Device, image sensor and method for examining samples using time-resolved fluorescence measurement

Abstract

Apparatus for examining samples, comprising: • an excitation light source for pulsed irradiation of a microarray having several test fields (T) with excitation light (L), by which a predetermined fluorophore can be excited; • an image sensor (10) with a sensor area (14) which is configured as a support for the microarray and comprises a plurality of SPAD arrangements (11) and a plurality of additional photodiodes (12), wherein the SPAD arrangements (11) each comprise at least one SPAD and are configured to detect a decay behavior of fluorescence light emitted as a result of the irradiation in a respective test field (T), wherein at least one of the additional photodiodes (12) is arranged between adjacent SPAD arrangements (11) and is configured to measure a respective intensity of the excitation light (L) incident on it, • computing electronics configured toto determine a standardized decay behavior of fluorescence light for one or more of the test fields (T) from the decay behavior recorded by the respective SPAD arrangement (11) and an intensity of excitation light (L) present at the respective SPAD arrangement (11), determined by interpolation of several intensities of excitation light (L) measured by the additional photodiodes (12).

Description

[0001] The present invention relates to a device and an image sensor for examining samples, in particular medical samples, by means of time-resolved fluorescence measurement. Furthermore, the invention relates to a method for examining a sample by means of time-resolved fluorescence measurement.

[0002] The targeted use of light, especially laser radiation, and its detection by image sensors to obtain information is known from various applications in both the micro and macro realms. For example, it can be used to make tiny objects visible, or to detect the properties, movements, or distances of objects.

[0003] US patent 2019 / 0281276 A1 concerns image sensors for capturing spatial information, particularly in the field of autonomous vehicle navigation. These image sensors contain pixels, each comprising several single-photon avalanche diodes (hereinafter referred to as "SPADs") and a pin diode (PPD) used as a time-to-charge converter. This allows for the scanning of objects illuminated by laser pulses using the time-of-flight method.

[0004] DE 10 2022 102 182 A1 relates to sensor devices, in particular LiDAR (Light detection and ranging) systems, which can also be used, for example, in autonomous vehicle systems. The associated pixels comprise photodiodes of different types arranged on a common substrate, e.g., SPADs for detecting position and reflectivity information on the one hand, and special photodiodes for capturing color information on the other.

[0005] US patent 2006 / 0124832A1 also discloses a photodetector that combines different types of diodes, in particular pin diodes with avalanche photodiodes operating in Geiger mode. This is intended to achieve a wide dynamic range with high sensitivity in optical detection applications.

[0006] From US patent 2022 / 0075171 A1, a light microscope is known which, in order to achieve high detection sensitivity, a large dynamic range, and a favorable signal-to-noise ratio, incorporates a photon-counting detector array with a plurality of detector elements, in particular SPADs. The photon counting rates of the detector elements are individually adapted to an expected light intensity distribution in order to account for an inhomogeneous distribution of the respective excitation light.

[0007] Further devices are known from WO 2018 / 218298 A1, GB 2557303 A, EP 0 350 595 A2, DE 195 28 198 A1, DE 10 2005 059 755 B3, EP 1 197 736 A2, DE 10 2016 006 960 A1 and DE 10 2005 052 752 A1.

[0008] Apart from devices that serve to directly detect the states, situations or external characteristics of an irradiated object, image sensors may be included in instruments designed to detect properties, in particular ingredients, of respective samples by means of their appropriate manipulation.

[0009] Fluorescence measurements can be performed using fluorescent dyes. A sample is analyzed to determine whether it contains one or more analytes. An analyte is the substance to be determined (DNA, proteins, etc.), which binds or activates a fluorescent dye. The intensity of the fluorescent dye is therefore proportional to the concentration of the analyte in the sample. During the analysis, so-called capture molecules are immobilized in test fields on a surface. These molecules bind specifically to the analyte. The sample is then irradiated with excitation light of a short wavelength (typically laser light). This excites the particles contained in the fluorescent dye to emit fluorescent light, which is then detected.The concentration of the analytes being sought can then be determined, for example, from the ratio of the intensity of the measured emitted light to a known intensity of the excitation light. In the field of medical diagnostics (e.g., in in-vitro diagnostics), proteins or DNA / genes are typically labeled with fluorescent dyes and detected accordingly.

[0010] Photodiodes are used to detect the fluorescence light. To separate the fluorescence light from the excitation light for detection, spectral filters are classically used, which filter out the excitation light based on its respective wavelength. The requirements for the spectral filters are high because the intensity of the excitation light is typically many times (e.g., a thousand times or more) the intensity of the emission light, and the respective wavelengths of excitation and fluorescence light are usually very close; the wavelength differences can, for example, be in the range of 25 nm to 35 nm.

[0011] The separation of excitation and fluorescence light can also be achieved based on their respective decay characteristics. Fluorescent dyes exhibit exponential decay with a time constant characteristic of the dye (typically on the order of a few nanoseconds). In this separation method, the excitation light source is switched off very quickly, allowing the decaying fluorescence light to be detected and analyzed immediately after the switch-off. The requirements for spectral filters used in this time-resolved fluorescence measurement are much lower than in the aforementioned classical approach; in the best-case scenario, no spectral filters are needed at all. The time constant of the exponential decay of the fluorescence light is characteristic of the fluorescent dye used. If multiple dyes are used, they can be distinguished by their time constant.The maximum / starting value corresponds to the concentration of the fluorescent dye and the resulting concentration of the analyte in the sample.

[0012] Single-photon avalanche diodes (SPADs) are particularly advantageous for time-resolved fluorescence measurements. These SPADs are special photodiodes that operate above their breakdown voltage in the Geiger region or Geiger mode, enabling them to detect individual photons with high temporal resolution, a process known as "time-correlated single-photon counting." Furthermore, SPADs can be readily integrated into the sensor areas of image sensors, allowing for very cost-effective setups for fluorescence detection without scanning mechanisms or spectral filters.

[0013] To test for multiple analytes simultaneously, microarrays are typically used. These consist of numerous test fields arranged in a matrix, each capable of detecting a different analyte. Typically, the test fields of such a microarray each have a diameter of a few hundred micrometers (e.g., up to 200 µm or up to 160 µm) and are spaced a few hundred micrometers apart (e.g., up to 300 µm or up to 280 µm). The number of test fields can vary depending on the application, for example, from 3x3, 12x24, 20x20, or more.

[0014] To measure the respective fluorescence in their test fields, the microarrays can be scanned or read out using an image sensor: In previously known systems, scanning involves mechanically moving the microarray under the light source and the detector, with each point being read out under identical conditions. This is associated with considerable mechanical complexity and control. The microarray is typically mounted on a glass slide.

[0015] In contrast, when reading the microarray using an image sensor, an image of the entire array is captured in previously known systems, eliminating the need for mechanical movement of the microarray. In these applications, the microarray is also mounted on a microscope slide, for example. However, the challenge here lies in achieving homogeneous illumination of the microarray by the light source: a laser typically produces a concentric distribution of light intensity, meaning that test areas in the center of the microarray are illuminated with higher intensity excitation light than those further out. Since the excitation light generally cannot be detected by SPADs (e.g., due to very high intensity and / or temporal proximity to the fluorescent light), it is conventionally homogenized artificially using complex optical setups.

[0016] The present invention is based on the objective of providing an improved technique for the precise examination of samples by means of time-resolved fluorescence measurement.

[0017] The problem is solved by a device according to claim 1, an image sensor according to claim 9 and a method according to claim 12. Advantageous embodiments are disclosed in the dependent claims, the description and the figures.

[0018] An apparatus according to the invention serves to examine samples, in particular samples in the form of a liquid or a gel. The apparatus comprises an excitation light source, preferably a laser light source, which is configured to irradiate a microarray having several test fields with pulsed excitation light.

[0019] The device further comprises an image sensor with a plurality of SPAD arrays and a plurality of additional photodiodes distributed across a sensor area. The sensor area of ​​the image sensor is also configured to support the microarray, such that the SPAD arrays are arranged within the area of ​​the microarray's multiple test fields.

[0020] The SPAD arrays each comprise at least one SPAD and are configured to detect the decay behavior of emitted fluorescence light during use of the device, i.e., the temporal progression or decrease in the intensity of the fluorescence light after each excitation pulse of the excitation light. Hereinafter, the use of laser light is preferably considered as the excitation light. Generally, however, the aim is to configure a fluorescence excitation light source for pulsed irradiation of a microarray comprising multiple test fields with excitation light, wherein the excitation light source is configured to excite a selected fluorophore. The test fields of the microarray are positioned on the image sensor such that each individual test field preferably covers an associated SPAD array, allowing the respective SPAD array to detect the fluorescence light emitted in that test field.In particular, the SPAD arrays are preferably configured to detect the respective decay behavior using time-correlated single-photon counting (TCSPC). The fluorescence light is emitted in the respective test fields of the microarray. It should be noted that the numerous test fields of the microarray are preferably applied directly to the sensor surface of the biosensor, for example, by pipetting a solution. Before the sample is introduced, the test fields contain, for example, dried capture molecules. Thus, the fluorescence radiation is emitted in the immediate vicinity of the SPADs, resulting in high sensitivity for detecting the fluorescence light. Alternatively, it is possible to overlay a grid of holes on the biosensor and introduce the sample into the holes to form the test fields.It is also conceivable to insert a transparent layer between the sensor area of ​​the image sensor and the test fields of the microarray, for example, if the image sensor needs to be protected from aggressive media. Preferably, the SPAD arrangements each include at least one time-to-digital converter (TDC) for measuring photon arrival times.

[0021] The additional photodiodes of the image sensor are configured to measure the respective intensity of the laser light striking them. In particular, they thus fulfill different measurement tasks than the SPAD arrangements.

[0022] The device further comprises computing electronics, which can be integrated with the image sensor or separate from it. The computing electronics are configured to determine a corresponding standardized decay behavior of fluorescence light for at least one of the SPAD arrangements when evaluating a respective test field of the microarray. This determination is based on the decay behavior detected by the respective SPAD arrangement and one or more of the excitation or laser light intensities measured by the additional photodiodes.

[0023] A method according to the invention serves to examine a (for example, medical) sample, in particular a sample in the form of a liquid or a gel.

[0024] The method comprises irradiating a microarray, which has a plurality of test fields impregnated with the sample and a respective fluorescent dye, with pulsed laser light. For this purpose, the microarray was applied to a sensor surface of an image sensor, preferably directly, i.e., without intermediate layers between the individual test fields and the image sensor. Particularly preferably, the image sensor is one of the embodiments described above.

[0025] According to the inventive method, a plurality of additional photodiodes are used to measure the respective intensity of the laser light (excitation radiation) striking each of the additional photodiodes.

[0026] For one or more of the microarray's test fields, the decay behavior of fluorescence light emitted by the respective test field as a result of irradiation (particularly after the end of a laser pulse) is detected by means of a SPAD array comprising at least one SPAD. It is understood that the respective test field lies within the detection range of the SPAD array. Preferably, the respective test field completely covers the associated SPAD array; that is, the test field is mounted directly on the SPAD array, and each test field of the microarray is preferably assigned exactly one SPAD array, with each SPAD array potentially comprising multiple SPADs.

[0027] The detection of the decay behavior (fluorescence radiation) and the measurement of the intensity of the excitation radiation are preferably carried out in close temporal proximity, particularly preferably almost simultaneously, so that it can be assumed that the measured intensity of the excitation radiation corresponds to the intensity that causes the fluorescence radiation emitted in the test field.

[0028] From the decay behavior recorded for a respective test field and one or more of the intensities of the laser light measured by the additional photodiodes, a normalized decay behavior is then determined (for the respective test field or for a SPAD arrangement in whose detection range the respective test field lies).

[0029] The SPAD arrangement and the additional photodiodes preferably belong to an image sensor according to one of the embodiments described above, which particularly preferably includes or is coupled to computing electronics that determine the normalized decay behavior(s). The image sensor (optionally with the computing electronics) can be designed as an application-specific integrated circuit (ASIC).

[0030] In particular, the method according to the invention can preferably be carried out by a device according to the invention, in accordance with one of the embodiments described above, or this device can be configured to carry out the method according to the invention.

[0031] By normalizing the decay behavior using the intensities of the laser light (excitation radiation) measured by the additional photodiodes, the present invention makes it possible to avoid the use of complex homogenization optics while still achieving precise time-resolved fluorescence measurement. This does not force (approximately) homogeneous illumination, but rather detects and computationally accounts for the existing inhomogeneity of the excitation light. In particular, this allows for targeted correction of the inhomogeneity, enabling the simultaneous examination and precise analysis of a large number of test fields within a given microarray.

[0032] The laser irradiation preferably occurs at a frequency of at least 80 MHz or at least 90 MHz. Preferably, each laser pulse has a duration of at most 5 ns or at most 4 ns. Such short pulses ensure that the excitation of the fluorescence is precisely time-limited; in particular, the time until the next pulse can be used to detect the respective decay behavior. Furthermore, this ensures that the afterglow time of the laser light source is shorter than the decay phase of the fluorescence and that switching off the laser does not affect the detection itself.

[0033] In a device according to the invention or a device used to carry out a method according to the invention, the SPAD arrangements and the additional photodiodes are preferably arranged in a common sensor area. In particular, at least one of the additional photodiodes of the image sensor can advantageously be arranged between two adjacent SPAD arrangements, and / or at least one of the SPAD arrangements can be arranged between two adjacent additional photodiodes.

[0034] The "detection area" of a SPAD arrangement is understood to be an area in which incoming photons, which are detected by the SPAD arrangement associated with the test field (usually located directly below the test field) due to the fluorescence triggered in the test field, can be detected in the device or when it is used in the process.

[0035] When applying the microarray to the sensor surface of the image sensor, care must be taken to ensure that the individual test fields are aligned so that they lie within the respective detection range of the SPAD arrays; that is, preferably, each test field covers all SPADs belonging to a SPAD array. It should be noted that the microarrays depend on the sample being examined and can be configured very differently. The general procedure for generating such microarrays is known to those skilled in the art and therefore does not need to be described in detail. In a further embodiment, a device according to the invention can already include a microarray mounted on the image sensor.

[0036] The additional photodiodes can each be arranged in areas located between adjacent test fields of the respective microarray.

[0037] The method according to the invention can analogously include such alignment or placement of the microarray on the sensor surface of the image sensor. In particular, preferably each test field of the microarray can lie at least partially within the detection area of ​​an associated SPAD arrangement.

[0038] When carrying out a method according to the invention, the microarray can preferably be arranged directly on the SPAD arrays. Alternatively, the optional holder of a device according to the invention can be configured to hold the microarray above the SPAD arrays in its intended orientation for use.

[0039] The laser light source used for irradiation can be arranged in an orientation of the device intended for use or preferably above the microarray when carrying out the method.

[0040] According to advantageous embodiments of the device according to the invention, the additional photodiodes are configured to repeatedly measure the respective intensity of the laser light incident on them, particularly for a plurality of laser pulses, for example in real time during irradiation with laser light, e.g., between two successive measurements of decay behavior by the SPAD arrangements. Similarly, according to the method according to the invention, the measurement of the respective intensity of the laser light incident on the additional photodiodes can be carried out repeatedly, particularly for a plurality of laser pulses, for example in real time during irradiation with laser light, e.g., between two successive measurements of decay behavior by the SPAD arrangements.The additional photodiodes preferably measure the intensity of the excitation radiation continuously, and normalization is then performed using the counts of the TCSPC histograms.

[0041] To determine the respective normalized decay behavior, the measured intensities of the laser light are used. This means that the determination of the respective decay behavior is based on a current state, so that, for example, heating effects do not distort the measurement result, since the normalization takes into account the continuously performed intensity measurements.

[0042] Determining the respective normalized decay behavior can be performed using at least one input parameter; the device according to the invention can accordingly include an input device for such an input parameter. In this way, sample- and / or analyte-specific properties can be taken into account, for example, those that cause saturation of the respective fluorescence in the sample.

[0043] According to advantageous embodiments of the device according to the invention, the computing electronics are configured to calculate (at least approximately) the laser light intensity present at the respective SPAD arrangement in order to determine the respective normalized decay behavior by interpolating several of the intensities measured by the additional photodiodes (preferably simultaneously). Similarly, determining the respective normalized decay behavior in the method according to advantageous embodiments of the invention comprises (at least approximately) calculating the laser light intensity present at the respective SPAD arrangement by interpolating several of the intensities measured by the additional photodiodes (preferably simultaneously). The computing electronics can be integrated into a chip / ASIC together with the image sensor.Alternatively, the computing electronics are formed by a unit spatially separate from the image sensor, for example, a PC or a microcontroller. The computing electronics can also be divided into multiple units.

[0044] In particular, the parallel measurements of the respective intensities of the laser light taken by the additional photodiodes can be used to interpolate the light intensity across the entire respective microarray.

[0045] The interpolated data provide at least an approximate representation of the excitation light intensity at the SPAD positions, thus enabling a precise correction of the measured fluorescence intensities when calculating the respective normalized decay behavior. In this way, the effects of the inhomogeneous excitation light distribution are corrected with particular accuracy, ensuring that the respective normalized decay behavior accurately reflects the actual lighting conditions, which can then be interpreted accordingly.

[0046] The recorded decay behavior can be compared to the laser light intensity present at the respective SPAD array, calculated by interpolation. For example, the recorded decay behavior values ​​corresponding to specific time points can be divided by the (interpolated) laser light intensity present.

[0047] In particular, to determine a normalized decay behavior for a given SPAD arrangement, an average value from several intensities of laser light can be used, which were measured by additional photodiodes arranged adjacent to the respective SPAD arrangement (preferably simultaneously with each other).

[0048] Preferably, the device according to the invention is configured to determine the concentration of a desired analyte in the sample from the standardized decay behavior(s) determined by the computer electronics. Similarly, in the method according to the invention, the concentration of an analyte in the sample is preferably determined from at least one previously determined standardized decay behavior. The determination of the concentration can preferably be at least partially automated. It can, in particular, include the application of a fitting algorithm. The analyte can, for example, be a protein, DNA, or a gene.

[0049] In particular, according to preferred embodiments of the present invention, the device is designed as a medical device for in-vitro diagnostics, or the sample is a medical sample that is analyzed as part of in-vitro diagnostics.

[0050] The method according to the invention can in particular include arranging the sample and / or the respective fluorescent dye in the test fields of the microarray prior to irradiation with excitation radiation.

[0051] According to further developed embodiments, the device according to the invention includes a display (e.g., in the form of a screen) and is configured to graphically represent the specific normalized decay behavior or at least one of the normalized decay behaviors. The method according to the invention can analogously include a graphical representation of at least one specific normalized decay behavior on a display. Based on the curves thus visualized, which reflect the characteristic decay behavior of the fluorophores and whose respective maximum is directly related to the intensity of the emitted fluorescence and to the concentration of the respective analytes in the respective test field of the microarray, a user can interpret the respective normalized decay behavior(s).

[0052] The present invention is explained below with reference to the drawings. Reference numerals for corresponding elements are used across all figures. It is understood that individual elements and components can also be combined differently than shown. For the sake of clarity, only some of the elements appearing multiple times in the figures are provided with reference numerals. The following are schematically illustrated: Fig. 1 a simplified sectional view of a section of an image sensor of an exemplary embodiment of the device according to the invention with test fields of a microarray arranged thereon; Fig. 2 a detailed view of a sensor area of ​​the image sensor; Fig. 3a the sensor area of ​​the image sensor with superimposed laser beam profile as excitation light; Fig. 3b: the intensity of the laser radiation at the image sensor of the Fig. 3a depending on the distance to the center of the irradiation; Fig. 3c: from selected SPAD arrangements of the image sensor of the Fig. 3a. Decay behavior of resulting fluorescence light recorded in each case for two different concentrations before normalization of the excitation radiation intensity; Fig. 3D: according to Fig. 3c recorded decay behavior of the fluorescence light after normalization of the intensity of the excitation radiation.

[0053] Fig. Figure 1 shows a simplified sectional view of a section of an image sensor 10 of a device according to the invention. The image sensor 10 has a sensor area 14. For use of the device or when carrying out the method according to the invention, a microarray with a plurality of test fields T, each containing a sample labeled with a fluorescent dye, is applied to the sensor area 14. The microarray formed from the test fields T is positioned such that the test fields T can be irradiated with laser light L and each lies within a detection area of ​​an associated SPAD arrangement 11. In particular, a number of test fields T of the microarray preferably correspond to a number of SPAD arrangements 11.

[0054] Fig. Figure 2 shows a detailed view of the sensor area 14 of the image sensor 10 of the device according to the invention. The sensor area 14 comprises a plurality of SPAD arrangements 11, each arranged between adjacent additional photodiodes 12.

[0055] The individual SPAD arrays 11 preferably comprise several SPADs for single-photon counting and several time-to-digital converters 13 arranged around and adjacent to the SPAD array 11 for measuring the arrival times of the photons. Each SPAD array 11 has a detection range. For fluorescence light emitted during use of the device, the SPAD arrays 11 can thus perform time-correlated single-photon counts and thereby detect the decay behavior of the fluorescence light.

[0056] In the Fig. Figure 2 shows only some of the SPAD arrays 11 and additional photodiodes 12. In particular, the sensor area 14 can, for example, comprise 12x12 (or 4x6 or similar) SPAD arrays 11, each of which can be arranged in spaces between 13x13 additional photodiodes 12 of the sensor area 14.

[0057] The device according to the invention, whose image sensor 10 comprises such a sensor area 14, is particularly suitable for examining microarrays with 12x12 test fields T, the respective diameters D and distances A from each other of which correspond to the respective distances and sizes of the respective SPAD arrangement 11. The corresponding microarray can be encompassed by the device according to the invention. As in the Fig. As shown in Figure 2, when using or carrying out the method according to the invention, the detection areas of the SPAD arrangements 11 preferably lie entirely within an associated test field T. In the present case, the test fields T are each circular disk-shaped, and their diameter D corresponds to the length of a diagonal of the SPAD arrangements 11.

[0058] The diameters D of the microarray test fields and / or the length of the diagonals of the SPAD arrangements 11 can advantageously be, for example, in the range of 120 µm to 180 µm or in the range of 140 µm to 160 µm. Within a row or column, the spacing A between the test fields T and / or the spacing between the SPAD arrangements can be, for example, in the range of 230 µm to 270 µm or in the range of 240 µm to 260 µm.

[0059] From the decay behavior recorded by the SPAD arrays 11, a standardized decay behavior can then be determined, as described in more detail below, using one or more intensities of laser light measured by the additional photodiodes to irradiate the microarray. This allows, in particular, the precise calculation of the concentration of an analyte located in the respective test field T in a sample, without requiring a complex device for homogenizing the irradiating laser light. This makes it especially possible to analyze medical samples for in-vitro diagnostics with high accuracy.

[0060] In the Fig. Figure 3a shows a schematic representation of the sensor area 14 of the image sensor 10. In this embodiment, the sensor area 14 comprises an arrangement of 9x9 SPAD arrays 11 and 8x8 additional photodiodes 12, each arranged between two of the SPAD arrays 11 or surrounded by four SPAD arrays 11.

[0061] The SPAD arrangements 11 are each configured for single photon counting. They each comprise at least one SPAD (not shown), preferably several SPADs; in particular, they can each, for example, comprise a square arrangement of a plurality of at least 3x3 or at least 5x5 SPADs.

[0062] The Fig. Figure 3a illustrates a situation in which a laser light source of the device according to the invention illuminates the sensor area 14 with (pulsed) laser light L; the illumination with the laser light L is schematically represented by three concentric circles, with the corresponding light intensity decreasing towards the outside. For better understanding, two cases within the context of the usual application of the device are considered below: In case 1, three samples / analytes with the same concentrations c1=c2=c3 are located at three test fields T, whose positions are here designated 1, 2, 3. In case 2, three samples / analytes with different concentrations c1' are located. <c2'<c3' an denselben drei Testfeldern T, deren Positionen hier mit 1', 2', 3' bezeichnet sind.

[0063] In the Fig. 3a In exemplary selected areas, which are marked by different circular lines (solid, dashed, dotted line), the test fields located there at positions 1 / 1', 2 / 2', 3 / 3' are each irradiated and excited by an intensity of laser light occurring at them.

[0064] Fig. Figure 3b shows a graph of the light intensity i of the laser radiation (excitation radiation) as a function of the distance from the center of illumination. In this example, the center of illumination is identical to the center of the image sensor. The light intensity of the excitation radiation decreases with increasing distance from the center of the image sensor, naturally independent of the concentration c of the analytes, whose positions 1 / 1', 2 / 2', 3 / 3' are included in the diagram for clarity. Fig. 3c and Fig. 3d shows measured values ​​or calculated values ​​that are recorded by the SPAD arrangements 11, which are located under the test fields at positions 1 / 1', 2 / 2', 3 / 3'.

[0065] The SPAD arrangements 11 are each designed to detect the respective decay behavior of fluorescence light emitted by the fluorescent dye contained in the test field assigned to the respective SPAD arrangement after a laser pulse.

[0066] The Fig. Figure 3c illustrates the decay behavior not normalized to the excitation intensity, namely the respective photon count rates n as a function of time t, which are generated by the SPAD arrays 11 at the in Fig. The test field positions 1 / 1', 2 / 2', and 3 / 3' shown in Figure 3a are recorded. The count rates are plotted logarithmically; a quantized representation is used for the time axis. The left figure of the Fig. Figure 3c shows the aforementioned case 1, i.e., equal concentrations c1=c2=c3 at test field positions 1, 2, 3. The right-hand representation of the Fig. 3c shows the above case 2, i.e., different concentrations c1' <c2'<c3' an den Testfeld-Positionen 1', 2', 3', wobei dieser Fall so gewählt ist, dass alle drei Abklingkurven denselben Verlauf zeigen, bei gleichem Rauschpegel. Es ist ersichtlich, dass ohne Normierung in Bezug auf die Anregungsintensität zwar eine absolute Messung der Zeitkonstanten möglich ist, jedoch eine korrekte Messung der unterschiedlichen Konzentrationen nicht oder nur mit Fehlern möglich wäre.

[0067] To compensate for this dependence of the detectable decay behavior on the position of the respective SPAD arrangement (relative to the center of the excitation radiation by the laser light L), the measured values ​​of the decay behavior are normalized according to the invention by including intensities of the laser light that were measured by one or more of the additional photodiodes 12.

[0068] The additional photodiodes 12, whose measured values ​​are thus incorporated into the normalized decay behavior determined for a respective SPAD arrangement 11, are preferably arranged adjacent to the respective SPAD arrangement 11 in the sensor area 14. In particular, the intensity of the exciting laser light present at the respective SPAD arrangement 11 can be calculated by interpolating several of the intensities measured by the additional photodiodes 12 and used in determining the associated normalized decay behavior.

[0069] With identical fluorescence behavior in the respective detection areas, the SPAD arrangements 11 exhibit corresponding normalized decay behaviors, as described in the Fig. It is illustrated in 3D. The left illustration of the Fig. Figure 3D again shows the measured values ​​at test field positions 1, 2, and 3 at the same concentration c1=c2=c3. Due to normalization, different noise levels occur, but this can be taken into account. The right-hand image of the Fig.Figure 3d shows case 2, i.e., different concentrations c1' <c2'<c3' an den Testfeld-Positionen 1', 2', 3', wobei alle drei Abklingkurven zwar einen ähnlichen Verlauf zeigen, aufgrund unterschiedlicher Rauschpegel aber aufgelöst werden können. Es ist ersichtlich, dass durch die Normierung in Bezug auf die Anregungsintensität weiterhin eine absolute Messung der Zeitkonstanten möglich bleibt und gleichzeitig eine korrekte Messung der unterschiedlichen Konzentrationen ermöglicht wird. Die auf der jeweiligen Position der SPAD-Anordnung beruhenden und aus der Inhomogenität der Laserlichtbestrahlung resultierenden Ungleichheiten sind damit nivelliert, so dass die Vorrichtung zur präzisen simultanen Untersuchung einer Vielzahl an Testfeldern eines Mikroarrays verwendet werden kann. Reference sign 1 / 1', 2 / 2', 3 / 3' Test field positions 10 Image sensor 11 SPAD arrangement 12 additional photodiodes 13 Time-to-Digital Converters 14 sensor area A distance between two adjacent test fields The diameter of a test field L Excitation light / Laser light T Test field QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] US 2019 / 0281276 A1

[0003] DE 10 2022 102 182 A1

[0004] US 2006 / 0124832A1

[0005] US 2022 / 0075171 A1

[0006] WO 2018 / 218298 A1

[0007] GB 2557303 A

[0007] EP 0 350 595 A2

[0007] DE 195 28 198 A1

[0007] DE 10 2005 059 755 B3

[0007] EP 1 197 736 A2

[0007] DE 10 2016 006 960 A1

[0007] DE 10 2005 052 752 A1

[0007]

Claims

Apparatus for examining samples, comprising: • an excitation light source for pulsed irradiation of a microarray having several test fields (T) with excitation light (L), by which a predetermined fluorophore can be excited;• an image sensor (10) with a sensor area (14) configured as a support for the microarray and comprising a plurality of SPAD arrangements (11) and a plurality of additional photodiodes (12), wherein the SPAD arrangements (11) each comprise at least one SPAD and are configured to detect the decay behavior of fluorescence light emitted as a result of irradiation in a respective test field (T), wherein the additional photodiodes (12) are configured to measure a respective intensity of the excitation light (L) incident on them, • computing electronics configured to determine a respective normalized decay behavior of fluorescence light for one or more of the test fields (T) from the decay behavior detected by the respective SPAD arrangement (11) and one or more of the intensities of the excitation light (L) measured by the additional photodiodes (12). Device according to claim 1, characterized in that the excitation light source is a laser light source. Device according to claim 1 or 2, characterized in that the computing electronics of the image sensor is configured to calculate an excitation light intensity present at the respective SPAD arrangement (11) by interpolating several of the intensities measured by the additional photodiodes (12) in order to determine the respective normalized decay behavior. Device according to one of claims 1 to 3, characterized in that it is configured to determine a respective concentration of at least one analyte in a sample to be examined from the or from at least one of the normalized decay behavior. Device according to one of claims 1 to 4, characterized in that it is designed as a medical device for in-vitro diagnostics. Device according to one of claims 1 to 5, characterized in that it comprises at least one display and is configured to graphically represent the normalized decay behavior on the display. Device according to one of claims 1 to 6, characterized in that the microarray with the plurality of test fields (T) can be applied directly to the sensor surface (14) of the image sensor (10), so that the test fields (T) are in direct contact with the SPAD arrangements (11). Device according to one of claims 1 to 6, characterized in that it comprises a holder which is configured to hold the microarray with the plurality of test fields (T) on the image sensor (10). Image sensor (10) with a plurality of SPAD arrangements (11) and a plurality of additional photodiodes (12), characterized in that: - the SPAD arrangements (11) each comprise at least one SPAD and are configured to detect a decay behavior of fluorescence light emitted as a result of pulsed irradiation with excitation light (L) in a respective detection area of ​​the SPAD arrangement (11); - the additional photodiodes (12) are configured to measure a respective intensity of the excitation light (L) incident on them. Image sensor (10) according to claim 9, characterized in that it comprises computing electronics which are configured to determine a respective normalized decay behavior of fluorescence light for one or more of the SPAD arrangements (11) from the decay behavior detected by the respective SPAD arrangement (11) and one or more of the intensities of the excitation light (L) measured by the additional photodiodes (12). Image sensor (10) according to claim 10, characterized in that it is designed as an application-specific integrated circuit (ASIC). A method for examining a sample, comprising the following steps: • Irradiating a microarray having a plurality of test fields (T) coated with the sample and a respective fluorescent dye with pulsed excitation light (L), wherein the microarray is mounted on a sensor surface (14) of an image sensor (10); • Measuring the intensity of the excitation light (L) incident on a plurality of additional photodiodes (12); and • Detecting the decay behavior of the fluorescent light emitted by the respective test field (T) as a result of the irradiation, using a SPAD arrangement (11) comprising at least one SPAD and within whose detection range the respective test field (T) is located, wherein the detection is performed for one or more of the test fields (T) of the microarray;• Determine at least one normalized decay behavior from the decay behavior or from one of the detected decay behaviors and from one or more of the excitation light intensities (L) measured by the additional photodiodes (12).; Method according to claim 12, wherein determining the at least one normalized decay behavior comprises calculating an intensity of the excitation light (L) present at the respective SPAD arrangement (11) by interpolating several of the intensities measured by the additional photodiodes (12). Method according to claim 12 or 13, characterized in that it comprises determining a respective concentration of at least one analyte in the sample from the at least one defined normalized decay behavior. Method according to one of claims 12 to 14, wherein the sample is a medical sample that is analyzed as part of in-vitro diagnostics.

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