the lower occupancy rate threshold, the sample quality signal may include an alarm signal.

[0012] Alarm signals may include: an indication that the sample signal occupancy is too low; a suggestion to increase the concentration of the fluorescently labeled sample solution; and / or an indication that no fluorescence peak is detected.

[0013] One or more processors can be configured to calculate the sample signal occupancy rate by: The detection signal is divided into multiple time intervals; For each time interval, if the signal level of the detected signal exceeds the sample identification threshold, then the time interval is determined to include the sample emission signal from the fluorescently labeled sample; and The occupancy rate of the sample signal is determined by dividing the number of time intervals including the transmitted signal by the total number of time intervals among these multiple time intervals.

[0014] One or more processors can be configured to: A peak detection algorithm is used to identify one or more burst peaks of the detection signal that correspond to the fluorescence emission from the fluorescently labeled sample; Determine the pulse width of each burst peak; and The occupancy rate of the sample signal is determined by dividing the sum of the pulse widths by the time period of the detection signal.

[0015] One or more processors can be configured to determine the fluorescence background level based on the proportion of the detected signal excluding the sample emission signal. One or more processors can be configured to output a sample quality signal based on the sample signal occupancy and the background fluorescence level.

[0016] One or more processors can be configured to: The detection signal is divided into multiple time intervals; For each time interval If the signal level of the detection signal exceeds the sample identification threshold, then the time interval is determined to include the sample emission signal from the fluorescently labeled sample; and If the signal level of the detected signal is less than or equal to the sample identification threshold, then the time interval is determined to include the background signal. The occupancy rate of the sample signal is determined by dividing the number of time intervals including the transmitted signal by the total number of time intervals among these multiple time intervals; The fluorescence background level is determined based on the average signal level of this time interval, which includes the background signal; and The sample quality signal is output based on the sample signal occupancy and the background fluorescence level.

[0017] One or more processors can be configured to: A peak detection algorithm is performed to identify one or more burst peaks of the detection signal corresponding to the fluorescence emission from the fluorescently labeled sample; and The background fluorescence level is determined based on the average signal level of the region excluding burst peaks of the detected signal.

[0018] Sample quality signals can include background fluorescence levels.

[0019] One or more processors can be configured to compare the fluorescence background level with a background level threshold. If the fluorescence background level is greater than the background level threshold, the sample quality signal may include a background alarm signal.

[0020] Background alarm signals may include indications of excessively high fluorescence background levels and / or recommendations to re-prepare the sample solution buffer.

[0021] The processor can be configured to output sample quality signals on a continuous or semi-continuous basis.

[0022] According to a second aspect of this disclosure, a method for determining the sample mass of a fluorescently labeled sample solution in a single-molecule fluorescence spectroscopy system is provided, the method comprising: Receive detection signals from the detectors in this system; Calculate the sample signal occupancy rate, which represents the proportion of time the detection signal includes the sample emission signal from the fluorescently labeled sample solution; and The sample quality signal is output based on the sample signal occupancy rate.

[0023] According to a third aspect of this disclosure, a computer-readable medium is provided that includes instructions that, when executed by one or more processors, cause one or more processors to perform any of the methods disclosed herein.

[0024] A computer program may be provided that, when executed on a computer, causes the computer to configure any device containing the circuitry, controllers, converters, or apparatuses disclosed herein, or to perform any of the methods disclosed herein. As a non-limiting example, the computer program may be a software implementation, and the computer may be considered any suitable hardware, including digital signal processors, microcontrollers, and implementations in read-only memory (ROM), erasable programmable read-only memory (EPROM), or electrically erasable programmable read-only memory (EEPROM). The software may be an assembler.

[0025] Computer programs may be provided on a computer-readable medium or may be implemented as a transient signal, which may be a physical computer-readable medium, such as an optical disc or storage device. This transient signal may be a network download, including an Internet download. One or more non-transitory computer-readable storage media may be provided storing computer-executable instructions that, when executed by a computing system, cause the computing system to perform any of the methods disclosed herein. Attached Figure Description

[0026] One or more embodiments will now be described by way of example only with reference to the accompanying drawings, in which: Figure 1 A simplified schematic diagram of a solution-based single-molecule fluorescence spectroscopy system according to an embodiment of the present disclosure is shown; Figure 2 Showing output to Figure 1 An example of a system's user interface for detecting signals; Figure 3 A method for determining the sample mass of a fluorescently labeled sample solution in a single-molecule fluorescence spectroscopy system according to embodiments of the present disclosure is demonstrated; and Figure 4 Another method for determining the sample quality of a fluorescently labeled sample solution in a single-molecule fluorescence spectroscopy system according to embodiments of the present disclosure is demonstrated. Detailed Implementation

[0027] FRET is a photophysical process that results in the transfer of excitation energy from a donor fluorophore to an acceptor fluorophore. The efficiency of this transfer is inversely proportional to the sixth power of the distance between the two fluorophores. Therefore, by measuring FRET efficiency (e.g., by observing the emission of the two fluorophores under donor excitation), spatial information in the 3 to 10 nm range can be determined, making FRET a "spectral ruler" well-matched to the size of biomolecules such as nucleic acids and proteins. In ensemble measurements, this can be used to detect on-off / relative distance changes, such as binding and cleavage in bimolecular interactions, or conformational changes (e.g., opening and closing) in monomolecular processes. At the monomolecular level, FRET is sensitive to heterogeneous subpopulations, can measure the kinetics of processes in equilibrium, and absolute FRET efficiency can be used to infer precise distances for biomolecular structure determination.

[0028] In confocal smFRET experiments, the sample consists of fluorescently labeled molecules allowed to diffuse freely in solution. An excitation laser is focused by an objective lens to a near-diffraction-limited spot, thus defining an excitation volume. Light emitted from this volume can be focused by a second objective (or the same objective) to a pinhole (which rejects light emitted outside the focal plane of the objective), thereby creating a detection volume in the sample from which fluorescent photons can be detected. The overlap of the excitation and detection volumes defines the confocal volume—the region in space where molecules can be excited and the resulting fluorescence emission detected. As the fluorescently labeled molecule diffuses into the confocal volume, it emits fluorescent emission radiation (i.e., light), which is collected and detected by the microscope's optics / detector. The intensity of the light detected as the molecule diffuses through the confocal volume (e.g., photon count or intensity value) can be analyzed to determine the distance between fluorophores attached to the molecule.

[0029] The target molecule is labeled with donor and acceptor fluorophores, and the light emitted from these fluorophores is counted on separate detectors. In some examples, light with wavelengths greater than 650 nm can be recorded on the acceptor detector, and light with wavelengths less than 650 nm can be recorded on the donor detector.

[0030] Confocal smFRET experiments can reveal different biomolecular conformations and corresponding conformational transition rates in (dynamic) equilibrium at timescales associated with key cellular processes such as protein (un)folding, transcription, and DNA replication and repair. Due to its simplicity in sample preparation, rapid data acquisition, and high temporal resolution, confocal experiments are also the preferred method for generating multiple smFRET constraints for integrated structural modeling.

[0031] Working at low concentrations of labeled substances (approximately 50 pM) ensures that only single molecules are detected most of the time. Therefore, sample quality control is a crucial aspect of smFRET experiments. Sample concentrations should be carefully controlled to ensure accurate results. If the sample concentration is too high, the probability of two (or more) molecules coexisting in the confocal volume increases. Such events will be indistinguishable from true single-molecule events and will bias any analytical results, providing inaccurate outcomes. When both types of signals are present, it is impossible to distinguish between single-molecule and multi-molecule signals. For example, if a single molecule has two conformations (e.g., open and closed), the multi-molecule signal may indicate an average conformation (which does not reflect physical reality) rather than two dissimilar conformations. If the sample concentration is too low, longer acquisition times are required, which can lead to sample evaporation and / or degradation. Furthermore, fluorescence contamination in the solution buffer can result in intolerable noise levels in single-molecule measurements. This contamination may manifest as increased background, reduced signal-to-noise ratio, or as spurious fluorescence bursts unrelated to the sample. These spurious fluorescence bursts may overlap with signals from the target molecule in the manner described above, thus reducing the quality of the collected data.

[0032] This disclosure provides a solution-based single-molecule fluorescence spectroscopy system (which may be simply referred to herein as "the system") that can advantageously analyze sample quality and provide a sample quality signal to the user.

[0033] Figure 1 A simplified schematic diagram of a solution-based single-molecule fluorescence spectroscopy system 100 according to an embodiment of the present disclosure is shown. In this example, the system is an smFRET system; however, other examples may include a two-color coincidence detection system.

[0034] System 100 includes: an excitation source 102 for irradiating an excitation volume of a fluorescently labeled sample solution 104 with excitation radiation; a detector 106 for detecting fluorescence emission radiation emitted from a detection volume of the fluorescently labeled sample solution 104; and a processor 108. Processor 108 is configured to: receive a detection signal 110 from detector 106; calculate a sample signal occupancy rate, representing the time proportion of the detection signal that includes the emission signal from the fluorescently labeled sample; and output a sample quality signal 150 to a user interface based on the sample signal occupancy rate.

[0035] The sample quality signal 150 output by system 100 can advantageously inform the user whether the sample has sufficient quality for single-molecule experiments and avoid inaccurate experiments due to poor sample quality. As discussed below, the sample quality signal may include: sample signal occupancy; alarms indicating that the sample signal occupancy is too high and / or the sample needs to be diluted; dilution recommendations; background fluorescence level; and / or alarms indicating that the background fluorescence is too high.

[0036] Detailed discussion Figure 1 In this example, the excitation source includes a laser 114 and a collimator 116. In other examples, the excitation source may include other optical irradiators, such as an LED. The laser 114 outputs excitation radiation (i.e., laser light) in the form of a diverging beam. The collimator 116 collimates the diverging excitation radiation into a collimated beam. In some examples, the excitation source 102 may include a multi-wavelength source for providing excitation radiation at different wavelengths. The excitation source 102 may include multiple lasers and / or may include lasers that can emit at different wavelengths. One or more wavelengths of the excitation source 102 may correspond to the excitation wavelength of the corresponding fluorescent label of the fluorescently labeled sample 104. In some examples, the system may include a dual-color coincidence detection system in which the excitation source simultaneously provides two excitation beams at different wavelengths. In some examples, the system may include an alternating laser excitation system in which the excitation source alternates between excitation wavelengths between a first wavelength and a second wavelength different from the first wavelength. In some examples, the excitation source may provide a single wavelength.

[0037] In some examples, the excitation source may emit one or more wavelengths in the visible light range to excite one or more corresponding fluorophores at the corresponding excitation wavelength. In some examples, the one or more wavelengths (and the corresponding excitation wavelengths) may include visible light, IR or UV wavelengths, or combinations thereof.

[0038] In this example, the excitation dichroic mirror 118 is arranged to reflect the collimated excitation radiation toward the objective lens 120. The excitation dichroic mirror 118 is configured to reflect radiation at one or more wavelengths of the excitation source and transmit radiation at one or more wavelengths of the fluorescence emission radiation emitted from the sample 104. The objective lens 120 focuses the collimated excitation radiation to a near-diffraction-limited spot, thereby defining an excitation volume 128 in the sample solution 104. It should be understood that different optical arrangements can be provided to focus the excitation radiation onto the excitation volume 128 of the sample solution 104.

[0039] The fluorescently labeled sample solution 104 contains a fluorescently labeled molecule 122, such as DNA or protein, in solution (free diffusion). The solution may contain a buffer, and the buffer may contain various buffer components (e.g., water, TRIS, MgCl2, phosphate, etc.). The fluorescently labeled molecule can be labeled with a donor fluorophore 124 and an acceptor fluorophore 126. The donor fluorophore 124 and acceptor fluorophore 126 can be localized at specific sites on the molecule to study the conformation of specific parts of the molecule. When the fluorescently labeled molecule diffuses into the excitation volume, excitation radiation can excite the donor fluorophore (and optionally the acceptor fluorophore). FRET occurs when the excitation energy is transferred from the donor fluorophore to the acceptor fluorophore, producing fluorescent emission radiation (i.e., emitted fluorescence), which includes both donor emission radiation (at a donor emission wavelength different from the acceptor emission wavelength) and acceptor emission radiation (at the acceptor emission wavelength). The efficiency of this transfer process from donor emission to acceptor emission is inversely proportional to the sixth power of the distance between the two fluorophores. Therefore, spatial information about a molecule can be determined by measuring FRET efficiency (e.g., by observing the emission ratio of the two fluorophores under donor excitation). In an alternating laser excitation system, the two wavelengths of the excitation source can correspond to the excitation wavelengths of the two fluorophores. Alternating excitation can provide: a first detection signal, which includes the FRET signal (donor and acceptor emission) generated by donor excitation; and a second reference signal, which corresponds to acceptor-only emission generated by acceptor excitation. Exciting the acceptor alone can provide confirmation of the presence of the acceptor fluorophore. In some examples, processor 108 can use the FRET signal and the second reference signal to calculate the FRET correction factor.

[0040] In this example, objective lens 120 captures a portion of the fluorescence emission radiation transmitted along an emission optics system, which includes an excitation dichroic mirror 118, a tube mirror 130, a pinhole 132, a second collimating lens 134, and a detection optics 136. The emission optics system defines a detection volume of the sample from which fluorescence emission radiation can be detected. The overlap of the excitation volume 128 and the detection volume defines a confocal volume within which molecules can be excited and their fluorescence emission detected. Fluorescence emission radiation from objective lens 120 passes through excitation dichroic mirror 118 and is focused by tube mirror 130 onto pinhole 132. Pinhole 132 rejects fluorescence emission radiation that is not emitted from the focal plane of objective lens 120. Fluorescence emission radiation passing through pinhole 132 is collimated by second collimating lens 134. Detector optics 136 couples the fluorescence emission radiation to sensor elements of one or more detectors 106, 140.

[0041] In this example, detector optics 136 includes an emission dichroic mirror 138 that reflects fluorescence emission radiation (at the donor emission wavelength) from the donor fluorophore to the first detector 106 via a first detector lens and transmits fluorescence emission radiation (at the acceptor emission wavelength) from the acceptor fluorophore to the second (acceptor) detector 140 via a second detector lens. Thus, the first detector 106 detects fluorescence emission radiation from the donor fluorophore, and the second detector 140 detects fluorescence emission radiation from the acceptor fluorophore. The first and / or second detectors 106, 140 may include single-photon counters. In this example, both detectors 106 and 140 include avalanche photodiodes (APDs). In some examples, one or more detectors may include photomultiplier tubes for detecting intensity levels rather than absolute photon counts. In some examples, system 100 may include a single detector capable of detecting multiple wavelengths (e.g., donor emission wavelength and acceptor emission wavelength). In some examples, each detector may also have a corresponding emission filter (not shown) to filter out any signal outside the fluorescence emission band of the corresponding donor / acceptor fluorescence emission radiation.

[0042] Detectors 106 and 140 output detection signal 110 to processor 108. The processor can output detection signal 110 to a display unit (not shown). Detection signal 110 may include information related to the (time-dependent) intensity level or photon count detected by detectors 106 and 140.

[0043] Figure 2 Showing output to Figure 1 The system's user interface 211 shows an example detection signal 210. An example detection signal 210 is shown for a 1-second window.

[0044] In this example, the molecules of the fluorescently labeled sample solution are labeled with: a donor fluorophore having an ATTO550 peak excitation wavelength of 550 nm, excited by a donor-excited laser including a 520 nm diode laser; and a donor emission wavelength of 576 nm; and an acceptor fluorophore having an ATTO647n peak excitation wavelength of 647 nm, excited by a 638 nm acceptor-excited laser; and an acceptor emission wavelength of 680 nm. A donor emission filter with an emission bandpass of 535 to 607 nm is placed in front of the first detector to filter out background radiation (including stray excitation radiation) outside the fluorescence emission band of the donor fluorophore. An acceptor emission filter with an emission bandpass is used in conjunction with a second detector to filter out background radiation (including stray excitation radiation) outside the fluorescence emission band of the acceptor fluorophore.

[0045] In this example, alternating laser excitation (at a rate of 20 kHz) is used. The resulting detection signal 210 includes: a donor-excitation-donor-emission (DXDE) signal 242; a donor-excitation-acceptor-emission (DXAE) signal 244; and an acceptor-excitation-acceptor-emission (AXAE) signal 246. As noted above, the acceptor excitation and the resulting AXAE signal 246 are optional and can be used as reference signals. The ratio of the DXDE signal 242 to the DXAE signal 244 can represent the FRET efficiency.

[0046] like Figure 2 As shown, detection signal 210 is at background levels for most of the 1-second window (for all three signals DXDE, DXAE, and AXAE). This corresponds to the time when there are no fluorescently labeled molecules within the confocal volume. A burst peak 248 can be observed corresponding to the following time periods (for all three signals): when a single molecule diffuses into the confocal volume, when excited by excitation radiation, and when fluorescent emission radiation is generated and detected by the detector to provide the detection signal. The burst peak can be referred to as the sample emission signal of the detection signal. The detection signal at other time periods can be referred to as the background signal.

[0047] The sample signal occupancy rate corresponds to the relative time portion (or proportion) of the detection signal 210, including the burst peak 248, to the sample emission signal (generated by fluorescence emission radiation). The sample signal occupancy rate can be defined as the percentage of time that at least one fluorescently labeled molecule is within the confocal volume (and produces detectable fluorescence emission radiation). The sample signal occupancy rate can correspond to a percentage or a ratio. It provides a relative measure of the sample concentration. As noted above, controlling the sample concentration is important for single-molecule systems to ensure that the detection signal truly corresponds to a single molecule and does not include synchronous signals from multiple molecules (which can produce seriously inaccurate data).

[0048] The system's processor is configured to calculate the sample signal occupancy from the detection signal 210. In some examples, the processor can divide the detection signal into multiple time intervals. In some examples, the processor can divide the detection signal into 1 ms time intervals, such as a 1000 x 1 ms time interval for a 1-second signal sample. For each time interval, the processor can determine that the time interval includes the sample emission signal if the signal level (e.g., intensity level or total photon count) of the detection signal 210 within the time interval exceeds a sample identification threshold. The sample detection threshold may include an intensity level threshold or a photon count threshold (e.g., 30 photons for a 1 ms time interval). The processor can determine that the time interval includes the background signal if the signal level within the time interval is less than or equal to the sample identification threshold. For a dual-detector system (one monitoring donor emission and one monitoring acceptor emission), the processor can: (i) determine that the time interval includes the sample emission signal if the signal level of the detection signal from either detector exceeds the corresponding sample identification threshold for each detector; and (ii) determine that the time interval includes the background signal if the signal levels of the detection signals from both detectors are less than or equal to the corresponding sample identification thresholds.

[0049] The processor can determine the sample signal occupancy rate by dividing the number of time intervals including the sample emission signal by the total number of time intervals in the multiple time intervals (e.g., 15 sample intervals / 1000 intervals in total = 1.5% sample signal occupancy rate).

[0050] In some examples, the processor may calculate the sample signal occupancy in different ways. For example, the processor may: perform a peak detection algorithm to identify each burst peak 248 corresponding to the fluorescence emission radiation; determine the pulse width of each burst peak; and determine the sample signal occupancy as the sum of the pulse widths divided by the time period of the detected signal sample (e.g., a 1-second time window).

[0051] In this example, the processor outputs a sample quality signal 250 to the user interface 210. In this example, the sample quality signal 250 includes: a sample signal occupancy rate 250-1, expressed as a percentage; and an alarm signal 250-2, which indicates that the sample signal occupancy rate is too high and suggests diluting the sample solution.

[0052] In some examples, the processor can determine if the sample signal occupancy is too high by comparing it to an upper occupancy threshold. It has been found that a 1% sample signal occupancy provides a reliable smFRET signal and produces a sufficiently small amount of multi-molecule signal to ensure that the detection signal reliably provides single-molecule results. In some examples, the upper occupancy threshold can be a value of 1%, or it can be a value greater than 1%, such as 1.1% or 1.2%. In some examples, the processor can set the upper occupancy threshold based on user input.

[0053] If the sample signal occupancy exceeds the upper occupancy threshold, the processor can output an alarm signal 250-2. Alarm signal 250-2 can include visual, auditory, and / or tactile signals. Visual alarm signals can include: an indication of excessive occupancy; a suggestion to dilute the sample solution; and / or a suggested dilution value. The processor can calculate the suggested dilution value based on the ratio of the sample signal occupancy to the target sample signal occupancy. For example, if the sample signal occupancy is 3% and the target sample signal occupancy is 1%, the processor can calculate the suggested dilution value as a 3-fold dilution.

[0054] In some examples, the system may include a motorized sample stage suitable for a multi-well plate comprising a sample solution in each well. Different wells (e.g., columns or rows of wells) of the multi-well sample may contain sample solutions of different concentrations (e.g., different amounts of buffer solution). If the sample mass signal 250 indicates that the sample signal occupancy is too high for the sample solution in the first well, the system may adjust the motorized sample stage to test a second well with a lower sample concentration. Thus, if the sample signal occupancy exceeds an upper occupancy threshold, the system may reduce the concentration of the fluorescently labeled sample solution.

[0055] In some examples, the system can be adapted to a microfluidic-based sample holder, wherein a first microfluidic supply line supplies a buffer solution to the sample well, and a second microfluidic supply line supplies fluorescently labeled molecules to the sample well. If the sample quality signal indicates that the sample signal occupancy is too high, the system can increase the flow rate from the first microfluidic supply line to reduce the concentration of the sample solution. Thus, if the sample signal occupancy exceeds an upper occupancy threshold, the system can reduce the concentration of the fluorescently labeled sample solution.

[0056] In some examples, the processor can compare the sample signal occupancy to a lower occupancy threshold. If the sample signal occupancy is less than the lower occupancy threshold, the sample quality signal can include an alarm signal. A low sample signal occupancy can indicate that the sample concentration is too low or that a problem has occurred in the fluorescent labeling process. The alarm signal can include: an indication that the sample signal occupancy is too high; a suggestion to increase the concentration of the fluorescently labeled sample solution; and / or an indication that no fluorescence peak was detected (if the sample signal occupancy is equal to zero).

[0057] In some examples, the sample quality signal 250 may include a background fluorescence level 250-3. The processor may calculate the background fluorescence level 250-3 based on a time interval that includes the background signal (in other words, a time interval that excludes the sample emission signal). In some examples, the processor may calculate the background fluorescence level 250-3 based on the average background signal over the background time interval. If burst detection is used, the processor may determine the background fluorescence level 250-3 by averaging the signal levels over the region of the detected signal that excludes burst peak 248. In this example, the background fluorescence level 250-3 is expressed as photon counts per second (Hz) and is 3.18 kHz. In some examples, the background fluorescence level 250-3 may be expressed as the average intensity level over the time interval that includes the background signal.

[0058] In this example, the sample quality signal includes the background fluorescence level 250–3. In some examples, the processor can determine if the background fluorescence level 250–3 is too high by comparing it to a background level threshold. Background fluorescence levels of 250–3 less than 10 kHz, and particularly less than 3 kHz, have been found to produce good smFRET results with high sensitivity. In some examples, the background level threshold may have a value between 3 kHz and 10 kHz. In some examples, the processor may set the background level threshold based on user input. If the background fluorescence level 250–3 exceeds the background level threshold, the sample quality signal may include a background alarm signal. The background alarm signal may include an indication of an excessively high background fluorescence level and / or a suggestion to re-prepare the sample solution buffer. Background fluorescence may be caused by fluorescent contamination in the buffer of the fluorescently labeled buffer solution. Contamination can originate from many sources, such as each buffer component of the buffer solution, a contaminated sample holder, the buffer solution container, personnel, etc.

[0059] The disclosed system and method can advantageously monitor sample solution 104 in real time and provide real-time sample quality signals to indicate: sample signal occupancy, background fluorescence level 250-3, and / or corresponding alarms for the user. In this way, the user can immediately identify whether the sample quality is insufficient and take remedial actions (dilution or re-preparation) before data collection.

[0060] Although the examples shown describe a processor calculating the sample signal occupancy and background fluorescence level 250-3 within a 1-second window for the detection signal 210, it should be understood that longer or shorter time windows can be used. In some examples, the processor may calculate the sample signal occupancy and / or background fluorescence level 250-3 and update and output the sample quality signal on a continuous or semi-continuous (periodic) basis. Providing the sample quality signal 250 (especially the sample signal occupancy) on a continuous or semi-continuous basis can advantageously provide an indication of changes in sample viability during the experiment (e.g., an increase in concentration due to buffer evaporation).

[0061] In some examples, the processor can calculate the sample signal occupancy and / or background fluorescence level 250⁻³ for each time window (e.g., a 1-second window). The processor can update and output the sample quality signal for each time window, or it can update and / or output the sample quality signal as an average of multiple time windows. For example, the processor can calculate the sample signal occupancy and / or background fluorescence level 250⁻³ for each 1-second time window and output the sample quality signal as a rolling 5-second average of 5 x 1-second time windows.

[0062] When a user runs a single-molecule fluorescence spectroscopy experiment, the disclosed system can analyze the arrival time of incoming photons and estimate the sample signal occupancy and average background count rate (background fluorescence level), outputting both parameters to the user interface 211. If either parameter is considered to be greater than that acceptable for reliable data analysis (>1% for sample signal occupancy; or >3 to 10 kHz for background count rate), the system can output a warning or alarm to the user interface 211.

[0063] One or more processors may be located locally on the system or remotely (e.g., on a server or in a cloud computing environment) or both. One or more processors may be part of a computing system that runs software used to control components of the system.

[0064] Figure 3 A method for determining the sample mass of a fluorescently labeled sample solution in a single-molecule fluorescence spectroscopy system according to embodiments of the present disclosure is demonstrated. This method can be performed by… Figure 1 The processor or more generally the system performs this.

[0065] The first step 360 includes receiving at least a portion (in this example, a 1-second sample) of detection signals from one or more detectors in the system.

[0066] The second step 362 includes dividing a portion of the detected signal into time intervals, in this example, these time intervals include a 1000 x 1 ms time interval.

[0067] Step 364 includes resetting the background photon counter. NBackgroundPhotons Background interval counter, NBackgroundBins ; and sample interval counter, NSampleBins .

[0068] Step 4, 366, includes: For each time interval, at the first sub-step 366-1, determining whether the signal level (photon count) of the detected signal in the time interval exceeds the sample identification threshold. The second sub-step 366-2 includes (for each time interval) if the signal level of the detected signal exceeds the sample identification threshold, then... NSampleBins Increment by one. The third sub-step 366-3 includes: (for each time interval) incrementing the background interval counter. NBackgroundBins Increment by one; and if the signal level of the detected signal is less than the sample recognition threshold, then increment the background photon counter. NBackgroundPhotons Increase the signal level (photon count) within the time interval.

[0069] Step 5, 368, includes: dividing the sample interval counter by the total number of time intervals (in this example) NSampleBins The sample signal occupancy is calculated by dividing the background photon counter NBackgroundPhotons by the background interval counter. NBackgroundBins To calculate the background fluorescence level.

[0070] The sixth decision step 370 includes determining whether the average sample signal occupancy is greater than a occupancy upper limit threshold, which in this example is 1%. If the average sample signal occupancy is greater than the occupancy upper limit threshold, the method proceeds to the seventh step 372, which includes outputting an alarm to the user interface before proceeding to the eighth step 374. If the average sample signal occupancy is less than the occupancy upper limit threshold, the method proceeds directly to the eighth step 374.

[0071] Step 8, 374, includes determining whether the fluorescence background level exceeds a background level threshold. If the fluorescence background level exceeds the background level threshold, the method proceeds to step 9, 376, which includes outputting a background alarm to the user interface before proceeding to step 10, 378. If the background fluorescence level does not exceed the background level threshold, the method proceeds directly to step 10, 378.

[0072] Step 378 includes receiving the next part of the detection signal and returning to step 362.

[0073] Figure 4 A method for determining the sample mass of a fluorescently labeled sample solution in a single-molecule fluorescence spectroscopy system according to embodiments of the present disclosure is demonstrated. This method can be performed by… Figure 1 The processor or more generally the system performs this.

[0074] The first step 480 includes receiving a detection signal from the system's detector. The second step 482 includes calculating a sample signal occupancy rate, which represents the time proportion of the detection signal that includes the sample emission signal from the fluorescently labeled sample solution. The third step 484 includes outputting a sample quality signal based on the sample signal occupancy rate.

[0075] Throughout this specification, descriptive terms relating to relative orientation and position, such as “horizontal,” “vertical,” “top,” “bottom,” and “side,” are used in the sense of the orientation of the system as presented in the accompanying drawings. However, such descriptors are not intended to limit in any way the intended use of the described or claimed invention.

[0076] It should be understood that any reference to “near,” “before,” “shortly before,” “after,” “shortly after,” “above,” or “below” can refer to the parameter in question being less than or greater than a threshold, or between two thresholds, depending on the context.

Claims

1. A solution-based single-molecule fluorescence spectroscopy system, comprising: An excitation source, which is used to irradiate the excitation volume of a fluorescently labeled sample solution with excitation radiation; A detector for detecting fluorescent emission radiation emitted from a detection volume of the fluorescently labeled sample solution; One or more processors are configured to: Receive detection signals from the detector; Calculate the sample signal occupancy rate, which represents the time proportion of the detection signal including the sample emission signal from the fluorescently labeled sample solution; as well as The sample quality signal is output based on the sample signal occupancy rate.

2. The system according to claim 1, wherein the sample quality signal includes the sample signal occupancy rate.

3. The system according to claim 1 or claim 2, wherein: The one or more processors are configured to compare the sample signal occupancy rate with an upper occupancy rate threshold. and If the sample signal occupancy exceeds the upper limit threshold, the sample quality signal includes an alarm signal.

4. The system of claim 3, wherein the alarm signal includes an indication of excessive sample signal occupancy and / or a suggestion to dilute the fluorescently labeled sample solution.

5. The system of claim 3 or claim 4, wherein the one or more processors are configured to calculate a proposed dilution value based on the sample signal occupancy, and wherein the alarm signal indicates the proposed dilution value.

6. The system according to any one of claims 3 to 5, wherein the system is configured to reduce the concentration of the fluorescently labeled sample solution if the sample signal occupancy exceeds the upper occupancy threshold.

7. The system of claim 6, wherein the system includes a microfluidic sample holder, and the system is configured to reduce the concentration of the fluorescently labeled sample solution by increasing the flow rate of a first microfluidic supply line supplying a buffer solution and / or decreasing the flow rate of a second microfluidic supply line supplying the fluorescently labeled molecules.

8. The system of claim 6, wherein the system is suitable for porous samples, wherein different pores of the porous sample comprise fluorescently labeled sample solutions of different concentrations, and wherein the system is configured to reduce the concentration of the fluorescently labeled sample solution by positioning pores having relatively low concentrations of sample solution within the excitation volume.

9. The method according to claim 3, wherein: The one or more processors are configured to compare the sample signal occupancy rate with an occupancy rate lower limit threshold. and If the sample signal occupancy rate is less than the lower occupancy rate threshold, then the sample quality signal includes an alarm signal.

10. The system of claim 9, wherein the alarm signal comprises: Indications of low sample signal occupancy; recommendations for increasing the concentration of the fluorescently labeled sample solution; And / or an indication that no fluorescence peak was detected.

11. The method according to any preceding claim, wherein the one or more processors are configured to calculate the sample signal occupancy rate by: The detection signal is divided into multiple time intervals; For each time interval, if the signal level of the detected signal exceeds the sample identification threshold, then the time interval is determined to include the sample emission signal from the fluorescently labeled sample; and The sample signal occupancy rate is determined by dividing the number of time intervals including the transmitted signal by the total number of time intervals among the plurality of time intervals.

12. The method according to any one of claims 1 to 10, wherein the one or more processors are configured to: A peak detection algorithm is used to identify one or more burst peaks of the detection signal that correspond to the fluorescence emission from the fluorescently labeled sample; Determine the pulse width of each burst peak; and The sample signal occupancy rate is determined by dividing the sum of the pulse widths by the time period of the detection signal.

13. The method according to any of the preceding claims, wherein: The one or more processors are configured to determine the fluorescence background level based on the signal level of the detection signal, excluding the proportion of the sample emission signal; and The one or more processors are configured to output the sample quality signal based on the sample signal occupancy and the background fluorescence level.

14. The method according to any preceding claim, wherein the one or more processors are configured to: The detection signal is divided into multiple time intervals; For each time interval If the signal level of the detected signal exceeds the sample identification threshold, then the time interval is determined to include the sample emission signal from the fluorescently labeled sample; and If the signal level of the detected signal is less than or equal to the sample identification threshold, then the time interval is determined to include background signals; The sample signal occupancy rate is determined by dividing the number of time intervals including the transmitted signal by the total number of time intervals among the plurality of time intervals; The fluorescence background level is determined based on the average signal level of the time interval, which includes the background signal; and The sample quality signal is output based on the sample signal occupancy and the background fluorescence level.

15. The method according to any one of claims 1 to 13, wherein the one or more processors are configured to: A peak detection algorithm is used to identify one or more burst peaks of the detection signal that correspond to the fluorescence emission from the fluorescently labeled sample; The background fluorescence level is determined based on the average signal level of the region excluding burst peaks of the detected signal.

16. The system according to any one of claims 13 to 15, wherein the sample quality signal includes the background fluorescence level.

17. The system according to any one of claims 13 to 16, wherein: The one or more processors are configured to compare the fluorescence background level with a background level threshold; and If the fluorescence background level is greater than the background level threshold, then the sample quality signal includes a background alarm signal.

18. The system of claim 17, wherein the background alarm signal includes an indication of an excessively high fluorescence background level and / or a suggestion to re-prepare the sample solution buffer.

19. The system according to any of the preceding claims, wherein the processor is configured to output the sample quality signal on a continuous or semi-continuous basis.

20. A method for determining the sample mass of a fluorescently labeled sample solution in a single-molecule fluorescence spectroscopy system, the method comprising: Receive detection signals from the detector of the system; Calculate the sample signal occupancy rate, which represents the time proportion of the detection signal including the sample emission signal from the fluorescently labeled sample solution; as well as The sample quality signal is output based on the sample signal occupancy rate.

21. A computer-readable medium comprising instructions that, when executed by one or more processors, cause the one or more processors to perform the method of claim 20.

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