Validation standards and methods utilizing raman activity of h2o

Abstract

A method for measuring with a primary reference standard and for potentially adjusting and / or calibrating a high-sensitivity spectrometer via a computer includes the following steps: using a high-purity H₂O solution as a primary standard stored in a container; exciting the standard using at least one excitation laser; collecting and measuring Raman scattering from the standard using a detector of the instrument, in the form of Stokes, anti-Stokes, or both; applying a correction method to correct for variations in Raman scattering caused by the container to improve measurement accuracy; measuring and integrating the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency; and potentially adjusting and / or calibrating the high-sensitivity spectrometer based on the calculated instrument optical efficiency.

Description

Technical Field

[0001] The disclosed invention relates to a method and system for adjusting and / or calibrating a high-sensitivity spectrometer using a primary reference standard. This invention belongs to the technical field of protein detection. Background Technology

[0002] Spectroscopic equipment typically requires periodic calibration and validation during its production and throughout its lifespan. Calibration standards are commonly used for these purposes and can nominally be divided into two categories within this technical field: The first Y-axis, or intensity, is calibrated using optical peak intensity calibration. The second X-axis, or wavenumber / wavelength calibration, is used to calibrate the peak position.

[0003] Intensity calibration can be performed by measuring a broad-spectrum reference material, such as SRM 2241, and fitting the resulting curve to a defined polynomial curve of the spectrum; or by measuring a defined standard with multiple peaks and measuring and fitting the relative peak ratio.

[0004] One example is NIST Standard Reference Material (SRM) 2241, which comprises optical glass with a broadband emission spectrum. After illumination with a 785 nm line, the resulting emission exhibits a spectral shape that can be defined by a polynomial fit.

[0005] On the other hand, wavenumber calibration can be performed by measuring a defined standard or solution with multiple peaks, and fitting the measured peaks to known peaks makes it possible to establish a calibration table.

[0006] The example chosen is a 1:1 mixture of toluene and acetonitrile, a solution with known Raman peak positions.

[0007] These examples are typical for the calibration and validation of various types of spectrometers, including Raman spectrometers. Below... Figure 2 It provides intuitive instructions for x-axis and y-axis calibration.

[0008] The calibration process for fluorescence imaging microscopes may include measuring individual optical detector paths to correct their alignment mapping.

[0009] An example is a polystyrene fluorescent dye bead, which is fluorescently dyed on both the outer and inner portions of the bead to achieve focusing by measuring and calibrating the detector and pixel positions for each detector path.

[0010] Other calibration procedures for flow cytometry may include alignment verification, measurement of counting accuracy, and sorting efficiency. Examples include alignment methods using polystyrene microspheres stained with fluorescence and counting methods using fluorescent microspheres sold as calibration suspensions for flow cytometer operation.

[0011] This leads to the task of optical identification and validation for next-generation instruments used in single molecule counting (SMC). The Raman standards mentioned above are most relevant to this application, and these standards offer many advantages, such as broad-spectrum standards with well-known intensity-to-wavelength ratios, narrow transitions and peaks that enable the calculation of peak ratios, many well-defined peak positions and consistent Raman cross sections that allow for x-axis calibration, and the absence of conventional photobleaching, unlike fluorescently labeled standards.

[0012] Raman standards are well-suited for their intended applications, but they also have some significant drawbacks, such as their generally high cost (SRM 2241). The analysis requires complex post-processing, and these standards are sensitive to preparation and require special sample fixation during use.

[0013] Therefore, such standards are not optimal for some high-sensitivity instruments, such as SMC instruments that use fixed fluorescence bands for detection rather than grating-based detection optical paths.

[0014] This provides the task of developing improved ways to apply Raman standards for optical identification and verification, and, where possible, providing cheaper Raman standards themselves. Summary of the Invention

[0015] This task can be addressed by a method for adjusting and / or calibrating a high-sensitivity spectrometer via computer using a primary reference standard. This method includes the following steps: using a high-purity H₂O solution as a primary standard stored in a container; exciting the standard using at least one excitation laser to induce Raman scattering; collecting and measuring the Raman scattering from the standard using the instrument's detector, in the form of Stokes, anti-Stokes, or both; applying a correction method to correct for variations in Raman scattering caused by the container to increase measurement accuracy; measuring and integrating the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency; and adjusting and / or calibrating the high-sensitivity spectrometer based on the calculated instrument optical efficiency. Using a pure H₂O solution as the primary standard provides robustness and minimizes measurement errors and processing problems associated with existing techniques. A broad Raman spectrum of the H₂O solution can be measured, and the signal can be measured at a fixed detection band. The improved accuracy and robustness of this method make it an alternative for the measurement and potential calibration and / or adjustment of high-sensitivity spectrometers.

[0016] Advantageous and therefore preferred further improvements to the invention are derived from the relevant dependent claims, as well as the description and drawings.

[0017] One of the preferred further improvements to the disclosed method involves using a highly sensitive spectroscopic instrument to detect the protein concentration in the prepared assay solution. This highlights the application of the method in protein concentration detection, emphasizing its suitability, sensitivity, and quantitative analytical capabilities in the context of prepared assay solutions.

[0018] Another preferred further improvement to the disclosed method includes protein detection via a single-molecule detection method. Single-molecule detection methods enhance the sensitivity of the detection method, improve quantification capabilities, enable the detection of rare events, expand the dynamic range, and provide valuable insights into protein behavior. This makes single-molecule detection a powerful tool for protein detection within the framework of the stated method.

[0019] Another preferred further improvement to the disclosed method involves using a fixed optical bandpass to collect Raman scattering from the H₂O solution. Utilizing a fixed optical bandpass for both collecting and filtering Raman scattering in this method results in an improved signal-to-noise ratio, increased specificity, reduced interference, improved instrument performance, and customizability. These collectively contribute to more accurate, specific, and reliable measurements in high-sensitivity spectroscopic instruments.

[0020] Another preferred further improvement to the disclosed method includes correcting measurements for background variations from the container used as a standard. Utilizing light radiation from the container, which serves as a standard, offers advantages such as simplified setup, cost-effectiveness, inherent alignment, reduced measurement errors, enhanced stability, and compatibility with various container types. These advantages collectively contribute to improving the accuracy, reliability, and usability of the method in spectroscopic instruments.

[0021] Another preferred further improvement to the disclosed method involves using a well plate container with a transparent bottom substrate to read validation standards from one or more wells of the well plate. Well plates are widely used in laboratory environments and provide a standardized form of sample handling. By using such a plate as a container, the method becomes compatible with existing laboratory workflows and equipment. In summary, utilizing a validation well plate with a transparent substrate as a container offers advantages such as versatility, multi-sample handling capability, and compatibility with automation. These advantages collectively enhance the efficiency, reliability, compatibility, and usability of the method in laboratory environments.

[0022] Another preferred further improvement to the disclosed method involves pipetting the standard into a well plate and scanning it through a transparent substrate at the bottom of the plate. This provides advantages such as: increased accuracy in standard preparation, prevention of contamination, ease of sample handling, enhanced scanning capability, flexibility of measurement techniques, and simplified experimental setup.

[0023] Another preferred further improvement to the disclosed method involves determining the instrument's optical efficiency in the form of parameters such as the alignment of the detection and excitation optical paths, the quantification of drift or optical loss, variations in excitation power, and variations in detector efficiency. These parameters enable comprehensive evaluation, including performance assessment and benchmarking, measurements of stability and reliability, optimization of excitation power, and evaluation of detector efficiency. These advantages collectively enhance measurement capabilities and ensure the instrument's performance, accuracy, and reliability in optical measurements, thereby ensuring optimal efficiency and quality control.

[0024] Another preferred further improvement to the disclosed system includes a fixed detection bandwidth of 670 nm to 704 nm in the Raman scattering spectrum. This provides advantages such as target measurement, reduced background noise, optimal sensitivity, minimized spectral interference, compatibility with specific analytes, measurement consistency, and simplified data analysis. These advantages collectively contribute to more accurate, selective, and reliable Raman scattering analysis within a specified wavelength range.

[0025] Another preferred further improvement to the disclosed system involves applying the correction method by a computer using a correction factor equation of the form MeasurementCorr = AvgH2O - Corr × AvgEmpty, for example, via internal hardware or external computation, where MeasurementCorr is the corrected background-removed Raman integral intensity, AvgH2O is the average integral intensity of the Raman scattering from the measured container containing the standard, AvgEmpty is the average integral intensity from the empty container, and CORR is the correction factor. Using the given correction factor equation for the correction method provides advantages including robust background removal, increased accuracy of Raman measurements, specificity, reproducibility, and traceability in measurements. These advantages collectively contribute to improving the quality and reliability of measurement data in Raman scattering measurement analysis.

[0026] Another preferred further improvement to the disclosed system involves a correction factor that is constant for a single instrument design but varies for different instrument designs, excitation bands, and / or detection bands. By having a constant correction factor for a single instrument design but allowing variation for different instruments, excitation bands, and detection bands, this provides advantages such as instrument-specific calibration, customization for different configurations, improved accuracy at specific bands, comparability between instruments, adaptability to instrument upgrades, validation and calibration processes, and improved data reproducibility. These advantages collectively contribute to accurate and reliable measurements, customized calibration methods, and enhanced comparability and reproducibility of data across different instrument configurations.

[0027] Another part of the claimed invention is a system for adjusting and / or calibrating a high-sensitivity spectrometer via computer using a primary reference standard, the system being configured to perform the following steps: using a high-purity H2O solution as a primary standard stored in a container; exciting the standard using at least one excitation laser; collecting and measuring Raman scattering from the standard using a detector instrument, in the form of Stokes, anti-Stokes, or both; applying a correction method to correct for variations in Raman scattering caused by the container to increase the accuracy of the measurement; measuring and integrating the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency; and adjusting and / or calibrating the high-sensitivity spectrometer based on the calculated instrument optical efficiency.

[0028] Another part of the disclosed invention is a computer program including instructions that cause the computer involved to perform the following method steps: using a high-purity H₂O solution as a primary standard stored in a container; exciting the standard using at least one excitation laser; collecting and measuring Raman scattering from the standard using a detector of the instrument, in the form of Stokes, anti-Stokes, or both; applying a correction method to correct for variations in Raman scattering caused by the container to increase the accuracy of the measurement; measuring and integrating the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency; and making possible adjustments and / or calibrations to the high-sensitivity spectrometer based on the calculated instrument optical efficiency. The program portion responsible for each method step runs on the corresponding computer portion. How the program itself is divided depends on the computer hardware involved. Main software running on one of the aforementioned computers or a separate computer controlling local client programs can be used. Other options include identical instances of software communicating with each other, etc.

[0029] The only requirement for the computer program to execute the entire described method is that the program and its corresponding hardware components are capable of executing the method completely and automatically. Such a program can then be stored on a computer-readable storage medium and / or data carrier signal, which enables the computer involved to perform the following method steps: using a high-purity H₂O solution as a primary standard stored in a container; exciting the standard using at least one excitation laser; collecting and measuring Raman scattering from the standard using a detector instrument, in the form of Stokes, anti-Stokes, or both; applying a correction method to correct for variations in Raman scattering caused by the container, to improve measurement accuracy; measuring and integrating the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency; and adjusting and / or calibrating a high-sensitivity spectrometer based on the calculated instrument optical efficiency. The storage medium can be stored on any suitable digital memory, such as a USB drive, hard disk, flash drive, etc. The software can also be transferred to its target hardware via a remote communication device using a suitable data carrier signal such as Ethernet, wired or wireless network, or any other suitable network transmission method. Attached Figure Description

[0030] Figure 1 This illustrates an example of the optical layout of a high-sensitivity spectrometer. Figure 2 This illustrates an example of using polystyrene spectroscopy to verify performance. Figure 3 The diagram shows an approximate emission spectrum generated by excitation from a pure H2O reference material used in this invention. Figure 4 This shows an approximate Raman spectrum and a possible detection window for a particular channel. Figure 5 The graph shows real-world data demonstrating a high correlation between SMC excitation and detection optical path faults and the integrated and calibrated Raman measurands scanned in the well plate from H2O standards. Figure 6 Displaying an orifice plate template for SMC next-generation instruments. Figure 7 A diagram illustrating the importance of the experimentally derived background correction and correction factor is provided. Figure 8 This diagram illustrates the workflow of scanning the verification board for SMC next-generation performance evaluation. Detailed Implementation

[0031] The invention will be explained in more detail by presenting a preferred exemplary embodiment.

[0032] The method of this invention can be applied to various high-sensitivity optical instruments, especially SMCs. Figure 1 An example of the optical path of a confocal detection instrument is shown in the figure.

[0033] Preferred embodiments of the present invention are described below, and the use and methods of H2O as a primary reference standard for high-sensitivity spectrometers are illustrated. It includes the following features: A high-purity H2O solution was used as a primary standard for Raman scattering measurements. This H2O standard is inexpensive and widely available. The standard was pipetted into a sample container, such as a well plate, allowing for measurement using typical instrumental procedures. This is a significant advantage of instruments measuring liquid samples, as the standard can be measured using the same overall measurement hardware and process.

[0034] Then, the instrument is used to excite the laser to excite the standard.

[0035] The instrument uses a detector to measure and collect Raman scattering—Stokes, anti-Stokes, or both. The instrument collects the scattering by detecting an optical bandpass filter in the optical path.

[0036] Correction methods are applied to correct for variations in the process, such as those not related to H2O, especially light radiation from the autofluorescence of the standard container, i.e., the perforated plate substrate, to increase the accuracy of the measurement.

[0037] Intensity measurements are collected over a fixed detection bandwidth of the detector and integrated to measure the instrument's optical efficiency.

[0038] The measurement intensity is directly related to the instrument's optical efficiency and thus presents a robust measurement of the instrument's conditions and performance: (1) alignment of the detection and excitation optical paths; quantification of drift or optical loss; (2) variation of excitation power; and (3) variation of detector efficiency.

[0039] Figure 3 The Raman spectrum of H2O is now highlighted (left), along with an image of the well plate used to scan the standard during the SMC process. Next-generation SMC instruments sample at approximately 600 to 1400 cm⁻¹ in the detection optical path. -1 The H2O standard was pipetted into the well plate, and the sample was scanned from the bottom using epifluorescence.

[0040] Figure 4 The method for calibrating the system described in this invention is helpful. A fixed band of the H2O Raman spectrum is sampled, and the integrated scattering intensity is collected, integrated, and used as a measure of the optical collection efficiency of the instrument system.

[0041] It is important to note that specific intensity-to-wavelength data is not required; only the integrated optical scattering intensity over the detection window is needed. This integrated intensity can be reported as energy (e.g., joules) or measured power, such as watts or even photons per second, over a fixed time interval. A relatively flat portion of the Raman scattering can be used, and lower sensitivity to variations in the filter band can be suggested.

[0042] The simplified formula for measuring the integral intensity of Raman scattering (Equation 1) can be described as follows: Among them I Raman It is the measured Raman integral intensity; I excitation It is the excitation power of the system, especially the laser power, σ Raman The Raman cross section of H2O is assumed to be fixed and invariant under uniform conditions; EFF collection It is the system's collection efficiency, which is derived from optical collection efficiency, percentage transmittance, numerical aperture of the objective lens, filter band and detector quantum efficiency, and the efficiency of optical-to-electrical signal conversion.

[0043] It is crucial for this process that the Raman cross section of H2O can be considered constant and invariant if the sample is kept under substantially uniform conditions (i.e., room temperature). This is a prerequisite for a consistent and traceable measurement standard.

[0044] The measured Raman integral intensity can be used to evaluate the performance of key factors that characterize common failure modes and therefore require periodic verification. These are: first, the excitation optical path, which includes the intensity of the laser and the alignment or blocking of the laser with the confocal volume; and second, the detection optical path, which includes the detection optical path and the alignment or blocking of the confocal volume, as well as the detector for quantum efficiency.

[0045] The obtained changes in the measured and excitation optical paths and the detection optical paths, i.e. drift, misalignment or general faults, have high linearity. Figure 5 This includes real experimental data from SMC's next-generation instruments and demonstrates high linearity, with a near 1:1 sensitivity ratio between the measured and excitation / detection optical paths. Any optical path faults shown here were induced during challenging research into this concept.

[0046] Any application of a liquid standard requires a container to hold the standard. This offers an advantage because many instruments in biological applications use liquid samples, and H2O solutions can be prepared and dispensed in the same sample containers that are typically used.

[0047] To apply H2O standards to SMC instruments, a so-called verification well plate is used. The standard is thus pipetted into the well plate and examined by scanning through the transparent substrate at the bottom of the plate. While this application is convenient because it reflects the preparation and scanning of SMC measurements, it does present challenges: the liquid is scanned through the transparent substrate at the bottom of the well plate, which unfortunately is not constant (consistent / uniform), and can significantly reduce measurement accuracy due to the following issues: a) The substrate exhibits autofluorescence, resulting in a noticeable and varied background.

[0048] b) The background will be incorporated into the Raman measurement and will be indistinguishable.

[0049] c) Autofluorescence is often inconsistent across the entire well plate or between different well plates and well plate batches.

[0050] d) Autofluorescence can change over time, for example, through photobleaching.

[0051] To address this issue, two solutions can be applied to the preferred example: 1. Measure the "empty" perforated plate holes (substrate only) simultaneously using the following steps: a. Measure the pore size near the H2O sample well to ensure similarity between material measurements and calibrations.

[0052] b. Measure multiple pores and H2O sample pores for statistical averaging.

[0053] c. Porosity can be measured before filling with H2O. However, this adds inconvenience and an extra process step.

[0054] 2. For the second solution (which is particularly preferred for the embodiments), a new subtraction formula is used to correct for large changes in the background, which has the following characteristics: a. This deduction formula uses a correction factor.

[0055] b. The correction factor is determined empirically to compensate for the difference between the void measurement and the H2O void measurement. Simply put, based on various factors including Fresnel reflection differences (COP-air interface vs. COP-H2O interface), the H2O voids are less affected by the substrate background of the perforated plate.

[0056] c. For a single instrument design, the correction factor can be considered constant, but it can be optimized for different instrument designs, excitation bands, and / or detection bands.

[0057] d. The correction factor improves the accuracy of subtraction and is especially necessary when there are significant changes in the substrate background.

[0058] Figure 6 The image shows a typical implementation of the validation board and correction factor method for SMC's next-generation instruments. Twelve wells are empty for background correction, and twelve wells are filled with H₂O.

[0059] The correction factor used for the background of the correction board substrate is expressed as Equation (Equation 2) as follows.

[0060] Measurement Corr It is the primary measurand in the verification process, with corrected background-removed Raman integral intensity; Avg H2O It is the average integrated intensity from the H2O sample pores, serving as a measure of the H2O Raman characteristic; Avg Empty It is the average integrated intensity from the voids, a measure of the substrate background, and CORR is a correction factor, where 0.85 is used for SMC next-generation instruments.

[0061] Figure 7 The importance of background correction and the modified correction factor discussed in Equation 2 is illustrated. When a correction factor of 0.85 is used (top), substrate variations caused by different well plates, photobleaching of substrate fluorescence, and other factors are almost eliminated, resulting in a flat Raman integral measurement over 4 months. When simple subtraction is performed without this 0.85 factor (middle), the slope trend increases sharply by 33-fold. When no correction / subtraction is used (bottom), the trend continues to increase, indicating the significant impact of background fluorescence variability on the measurement results.

[0062] This type of sensitivity and calibration may not be necessary for all spectroscopic instruments, but it is important for sensitive measurement systems such as... Figure 1 The SMC system shown is very important.

[0063] The following describes specific preferred operating modes with reference to preferred embodiments of the invention. It describes the use of the invention in the field for verification. Figure 1 This illustrates the general applications of SMC's next-generation instruments. (For example...) Figure 6 As shown, a validation plate was prepared by pipetting a 12-well H2O solution into the well plate. The H2O and empty wells were scanned, and a background-corrected integrated Raman measurement was calculated using a background correction algorithm (see Equation 2). This measurand was then compared to the acceptance limits to determine whether the instrument's optical performance was acceptable or unacceptable.

[0064] The workflow of this process is as follows: Figure 8 As shown in the image.

[0065] The following shows typical pass / fail limits for SMC's next-generation red laser scanning. Note that for more stringent test specifications, such as when performing end-of-line testing, multiple detectors, such as Stokes and anti-Stokes Raman intensities, may be checked for measurement and specification limits.

[0066] This method has many advantages, the most important of which are as follows: 1. A simplified and cost-effective solution. Using H2O in solution as a Raman standard offers a streamlined solution for high-sensitivity spectroscopic instruments. H2O is a biocompatible liquid and can be prepared in forms similar to those used in many instrument types, such as by pipetting into well plates. High-purity H2O is readily available and inexpensive, making it suitable for numerous applications, including cell biology, buffer preparation, and chromatography.

[0067] As a comparison—considering the given preferred embodiment, using high-purity water (EMD #W4502) as another example, and comparing it to using NIST SRM #2241 (a common Raman intensity standard). In SMC applications, less than 1 mL of H2O solution is required per validation plate, costing less than $0.10 per plate in bulk reagent. This solution can be directly pipetted into the well plates for measurement using standard hardware and procedures. For many applications, including SMC, SRM #2241 requires custom fixtures and measurement procedures, often costing thousands of dollars. Clearly, H2O is disposable after use, eliminating concerns about contamination and storage, while SRM #2241 requires careful storage and reuse, and is likely only used as a high-level traceability standard.

[0068] 2. Robust solution with background correction A typical confocal volume size of 10 cubic micrometers would contain approximately 33.5 billion H2O molecules in a primary aqueous solution. High-purity yet relatively inexpensive H2O can provide trace impurities of less than one part per billion, resulting in highly averaged measurements where the proportion of detected H2O to non-H2O molecules is very high. Combined with the consistent Raman cross section of H2O (at room temperature), this provides robust Raman integration intensity measurements and thus offers robust and reproducible standards.

[0069] The disclosed background subtraction method significantly reduces the impact of the perforated plate substrate background on the process. The perforated plate substrate background is particularly important because it can vary between different batches of perforated plates due to substrate chemistry and impurities, and also undergoes photobleaching over time due to repeated scanning. See Equation 2 and... Figure 7 .

[0070] 3. High linearity of optical efficiency, including excitation / detection optical path conditions. As stated in Equation 1 and Figure 5 As shown, the present invention exhibits high sensitivity and linearity (close to 1:1) to the optical efficiency of the instrument. Detailed challenge experiments have been conducted, demonstrating that faults in the excitation and detection optical paths of the instrument are easily identifiable in the measured quantity.

[0071] 4. Can be used as an end-of-line manufacturing tool and service verification tool. As previously mentioned, this solution can be easily deployed using standard procedures by pipetting H2O into a well plate or any other standard container and subsequently performing an instrument scan. Because Figure 7 With visible background subtraction, the resulting measurements are robust and sensitive to major instrument failure modes. See [link to documentation]. Figure 5 This provides a tool with low false negatives and high confidence levels for evaluating instrument optical efficiency and thus overall system performance.

[0072] This tool is robust and can be easily deployed as an "end-of-line" test to ensure the instrument's operation after assembly and before it is stored in warehouses. It is also a valuable tool for field personnel and / or scientist customers to continuously measure the instrument's performance throughout its lifespan.

Claims

1. A method for adjusting and / or calibrating a high-sensitivity spectrometer using a primary reference standard via a computer, comprising the following steps: - Use high-purity H2O solution as the primary standard stored in the container. - The standard is excited using at least one excitation laser that induces Raman scattering. - The Raman scattering of the standard object is collected and measured using the instrument's detector, in the form of Stokes, anti-Stokes, or both. - A correction method is applied to correct for changes in Raman scattering caused by the container, thereby improving measurement accuracy. - Measure and integrate the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency. - Adjust and / or calibrate the high-sensitivity spectrometer based on the calculated instrument optical efficiency.

2. The method according to claim 1, wherein the high-sensitivity spectrometer is used to detect the protein concentration in the prepared assay solution.

3. The method according to claim 2, wherein the protein detection is performed by a single-molecule detection method.

4. The method of claim 1, wherein a fixed optical bandpass is used to collect Raman scattering from the H2O solution.

5. The method of claim 1, wherein the measurement is corrected for background changes from the container serving as a standard container.

6. The method of claim 1, wherein the perforated plate having a bottom transparent substrate is used as a container.

7. The method of claim 6, wherein the standard is pipetted into the well plate and inspected by scanning through a transparent substrate at the bottom of the plate.

8. The method of claim 1, wherein the optical efficiency of the instrument is determined in the form of parameters such as alignment, drift, or quantification of optical loss of the detection and excitation optical paths, changes in excitation power, and changes in detector efficiency.

9. The method according to claim 1, wherein the fixed detection bandwidth in the Raman scattering spectrum is 670 nm to 704 nm.

10. The method of claim 1, wherein the correction method is implemented by the computer using a correction factor equation of the form: Measurement Corr =Avg H2O - Corr xAvg Empty , Measurement Corr It is the corrected Raman integral intensity after background removal, Avg H2O It is the average integrated intensity of Raman scattering from a container holding a standard, Avg. Empty is the average integral intensity of the empty container, and CORR is the correction factor.

11. The method of claim 10, wherein the correction factor is constant for a single instrument design, but varies for different instrument designs, excitation bands, and / or detection bands.

12. A system for adjusting and / or calibrating a high-sensitivity spectrometer using a primary reference standard via a computer, the system being configured to perform the following steps: - Use high-purity H2O solution as the primary standard stored in the container. - Excite the standard object using at least one excitation laser. - The detector of the instrument is used to collect and measure Raman scattering from the standard, which is of the form of Stokes, anti-Stokes, or both. - A correction method is applied to correct for changes in Raman scattering caused by the container, thereby improving measurement accuracy. - Measure and integrate the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency. - Adjust and / or calibrate the high-sensitivity spectrometer based on the calculated instrument optical efficiency.

13. A computer program comprising instructions that cause the computer concerned to control hardware components of a corresponding configuration to perform the following method steps: - Use high-purity H2O solution as the primary standard stored in the container. - Excite the standard object using at least one excitation laser. - The detector of the instrument is used to collect and measure Raman scattering from the standard, which is of the form of Stokes, anti-Stokes, or both. - A correction method is applied to correct for changes in Raman scattering caused by the container, thereby improving measurement accuracy. - Measure and integrate the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency. - Potential adjustments and / or calibrations can be made to high-sensitivity spectrometers based on the calculated instrument optical efficiency.

14. A computer-readable storage medium and / or data carrier signal, having stored thereon a computer program according to claim 13, the computer program causing the computer involved to control correspondingly configured hardware components to perform the following method steps: - Use high-purity H2O solution as the primary standard stored in the container. - Excite the standard object using at least one excitation laser. - The detector of the instrument is used to collect and measure Raman scattering from the standard, which is of the form of Stokes, anti-Stokes, or both. - A correction method is applied to correct for changes in Raman scattering caused by the container, thereby improving measurement accuracy. - Measure and integrate the intensity over a fixed detection bandwidth of the Raman scattering spectrum to calculate the instrument's optical efficiency. - Potential adjustments and / or calibrations can be made to the high-sensitivity spectrometer based on the calculated instrument optical efficiency.

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