Method and measurement setup for the investigation of organic material

Abstract

The invention relates to a method for examining organic material, wherein the organic material is irradiated with excitation radiation (3), and wherein the intensity of fluorescent radiation (14) excited by the irradiation is determined. The organic material is additionally irradiated with electromagnetic scattered radiation (16), in which scattering occurs on the material and the scattered radiation (19) is absorbed. Furthermore, the invention relates to a corresponding measuring arrangement.

Description

[0001] Method and measuring arrangement for the investigation of organic material

[0002] This application claims priority from German patent application DE 10 2022 107 746.2 of March 31, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] The invention relates to a method for examining, in particular for determining the vitality of organic material and to a measuring arrangement for carrying out the method according to the invention.

[0004] Knowledge of the vitality of organic material, i.e., the state of cells in nutrient solutions, for example, is advantageous in a variety of possible applications. For example, in the food industry, it may be of interest to monitor fermentation processes in situ with regard to the activity of the organisms involved. The same applies to the production of pharmaceuticals or the analysis of cell cultures in the medical field.

[0005] It is known that the presence of NADH (nicotinamide adenine dinucleotide) can be used as an indicator of cell vitality. NADH exhibits fluorescence in the range of 450 nm when irradiated with electromagnetic radiation with a wavelength of approximately 340 nm. However, the presence of fluorescence alone is often insufficient to make quantitative statements about the state of organic material, especially living cells.

[0006] The object of the present invention is to provide a method and a measuring arrangement which allows an improved determination of the state of living cells.

[0007] This object is achieved by a method having the features of independent claim 1 and a measuring device having the features of independent claim 15. The subclaims relate to advantageous developments and variants of the invention. In a method for examining, in particular for determining the vitality of organic material, the organic material is irradiated with excitation radiation, for example in the wavelength range of 340 nm, and the intensity of the fluorescence radiation excited by the irradiation is determined. According to the invention, the organic material is additionally irradiated with electromagnetic scattered radiation, during which scattering occurs on the material, and the scattered radiation is subsequently recorded. Scattered radiation in the sense of the present application is therefore to be understood as radiation irradiated onto the material and not the scattered radiation mentioned above.The scattering measurement allows statements to be made about the concentration of organic material in the sample volume, so that the measured fluorescence radiation can be better interpreted using this additional information.

[0008] In particular, the scattered radiation and / or the fluorescent radiation can be recorded at at least two different angles relative to an irradiation direction, wherein a first angle can be between 160° and 200°, in particular 180°.

[0009] Furthermore, a second angle can be between 70° and 110°, in particular 90°; a third angle can be between 0° and 40°, in particular 20°.

[0010] This angular distribution makes it possible to make statements about vitality, for example, over a wide range of turbidity levels and thus concentrations of organic material.

[0011] In particular, the organic material can be irradiated alternately with excitation radiation and scattered radiation, so that a simple assignment of excited fluorescence and scattering is possible.

[0012] In cases where the scattered radiation has a wavelength in the range of 450nm, a single detector type can be used advantageously.

[0013] Alternatively, an apparatus can be constructed in which the radiation source and detectors are exchanged so that one detector and multiple radiation sources generate the same excitation / emission geometry from different angles.

[0014] The excitation radiation can be in the 340 nm range to determine cell viability. If the organic material is irradiated with excitation radiation in the 260-280 nm wavelength range, the protein content in the sample volume can be determined using an additional fluorescence measurement.

[0015] Furthermore, the organic material can be irradiated with scattered radiation in the wavelength range of 650nm or 850nm.

[0016] To determine the vitality of the organic material, the intensities of scattered radiation and fluorescence radiation can advantageously be mathematically related to each other; for example, a quotient can be formed. It is also conceivable to use more complex analysis models, such as adapted multivariate analysis or algorithms with artificial intelligence, to link the measurements in such a way that the vitality parameter can be determined in a self-learning manner.

[0017] By directing the excitation radiation and the scattered radiation onto the sample volume at least in sections along the same path, the significance of the measurement can be further improved.

[0018] A measuring arrangement according to the invention for the optical examination of organic material comprises

[0019] - a sample volume to hold the material

[0020] - at least one first electromagnetic radiation source for irradiating the material in one irradiation direction with excitation radiation, in particular with a wavelength of 340 nm or 260-280 nm

[0021] - at least one excitation detector which is configured to detect fluorescence radiation excited by the excitation radiation, in particular only electromagnetic radiation in a wavelength range of 430 nm to 470 nm, preferably in a wavelength range of 450 nm, or in a wavelength range of 280 nm to 350 nm - at least one second radiation source which is configured to additionally irradiate the organic material with scattered electromagnetic radiation

[0022] - at least one detector designed to detect the scattered radiation.

[0023] The measuring arrangement can be realized in a simple manner by using the detector for detecting the scattered radiation as the excitation detector.

[0024] It is advantageous if the second radiation source is suitable for emitting electromagnetic radiation in a wavelength range of 450nm or 650nm or 850nm.

[0025] In particular, the sample volume can be arranged in a traditionally manufactured and cleanable flow cell made of classic materials (stainless steel, PEEK, Teflon, sapphire, etc.) or in the form of a single-use flow cell with an inlet and outlet for the material to be analyzed; the realization of the measuring arrangement according to the invention as a probe, in particular as an immersion probe, is also conceivable.

[0026] In a further advantageous embodiment of the invention, a further radiation source is provided which is configured to emit only electromagnetic radiation with a wavelength of 260 nm to 280 nm; furthermore, at least one detector is provided which is configured to detect only electromagnetic radiation in a wavelength range of 280 nm to 350 nm.

[0027] The transmission direction of a radiation source and the reception direction of a corresponding detector can in particular enclose an angle of 170°-200°, in particular of 180°.

[0028] Furthermore, the transmission direction of a radiation source and the reception direction of a corresponding detector can enclose an angle of 70°-110°, in particular 90°. Additionally or alternatively, the transmission direction of a radiation source and the reception direction of a corresponding detector can enclose an angle of 0°-40°, in particular 20°.

[0029] In an advantageous variant of the invention, a further radiation source is provided, which is configured to emit only electromagnetic radiation with a wavelength of, for example, 650 nm or 850 nm, and at least one detector configured to detect only electromagnetic radiation in a wavelength range of 650 nm or 850 nm. In particular, when using scattered radiation in the 850 nm range, any fluorescence artifacts can be avoided.

[0030] By providing a deflection element which is designed to direct at least a portion of the electromagnetic radiation emitted by the radiation source directly onto a detector, a reference measurement can be carried out.

[0031] The deflection element on the light source(s) can, in particular, be a beam splitter. The beam splitter can, for example, comprise a quartz glass film tilted relative to the direction of incidence of the radiation; the quartz glass film can be tilted by approximately 45° relative to the direction of incidence of the radiation. Furthermore, an optical element, such as a dichroic mirror, can be present, which is designed to direct the excitation radiation and the scattered radiation, at least in sections, along the same path onto the sample volume.

[0032] In the following, embodiments and variants of the invention are explained in more detail with reference to the drawings.

[0033] Figure 1 shows a possible embodiment of a measuring arrangement according to the invention.

[0034] Figure 1 shows a schematic representation of a first possible embodiment of a measuring arrangement according to the invention. It shows a sample volume 1, which may, for example, contain a nutrient solution to be examined containing organic material, in particular a number of cells. It goes without saying that, in a real-world application, the sample volume 1 may be arranged, for example, in a flow-through measuring cell, an immersion probe, or a cuvette.

[0035] In the example shown, the vitality of the cells in the sample volume is determined by emitting excitation radiation 3 toward the sample volume 1 using an excitation radiation source 2, which can be configured, for example, as an LED. The direction of irradiation is indicated in the figure by an arrow. The excitation radiation 3 can, in particular, be in a wavelength range of approximately 340 nm - 360 nm or also in the range of approximately 260 - 280 nm.

[0036] It first passes through an optical element designed as a dichroic mirror 4, which is designed to largely transmit the excitation radiation 3. The excitation radiation 3 then passes through a deflection element designed as a beam splitter 5, in which it is split into a reference portion 6 and a measurement portion 7. The measurement portion 7 then reaches the sample volume 1, where it interacts with the nutrient solution and the biological material contained therein.

[0037] As long as the biological material is vital, i.e. living material, fluorescence at approx.

[0038] 450nm, which can then be recorded by three detectors 8,9,10 at different angles.

[0039] For a measurement of FAD (flavin adenine dinucleotide), excitation radiation in the range of 470 nm can be used; the corresponding fluorescence radiation is in the range of 420 nm. Particularly in the case of a combined measurement of interrelated indicators such as NADH and FAD, a change statement can be generated via the quotient of the two signals, where the quotient is invariant with respect to the absolute intensities.

[0040] In the example shown, the detectors 8, 9, 10 are arranged at angles of 180°, 90°, and 20° to the incident excitation radiation 3 and are provided with filters 11, 12, 13 that transmit only for a wavelength of 450 nm, so that only the excited fluorescence radiation 14 can pass through and reach the detectors 8, 9, 10. The filters can, of course, be adapted accordingly with regard to their transmission properties or be designed to be switchable for other measurement variants.

[0041] The described fluorescence measurement can be made more meaningful by performing a scattered light measurement in parallel or alternatively. The scattered light measurement allows conclusions to be drawn about the degree of turbidity in the sample volume and thus about the concentration of cells in the nutrient solution based on the measured scattering of incident radiation. In the embodiment shown, a further radiation source 15 is present to provide the radiation used for this purpose, referred to below as scattered radiation. This radiation emits electromagnetic radiation at a wavelength in the range of 450 nm. After emerging from the radiation source 15, this scattered radiation 16 first strikes the dichroic mirror 4, where it is reflected in the direction of the sample volume 1 due to the transmission properties of the dichroic mirror 4.It is advantageous if the scattered radiation 16 is reflected in that region of the dichroic mirror 4 in which the excitation radiation 3 also passes the mirror 4, since in this case largely identical conditions are created for the geometric beam path of the scattered radiation 16 and the excitation radiation 3.

[0042] Like the excitation radiation 3, the scattered radiation 16 also passes through the beam splitter 5, where a reference component 17 is also branched off from the scattered radiation 16. The reference components 6 and 17 of the excitation and scattered radiation 3 and 16 are recorded on a reference detector 18 and serve, for example, to detect intensity fluctuations in the radiation emitted by the radiation sources 2 or 15 and to take them into account when evaluating the measurements.

[0043] The measured portion of the scattered radiation 16 then enters the sample volume 1, where scattering occurs at the cells present in the sample volume 1 as scattering centers, so that the radiation 16 passing through unscattered and the scattered radiation 19 can be recorded by the aforementioned detectors 8, 9, 10 at different angular ranges. The extent to which scattering occurs will depend on the degree of turbidity of the nutrient solution, and therefore in particular on the concentration of cells present in the nutrient solution. With a low concentration of cells in the nutrient solution, it can be assumed that a significant portion of the scattered radiation 16 passes through the nutrient solution unscattered, thus reaching the detector 8 at 180°. As the concentration of cells in the nutrient solution increases, the intensity of the scattered radiation 19 will increase at the detectors 9 and 10 arranged at 90° and 20°, respectively.

[0044] It goes without saying that the sample volume 1 is advantageously irradiated either with excitation radiation 3 or with scattered radiation 16. During irradiation with excitation radiation 3, the vitality of the cells present in the nutrient solution is determined by detecting the excited fluorescence. The significance of this measurement is increased according to the present invention by determining the concentration of cells in the nutrient solution by means of the scattering measurement. A high measured intensity of the fluorescence radiation 14 with a low concentration of cells in the nutrient solution is an indicator of a high proportion of vital cells. Conversely, a low intensity of the fluorescence radiation 14 is an indicator of low cell vitality.

[0045] The arrangement shown in the figure is merely exemplary. It goes without saying that the invention can also be implemented by arrangements in which components have been omitted or replaced by others compared to Figure 1. Likewise, the angles shown are purely exemplary; the invention is not limited to the angle values ​​shown.

[0046] It is also conceivable to use the combination measurement for other applications in laboratory and process measurement technology - (turbidity and fluorescence, absorption and scattered light, especially

[0047] • Absorption for dissolved substances

[0048] • Scattered light for angle-resolved particle detection • Fluorescence for vitality / other applications in laboratory and process measurement technology

[0049] It would also be possible to use such a solution in three or more dimensions.

[0050] For example, two or more of the following measurements could be linked together:

[0051] • Fluorescence at different angles

[0052] • Scattering at different angles

[0053] • Absorption with different path lengths

[0054] • Raman scattering

[0055] The invention enables the simultaneous analysis of dissolved substances and particles in a liquid. Particle scattering, fluorescence, or absorption can be detected forward, sideways, or backward.

[0056] Whether scattered radiation is detected forward, sideways, or backward depends on the amount of particles or the amount of absorption caused by the fluorescent solutes.

[0057] The measuring range of the scattered light measurement of particles can be extended in particular by measuring in an angle-dependent manner, for example first forwards, then sideways and finally, when no more intensity can be measured in the forward direction, backwards.

[0058] The combined measurement of absorption (forward only) and scattered light (sideways and backward) allows a simple correction of particle scattering of low concentration, which scatters only slightly.

[0059] In particular, the combined and angle-dependent measurement of scattered light and fluorescence can be used to simultaneously measure scattered light and fluorescence of particles

[0060] - to compensate for the proportion of scattering particles when measuring fluorescent particles

[0061] - to simultaneously measure and compensate particle-scattered light and fluorescence of substances dissolved in liquid

[0062] - to determine the difference between fluorescent and non-fluorescent particles depending on the angle, if the scattering behavior of fluorescent and non-fluorescent particles is different, for example due to different size or shape

[0063] It may be particularly advantageous to measure fluorescence and scattering at different angles to obtain the largest possible signal.

[0064] For absolute measurements, calibration using concentration series is advantageous.

[0065] The described concept of angle-dependent measurement and thus measurement range extension can be applied to all spectroscopic methods, such as Raman, luminescence, etc., especially in cases where the light to be detected has a Lambertian character, i.e., radiates evenly in all directions. In this case, the radiation excited by the effects described above, i.e., in particular, Raman radiation and luminescence radiation, would replace or be added to the fluorescence radiation described above.

[0066] List of reference symbols

[0067] 1 sample volume

[0068] 2 Radiation source

[0069] 3 Excitation radiation

[0070] 4 Optical element

[0071] 5 Deflection element

[0072] 6 Reference portion

[0073] 7 Measurement component

[0074] 8,9,10 detectors

[0075] 11,12,13 filters

[0076] 14 Fluorescence radiation

[0077] 15 Radiation source

[0078] 16 Scattered radiation

[0079] 17 Reference portion

[0080] 18 Reference detector

[0081] 19 Scattered radiation

Claims

Claims: A method for examining organic material, wherein the organic material is irradiated with excitation radiation (3), and wherein the intensity of fluorescence radiation (14) excited by the irradiation is determined, wherein the organic material is additionally irradiated with electromagnetic scattering radiation (16), during which scattering occurs at the material and the scattered radiation (19) is recorded, characterized in that the scattered radiation (19) and / or the fluorescence radiation (14) are recorded at at least two different angles relative to an irradiation direction. A method according to claim 1, characterized in that a first angle is between 160° and 200°, in particular 180°. A method according to claim 1 or 2, characterized in that a second angle is between 70° and 110°, in particular 90°.A method according to any one of claims 1 to 3, characterized in that a third angle lies between 0° and 40°, in particular at 20°. A method according to any one of the preceding claims, characterized in that the organic material is irradiated alternately with excitation radiation (3) and scattered radiation (16). A method according to any one of the preceding claims, characterized in that the scattered radiation (16) has a wavelength in the range of 450 nm. A method according to any one of the preceding claims, characterized in that the organic material is irradiated with excitation radiation (3) in the wavelength range of 260 nm to 280 nm. A method according to any one of the preceding claims, characterized in that the organic material is irradiated with scattered radiation (16) in the wavelength range of 650 nm or 450 nm. A method according to any one of the preceding claims, characterized in that, to determine the viability of the organic material, the intensity of the scattered radiation (19) and the intensity of the fluorescence radiation (14) are mathematically related to each other. A method according to claim 9, characterized in that the mathematical relationship is a quotient.A method according to any of the preceding claims, characterized in that the excitation radiation (3) and the scattered radiation (16) are directed at least sectionally along the same path onto the sample volume. A method according to any of the preceding claims, characterized in that. Excitation radiation in the wavelength range of 340 nm is used. A method according to one of the preceding claims, characterized in that Excitation radiation in the wavelength range of 260 nm–280 nm is used. Measuring setup for the optical investigation of organic material, comprising - a sample volume (1 ) to receive the material - at least one first electromagnetic radiation source (2) for irradiating the material in an irradiation direction with excitation radiation (3) - at least one excitation detector (8, 9, 10) which is configured to detect fluorescence radiation (14) excited by the excitation radiation - at least one second radiation source (15) which is designed to additionally irradiate the organic material with electromagnetic scatter radiation (16) - at least one detector (9, 10) configured to detect the scattered radiation (19), characterized in that the measuring arrangement is configured to detect the scattered radiation (19) and / or the fluorescence radiation (14) at at least two different angles relative to an irradiation direction. Measuring arrangement according to claim 14, characterized in that the detector for detecting the scattered radiation is the excitation detector (9, 10). Measuring arrangement according to claim 14 or 15, characterized in that the second radiation source (15) is suitable for emitting electromagnetic radiation in a wavelength range of 450 nm or 650 nm. Measuring arrangement according to one of claims 14 to 16, characterized in that - another radiation source is present, which is designed to emit only electromagnetic radiation with a wavelength of approximately 260-280nm. - at least one detector is provided which is configured to detect only electromagnetic radiation in a wavelength range of approximately 280-350 nm. Measuring arrangement according to any one of claims 14 to 17, characterized in that the transmitting direction of a radiation source (2) and the receiving direction of a corresponding detector (8) enclose an angle of 160°-200°, in particular in the range of 180°. Measuring arrangement according to any one of claims 14 to 18, characterized in that the transmitting direction of a radiation source (2) and the receiving direction of a corresponding detector (9) enclose an angle of 70°-110°, in particular in the range of 90°. Measuring arrangement according to one of claims 14 to 19, characterized in that the transmitting direction of a radiation source (2) and the receiving direction of a corresponding detector (10) enclose an angle of 0°- 40°, in particular in the range of 20°.Measuring arrangement according to one of claims 14 to 20, characterized in that. - another radiation source is present, which is designed to emit only electromagnetic radiation with a wavelength of 650nm - at least one detector is configured to detect only electromagnetic radiation in a wavelength range of 650 nm. Measuring arrangement according to one of claims 14 to 21, characterized in that - a deflection element (5) is provided which is configured to direct at least a portion of the electromagnetic radiation (3, 16) emitted by a radiation source (2, 15) directly onto a detector (18). Measuring arrangement according to claim 22, characterized in that the deflection element (5) is a beam splitter. Measuring arrangement according to claim 23, characterized in that the beam splitter (5) comprises a quartz glass film tilted relative to the direction of incidence of the radiation (3, 16). Measuring arrangement according to claim 24, characterized in that the quartz glass film is arranged tilted at approximately 45° relative to the direction of incidence of the radiation (3, 16). Measuring arrangement according to any one of claims 14 to 25, characterized in that an optical element (4) is provided which is configured to direct the excitation radiation (3) and the scattered radiation (16) at least section by section along the same path onto the sample volume (1).Measuring arrangement according to claim 26, characterized in that the optical element (4) is a dichroic mirror.

28. Measuring arrangement according to one of claims 14 to 27, characterized in that the sample volume is arranged in the detection range of a process probe.

29. Measuring arrangement according to one of claims 14 to 27, characterized in that the sample volume is arranged within a flow cell.

30. Measuring arrangement according to claim 29, characterized in that the flow cell is designed as a single-use flow cell.

31. Measuring arrangement according to one of claims 14 to 30, characterized in that at least two radiation sources are present which emit with the same wavelength and a detector which is reached by radiation emitted or excited by both radiation sources.