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DE-102024133072-A1 - Methods for determining measurement settings of spectral parameters for chemical and/or biological samples in a microplate, methods for measuring samples and microplate measurement systems

DE102024133072A1DE 102024133072 A1DE102024133072 A1DE 102024133072A1DE-102024133072-A1

Abstract

The invention relates to a method for determining measurement settings of spectral parameters and preferably further measurement settings for chemical and/or biological samples (3a) in a microplate (2) in a microplate reader (1) by means of an optimization algorithm, wherein at least one sample (3a) to be analyzed is provided in a well (3) of the microplate (2) and wherein the optimization algorithm varies as spectral parameters at least an emission wavelength of the at least one sample (3a), an excitation wavelength of a light source and/or a bandwidth of the emission wavelength and/or excitation wavelength, comprising the following process steps: A. Setting and saving initial values of the spectral parameters; B. Measuring and storing intensities of at least one emission of the sample (3a) at the initial values set in process step A; C. Determining and storing the value of a quantity to be optimized from the measurement results and evaluating termination conditions of the optimization algorithm; D. Calculating values of the spectral parameters according to the optimization algorithm, in particular depending on previously stored values of the quantity to be optimized, and preferably depending on previously stored values of the spectral parameters; E. Setting the values of the spectral parameters calculated in process step D, as well as measuring and storing the emission intensities of the sample, wherein process steps C, D, and E are optionally repeated until at least one of the termination conditions evaluated in process step C is met. The invention further relates to a method for measuring chemical and/or biological samples with a microplate reader (1) and a microplate measuring system.

Inventors

  • Mario Schneider

Assignees

  • BMG LABTECH GMBH

Dates

Publication Date
20260513
Application Date
20241112

Claims (17)

  1. Method for determining measurement settings of spectral parameters and preferably further measurement settings for chemical and/or biological samples (3a) in a microplate (2) in a microplate reader (1) by means of an optimization algorithm, in which at least one sample (3a) to be analyzed is provided in a well (3) of the microplate (2) and in which the optimization algorithm varies at least one emission wavelength of the at least one sample (3a), one excitation wavelength of a light source and/or one bandwidth of the emission wavelength and/or excitation wavelength as spectral parameters, comprising the following process steps: A. Setting and storing initial values of the spectral parameters; B. Measuring and storing intensities of at least one emission of the sample (3a) at the initial values set in process step A; C. Determining and storing the value of a quantity to be optimized from the measurement results and evaluating termination conditions of the optimization algorithm; D. Calculating values of the spectral parameters according to the optimization algorithm, in particular depending on previously stored values of the quantity to be optimized, and preferably depending on previously stored values of the spectral parameters; E. Setting the values of the spectral parameters calculated in process step D, as well as measuring and storing the emission intensities of the sample, where process steps C, D, and E are repeated as necessary until at least one of the termination conditions evaluated in process step C is met.
  2. Procedure according to Claim 1 , characterized in that the optimization algorithm corresponds to a sequential simplex algorithm and in particular in procedure step B p+1 p-tuples of initial values of the spectral parameters are set, where p is the number of spectral parameters and procedure step C is carried out for each of the p-tuples.
  3. Method according to one of the preceding claims, characterized in that a further method step A01 is carried out before method step A, in which at least one boundary condition with respect to the spectral parameters is entered, and that in method step A the initial values of the spectral parameters are set taking into account the at least one boundary condition.
  4. Method according to one of the preceding claims, characterized in that a further method step A02 is carried out before method step A, in which parameters and/or additional termination conditions of the optimization algorithm are entered.
  5. Method according to one of the preceding claims, characterized in that the microplate reader (1) comprises an excitation light source as a light source, and that in method step C the determination of the value of a quantity to be optimized comprises a correction which depends on the excitation wavelength of the excitation light source.
  6. Procedure according to Claim 5 , characterized in that in process step C the correction is carried out using a reference signal.
  7. Procedure according to Claim 5 or Claim 6 , characterized in that in process step C the correction is carried out using a table in which spectral data of the excitation light source are stored.
  8. A method according to one of the preceding claims, characterized in that the method is carried out with at least one sample (3a) and at least one reference sample, and in that the quantity to be optimized depends on a measured intensity of emitted light of the at least one sample (3a) and a measured intensity of emission of the at least one reference sample, preferably that the quantity to be optimized depends on a functional relationship between at least one sample (3a) and at least one reference sample, in particular preferably that the quantity to be optimized depends on the ratio of a measured intensity of emission of the at least one sample (3a) and a measured intensity of emission of the at least one reference sample.
  9. Method according to one of the preceding claims, characterized in that the method is carried out with at least one luminophore sample as sample (3a).
  10. Method according to one of the preceding claims, characterized in that the reference sample is formed by a blank sample (3c).
  11. Method according to one of the preceding claims, characterized in that, after a termination condition in process step C is fulfilled, a process step F is carried out, in which the stored values of the quantity to be optimized are compared with each other and the values of the associated measurement settings are adjusted.
  12. Method according to one of the preceding claims, characterized in that in method step A the initial values of the spectral parameters depend on results of a preliminary measurement or a previous execution of the method.
  13. A method for measuring chemical and/or biological samples using a microplate reader (1), comprising a receiving device (4) for receiving a microplate (2) having a plurality of wells (3) in which samples (3a) to be analyzed are arranged, and an optical detector (5) for detecting emission at each of the wells of the microplate (2) received in the receiving device (4), wherein the receiving device (4) and/or the optical detector (5) are movably arranged relative to each other in order to position the received microplate (2) with respect to the optical detector (5) for successive measurements at different wells (3), wherein the method comprises the following steps: V1. Performing the method for determining measurement settings of spectral parameters according to one of the Claims 1 until 12 ; V2. Setting the optimized measurement settings of the spectral parameters; V3. Performing at least one measurement of spectral properties of at least one sample (3a) using the microplate reader (1).
  14. Microplate measuring system (10) for chemical and/or biological samples (3a) comprising a microplate reader (1), a computing unit (6) and an output interface (8), wherein the microplate reader (1) comprises a receiving device (4) for receiving a microplate (2) having a plurality of wells (3) in which wells (3) to be analyzed can be arranged, and an optical detector (5) for detecting an emission at each of the wells of the microplate (2) received in the receiving device (4), wherein the receiving device (4) and/or the optical detector (5) are movably arranged relative to each other in order to position the received microplate (2) with respect to the optical detector (5) for successive measurements at different wells (3), wherein the microplate reader (1) further comprises an input interface (7) for setting spectral parameters and for executing control commands, the microplate reader (1) and the computing unit (6) are interconnectable via data processing, and wherein the computing unit (6) is interconnectable via data processing with the output interface (8), characterized in that the computing unit (6) is configured to perform a method for determining measurement settings of spectral parameters according to one of the Claims 1 until 12 to be carried out and is set up to interact with the output interface (8) in such a way that calculation results are accessible to the computing unit (6).
  15. microplate measuring system (10) according to Claim 14 , characterized in that the microplate reader (1), the computing unit (6) and the output interface (8) are designed to work together as a single device.
  16. microplate measuring system (10) according to one of the Claims 14 or 15 , characterized in that the microplate reader (1), the computing unit (6), the input interface (7) and/or the output interface (8) are designed as units that are physically and technically separable from one another.
  17. microplate measuring system (10) according to one of the Claims 14 until 16 , characterized in that the computing unit (6) executes software which is configured to transmit user inputs via a data connection between the computing unit (6) and the microplate reader (1) to the input interface (7) of the microplate reader (1), and the input interface (7) of the microplate reader (1) is configured to interpret the transmitted user inputs.

Description

The invention relates to a method for determining measurement settings of spectral parameters for chemical and/or biological samples in a microplate in a microplate reader by means of an optimization algorithm, a method for measuring chemical and/or biological samples with a microplate reader and a microplate measurement system for chemical and/or biological samples. Microplate readers are well-known from the prior art. They are used to examine biological and/or chemical samples placed in microplates. A microplate typically has several wells for holding sample material. When examining chemical and/or biological samples with a microplate reader, the microplate is first prepared with the sample material. The prepared microplate is placed into a designated well of the microplate reader, and the samples within are examined for their optical properties. Commonly examined properties include absorption and various forms of luminescence, such as fluorescence or phosphorescence. For this purpose, the microplate reader has at least one optical detector that measures light emitted by or transmitted through the sample. Finally, the light intensity or a recorded light spectrum is analyzed to determine, for example, the biochemical composition of the sample. For the optical analysis of a sample's emission light, those wavelengths of the emission spectrum are typically selected for which the measurement is most sensitive. Fluorescent samples are irradiated with light to optically excite them. Since the spectrum of the excitation light affects the emission spectrum, the sensitivity in this case also depends on the selected wavelength of the excitation light. These spectral parameters—in the case of fluorescence measurements, in particular the excitation and emission wavelengths and their bandwidths—are currently set manually and successively varied to find the sensitivity optimum. This method is time-consuming and prone to errors. If the sample composition is completely unknown, it may be necessary to perform measurements across the entire adjustable wavelength range of the spectral parameters. A system for the automated adjustment of spectral parameters is, for example, from the US 8,723,139 B2 This is known. This shows a system for the spectral analysis of fluorophores using a spectrofluorometer. A user can specify an interval for the emission and excitation wavelengths, as well as a wavelength step size, via an input interface. Starting with the lowest excitation/emission wavelength pair, these values are sent to a control module, which instructs the spectrofluorometer to measure the fluorescence intensities of a sample at these wavelengths. Then, one of the two wavelengths is changed by a step size and measured again. The sample is measured successively at all possible excitation and emission wavelengths. The entire procedure is also performed for a blank sample. For each measurement at an excitation/emission wavelength pair, the ratio of the intensities of the two samples is determined. This ratio serves as a measure of the optimal sensitivity. By finally determining the largest value for the ratio of intensities, the optimal spectral parameters are obtained. A disadvantage of this method is that a large number of measurements must be performed. Determining the optimal spectral parameters for optical measurements, especially of uncharacterized samples, is a complex process using known methods. For example, with fluorescent samples, either emission spectra for many wavelengths of the excitation light or excitation spectra (spectra of the excitation light) for many emission wavelengths are generated. Depending on these two parameters, a quantity to be optimized can be defined and calculated. Each additional spectral parameter increases the dimensionality of the parameter space spanned by the spectral parameters, thus exponentially increasing the complexity of the procedure. Therefore, a large number of measurements must be performed, making the known method for determining optimal measurement settings time-consuming and generating a large amount of data. The object of the present invention is therefore to improve the previously known method for determining optimal measurement settings of spectral parameters for chemical and/or biological samples in a microplate in a microplate reader. The problem is solved by a method for determining measurement settings of spectral parameters using an optimization algorithm according to claim 1, by a method for measuring chemical and/or biological samples with a microplate reader according to claim 13. as well as by a microplate measuring system according to claim 14. Advantageous embodiments of the method for determining measurement settings of spectral parameters using an optimization algorithm are set out in claims 2 to 12. Preferred configurations of the microplate measurement system are shown in claims 15 to 17. The aforementioned problem, as well as further problems, are solved b