CN-122029418-A - Single molecule fluorescence detection system and method

Abstract

A solution-based single molecule fluorescence spectroscopy system (100) includes an excitation source (102) for illuminating an excitation volume of a fluorescently labeled sample solution (104) with excitation radiation, a detector (106) for detecting fluorescent emission radiation emitted from a detection volume (128) of the fluorescently labeled sample solution, one or more processors (108) configured to receive detection signals from the detector, calculate a sample signal occupancy representing a proportion of time that the detection signals include sample emission signals from the fluorescently labeled sample solution, and output a sample mass signal based on the sample signal occupancy.

Inventors

• Timothy David Kregs
• Benjamin Mark Ambrose
• Elliott McCallum Steele

Assignees

• 谢菲尔德大学

Dates

Publication Date

20260512

Application Date

20230929

Claims (20)

1. 1. A solution-based single molecule fluorescence spectroscopy system, comprising: An excitation source for irradiating an excitation volume of a fluorescently labeled sample solution with excitation radiation; A detector for detecting fluorescence emission radiation emitted from a detection volume of the fluorescence labelled sample solution; one or more processors configured to: receiving a detection signal from the detector; Calculating a sample signal occupancy representing a proportion of time that the detection signal comprises a sample emission signal from the fluorescently labeled sample solution, and A sample mass signal is output based on the sample signal occupancy.
2. 2. The system of claim 1, wherein the sample mass signal comprises the sample signal occupancy.
3. 3. The system of claim 1 or claim 2, wherein: The one or more processors are configured to compare the sample signal occupancy to an occupancy upper threshold, and The sample quality signal includes an alarm signal if the sample signal occupancy exceeds the occupancy upper threshold.
4. 4. A system according to claim 3, wherein the alarm signal comprises an indication of an excessive occupancy of the sample signal and/or a suggestion of dilution of the fluorescently labeled sample solution.
5. 5. The system of claim 3 or claim 4, wherein the one or more processors are configured to calculate a suggested dilution value based on the sample signal occupancy, and wherein the alarm signal indicates the suggested dilution value.
6. 6. The system of any one of claims 3 to 5, wherein the system is configured to decrease the concentration of the fluorescently labeled sample solution if the sample signal occupancy exceeds the occupancy upper threshold.
7. 7. The system of claim 6, wherein the system comprises a microfluidic sample holder and the system is configured to reduce the concentration of the fluorescently labeled sample solution by increasing a flow rate of a first microfluidic supply line supplying a buffer solution and/or decreasing a flow rate of a second microfluidic supply line supplying a fluorescently labeled molecule.
8. 8. The system of claim 6, wherein the system is adapted for a porous sample, wherein different wells of the porous sample comprise different concentrations of a fluorescently labeled sample solution, and wherein the system is configured to reduce the concentration of the fluorescently labeled sample solution by positioning wells having a relatively low concentration of sample solution in the excitation volume.
9. 9. A method according to claim 3, wherein: The one or more processors are configured to compare the sample signal occupancy to an occupancy lower threshold, and The sample mass signal comprises an alarm signal if the sample signal occupancy is less than the occupancy lower threshold.
10. 10. The system of claim 9, wherein the alarm signal comprises 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.
11. 11. The method of any preceding claim, wherein the one or more processors are configured to calculate the sample signal occupancy by: Dividing the detection signal into a plurality of time intervals; For each time interval, determining that the time interval includes a sample emission signal from the fluorescently labeled sample if the signal level of the detection signal exceeds a sample identification threshold, and The sample signal occupancy is determined as the number of time intervals comprising the transmitted signal divided by the total number of time intervals of the plurality of time intervals.
12. 12. The method of any one of claims 1 to 10, wherein the one or more processors are configured to: Performing a peak detection algorithm to identify one or more burst peaks of the detection signal corresponding to fluorescent emissions from the fluorescently labeled sample; Determining the pulse width of each burst peak, and The sample signal occupancy is determined as the sum of the pulse widths divided by the time period of the detection signal.
13. 13. The method of any preceding claim, wherein: The one or more processors are configured to determine a fluorescence background level based on a signal level of the detection signal that does not include a proportion of the sample emission signal, and The one or more processors are configured to output the sample mass signal based on the sample signal occupancy and the background fluorescence level.
14. 14. The method of any preceding claim, wherein the one or more processors are configured to: Dividing the detection signal into a plurality of time intervals; for each time interval of time it is possible, Determining that the time interval includes a sample emission signal from the fluorescently labeled sample if the signal level of the detection signal exceeds a sample identification threshold, and Determining that the time interval includes a background signal if the signal level of the detection signal is less than or equal to the sample identification threshold; Determining the sample signal occupancy as a number of time intervals comprising the transmitted signal divided by a total number of time intervals of the plurality of time intervals; determining a fluorescence background level based on an average signal level of the time interval including the background signal, and The sample quality signal is output based on the sample signal occupancy and the background fluorescence level.
15. 15. The method of any one of claims 1 to 13, wherein the one or more processors are configured to: Performing a peak detection algorithm to identify one or more burst peaks of the detection signal corresponding to fluorescent emissions from the fluorescently labeled sample; A background fluorescence level is determined based on an average signal level of a region of the detection signal that does not include a burst peak.
16. 16. The system of any one of claims 13 to 15, wherein the sample mass signal comprises the background fluorescence level.
17. 17. The system of any of claims 13 to 16, wherein: the one or more processors are configured to compare the fluorescence background level to a background level threshold, and The sample quality signal comprises a background alarm signal if the fluorescent background level is greater than the background level threshold.
18. 18. The system of claim 17, wherein the background alarm signal comprises an indication of an excessive level of the fluorescent background and/or a suggestion to re-prepare sample solution buffer.
19. 19. The system of any preceding claim, wherein the processor is configured to output the sample mass signal on a continuous or semi-continuous basis.
20. 20. A method of determining the sample mass of a fluorescently labeled sample solution in a single molecule fluorescence spectroscopy system, the method comprising: Receiving a detection signal from a detector of the system; Calculating a sample signal occupancy representing a proportion of time that the detection signal comprises a sample emission signal from the fluorescently labeled sample solution, and A sample mass signal is output based on the sample signal occupancy.

Description

Single molecule fluorescence detection system and method Technical Field The present disclosure relates to a solution-based single-molecule fluorescence spectroscopy system and a method for analyzing sample mass of a sample in the system. Background Single molecule forster resonance energy transfer (smFRET) is a fluorescence technique used to study single molecules. smFRET can enable the determination of the different biomolecular conformations present in the (dynamic) equilibrium and the corresponding conformational transition rates on a time scale associated with critical cellular processes such as protein (unfolding), transcription, and DNA replication and repair. Confocal experiments are also the preferred method for generating multiple smFRET constraints for integrated structural modeling due to simple sample preparation, fast data acquisition, and high temporal resolution. Disclosure of Invention According to a first aspect of the present disclosure, there is provided a solution-based single molecule fluorescence spectroscopy system comprising: An excitation source for irradiating an excitation volume of a fluorescently labeled sample solution with excitation radiation; A detector for detecting fluorescence emission radiation emitted from a detection volume of the fluorescence labelled sample solution; One or more of the processors of the present invention, the one or more processors are configured to: receiving a detection signal from the detector; calculating a sample signal occupancy representing a time proportion of the detection signal including a sample emission signal from the fluorescently labeled sample solution, and A sample quality signal is output based on the sample signal occupancy. The sample mass signal may comprise a sample signal occupancy. The one or more processors may be configured to compare the sample signal occupancy to an occupancy upper threshold. The sample quality signal may include an alarm signal if the sample signal occupancy exceeds an occupancy upper threshold. The alarm signal may include an indication of too high a sample signal occupancy and/or a suggestion to dilute the fluorescently labeled sample solution. The one or more processors may be configured to calculate a recommended dilution value based on the sample signal occupancy, and wherein the alarm signal indicates the recommended dilution value. The system may be configured to decrease the concentration of the fluorescently labeled sample solution if the sample signal occupancy exceeds an occupancy upper threshold. The system may include a microfluidic sample holder. The system may be configured to reduce the concentration of the fluorescently labeled sample solution by increasing the flow rate of a first microfluidic supply line that supplies a buffer solution and/or decreasing the flow rate of a second microfluidic supply line that supplies fluorescently labeled molecules. The system may be adapted for use with a porous sample, wherein different wells of the porous sample comprise different concentrations of the fluorescently labeled sample solution. The system may be configured to reduce the concentration of the fluorescent-labeled sample solution by positioning an aperture having a relatively low concentration of the sample solution in the excitation volume. The one or more processors may be configured to compare the sample signal occupancy to an occupancy lower threshold. The sample quality signal may include an alarm signal if the sample signal occupancy is less than the occupancy lower threshold. The alarm signal may include an indication that the occupancy of the sample signal 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. The one or more processors may be configured to calculate the sample signal occupancy by: Dividing the detection signal into a plurality of time intervals; For each time interval, determining that the time interval includes a sample emission signal from the fluorescently labeled sample if the signal level of the detection signal exceeds a sample identification threshold, and The sample signal occupancy is determined as the number of time intervals comprising the transmitted signal divided by the total number of time intervals of the plurality of time intervals. The one or more processors may be configured to: performing a peak detection algorithm to identify one or more burst peaks of the detection signal corresponding to fluorescent emissions from the fluorescently labeled sample; Determining the pulse width of each burst peak, and The sample signal occupancy is determined as the sum of the pulse widths divided by the time period of the detection signal. The one or more processors may be configured to determine the fluorescence background level based on a signal level of the detection signal that does not include a proportion of the sample emission signal. The one or mor