Method for adapting fluorescence image intensity and fluorescence microscope

Abstract

A method for adapting fluorescence image intensities for different fluorescence microscopes and for different points in time of image acquisition by a fluorescence microscope is disclosed. It comprises: - acquiring a predetermined system configuration of a reference fluorescence microscope, - generating a shading correction by the reference fluorescence microscope, - acquiring a reference fluorescence image of an artificial reference calibration sample, - determining an average reference fluorescence image intensity of the fluorescent radiation generated in a fluorescence channel of the reference fluorescence microscope, - generating an adapted shading correction by a fluorescence microscope to be adapted by the shading reference sample, - acquiring an adapted fluorescence image of the artificial reference calibration sample, - determining an average adapted fluorescence image intensity of the fluorescent radiation generated in a fluorescence channel of the fluorescence microscope to be adapted, - calculating and storing a correction factor for the fluorescence channel depending on the average reference fluorescence image intensity and the average adapted fluorescence image intensity. Furthermore a fluorescence microscope for adaptation by the method is provided.

Description

Technical Field

[0001] This invention relates to a method for adapting fluorescence image intensity and a fluorescence microscope. Background Technology

[0002] Fluorescence microscopy is a complex system with many components that affect the intensity of the acquired sample image. These components include the sample itself, as well as factors such as the illumination power of the light source used or the quantum efficiency of the detector used.

[0003] For specific applications, it is important that for a given sample, the same or at least comparable image intensities are produced at different microscopes and / or at different time points within the same microscope. In other words, for a single sample, the same or at least comparable measurements should be detected as closely as possible at different microscopes and at different times of microscope use. One known method for setting the system to subjectively comparable illumination characteristics is to measure the illumination power in the sample plane and set the illumination power accordingly. However, this method ignores the effects of the detection path, detector characteristics, and the sensor surface of the detector as a local region of the illuminated surface.

[0004] DE 10 2014 112 002 A1 describes a method and microscope for imaging samples, wherein systematically induced inhomogeneities and / or undesirable brightness fluctuations in the acquired images are reduced. This is primarily achieved through shading correction, where a brightness-corrected image is created to compensate for inhomogeneities in illumination and imaging. The brightness-corrected image is obtained from reference measurements of different regions of one or more comparative samples. The acquired sample image is then corrected based on this brightness-corrected image. Correction can be performed continuously, where the acquired image is used as a reference image to compensate for time-varying inhomogeneities in illumination. The effects of separately studied samples are not considered. It is also not possible to adapt this method to multiple systems. Correction is accomplished by pixel manipulation of the acquired raw image data. However, altering the raw image data is generally undesirable for the client and should be avoided.

[0005] US 9366628 B2 discloses a system and a method for standardizing a fluorescence microscope system. Absolute calibration of a fluorescence microscope system, i.e., calibration relative to an absolute sensitivity reference, is very difficult, even impossible, in practice because there is no stable, calibrated reference standard. Therefore, US 9366628 B2 proposes separating the effects of drift and normalization to standardize the fluorescence microscope system. To normalize the imaging system, a first normalized image of the normalized sample is acquired using the imaging system to be normalized. A reference normalized image of the same normalized sample is acquired using a reference imaging system. The first normalized image and the reference normalized image are then compared, and the gamma value and offset value of the imaging system to be normalized are determined based on this comparison. Furthermore, a drift measurement is performed as the ratio of the average pixel intensity in the acquired image to the exposure time. Finally, for normalization, the pixel intensity of each pixel in the acquired digitized image is changed according to the determined gamma value, offset value, and drift measurement. A disadvantage is the additional overhead required to perform normalization and drift correction. This also involves operations on the raw image data.

[0006] US patent 20160061654 A1 discloses a calibration apparatus and related method for calibrating microscopes. Using a calibration plate with two or more fixed fluorescent reference slides, the intensity and shape of the illumination field are measured for each combination of fluorescence channels (e.g., DAPI, FITC, CY3, CY5) and objectives (e.g., 2x, 10x, 20x, 40x) in a microscope system to be calibrated. To achieve consistent image intensity across multiple microscopes, for intensity calibration, the average intensity of each objective / channel combination is determined using the fixed fluorescent reference slides (red, green, blue) of the calibration plate, thus enabling scaling of exposure time during acquisition. However, for laboratories wishing to calibrate their microscopes in this manner, the construction and use of the calibration plate incur additional costs and expenses. This method also assumes that all microscopes used are compatible with the calibration plate and support the required automation. Furthermore, calibration must be repeated periodically to account for changes in the microscope over time.

[0007] US 20130310268 A1 describes a method and system for standardizing quantitative measurements of biological samples examined by a microscope system. To this end, light source correction values ​​are determined to compensate for fluctuations in excitation source intensity, and intrinsic device factors are determined to correct for variations in the optical system used. The correction values ​​are applied to the measured data, i.e., images of the examined biological sample, to obtain standardized and comparable results across different microscope systems. The method in US 20130310268 A1 requires special calibration elements, such as calibration cubes, which increases the cost of the measurement. The correction factor is applied to the acquired images but is not used to calibrate the microscope system itself.

[0008] JP 2022122181 A discloses a method for setting calibration parameters and a data correction method to prevent differences in detection results due to individual variations between microscopes. A calibration parameter p is calculated for a sample using emission spectral data from a first reference microscope and a second microscope, so that the emission spectral data of the second microscope for each individual sample can be subsequently corrected. A drawback is that this method assumes the existence of a reference sample with the same emission intensity distribution as the sample to be measured, which limits the applicability of the method.

[0009] US 20040051050 A1 describes a reference device, method, and apparatus for evaluating the performance of a confocal laser scanning microscope for two-dimensional quantitative fluorescence measurements. The reference device comprises a substrate having a predetermined spatial distribution of reference fluorescent pigment on its surface. This allows characterization of the following properties of the microscope: a) quantitative signal detection sensitivity, b) quantitative signal detection limit, c) uniformity of the confocal volume within the field of view, d) spatial resolution of the scanning process, and e) dynamic behavior of the measurement signal within the field of view. The method involves scanning the reference device with the microscope to be evaluated to obtain measurements, which are then used to determine correction factors. These correction factors can then be applied to correct measurements of the sample under study, such as a DNA binding array. No adjustment is made to the intensity of the laser source of the laser scanning microscope.

[0010] US 11747280 B2 describes a system and method for calibrating a microscope or imaging system before acquiring sample image data. The calibration consists of several individual steps, such as performing power output calibration to calibrate reproducibility measurements taken using the imaging device, performing image intensity calibration to calibrate reproducibility measurements taken using the imaging device, and calibrating differences in detection efficiency between channels. To obtain comparable results for the acquired images, the image data acquired using the microscope or imaging system is multiplied pixel-wise by an image correction factor for each pigment or marker. The acquired images are then calibrated. Summary of the Invention

[0011] In view of the shortcomings mentioned in the prior art, it is desirable to provide a method that can adapt fluorescence intensity for different time points and / or different microscopes so that the acquired fluorescence intensities are comparable to each other. When using the same imaging characteristics, such as camera pixel binning, exposure time, filter group, etc., samples (with constant characteristics) detected at different measurement time points on the same microscope or on different systems should produce the same image intensity.

[0012] The technical problem is solved by a method for adapting fluorescence image intensity to different fluorescence microscopes and to different time points of image acquisition using a fluorescence microscope. According to the present invention, the method for adapting fluorescence image intensity to different fluorescence microscopes and to different time points of image acquisition using a fluorescence microscope includes the following steps:

[0013] - Obtain the predetermined system configuration for the reference fluorescence microscope.

[0014] - Shadow correction is generated using a shadowed reference sample via a reference fluorescence microscope.

[0015] - Under a predetermined system configuration and with previously generated shading correction applied, reference fluorescence images of the artificial reference calibration sample are acquired using a detector from a reference fluorescence microscope.

[0016] - Determine the average reference fluorescence image intensity of the fluorescence radiation generated in the artificial reference calibration sample within the fluorescence channel of the reference fluorescence microscope. ,

[0017] - Adaptive shadow correction is generated using the fluorescence microscope to be adapted, with the aid of this shadow reference sample or another shadow reference sample.

[0018] - Under a predetermined system configuration and with the application of previously generated adaptation shading correction, an adaptation fluorescence image of the artificial reference calibration sample is acquired through the detector of the fluorescence microscope to be adapted.

[0019] Determine the average fitted fluorescence image intensity of the fluorescence radiation generated in the artificial reference calibration sample within the fluorescence channel of the fluorescence microscope to be fitted. ,

[0020] according to From the average reference fluorescence image intensity and average fitted fluorescence image intensity Calculate and store the correction factor K for this fluorescence channel. FI Among them, with the help of correction factor K FI It can correct each adjustable system parameter that is linearly related to the fluorescence image intensity of the fluorescence microscope to be adapted, thereby adapting to the fluorescence image intensity, wherein,

[0021] - The artificial reference sample is designed as a uniformly fluorescent glass substrate with an emission band of 400nm to 850nm.

[0022] A predetermined system configuration is acquired at the reference fluorescence microscope, i.e., settings for objective lens selection, magnification, camera gain, etc., are made and stored at the microscope. These settings are ultimately used to examine samples in practical studies. The system configuration is identical for both the reference fluorescence microscope and the microscope to be adapted; otherwise, an error message will be output to the user or adaptation / calibration will not be performed. Subsequently, shading correction is performed using the reference fluorescence microscope with the aid of a shading reference sample, so that brightness differences at the edges of the microscope's field of view can be subtracted before examining the real sample image. The shading reference sample is understood as a real, standardized sample used to improve image quality by correcting brightness differences caused by various factors such as illumination, objective lens errors, or the characteristics of the object / sample itself. Under the predetermined system configuration and with the previously generated shading correction applied, a reference fluorescence image of the artificial reference calibration sample is acquired through the detector of the reference fluorescence microscope. The average reference fluorescence image intensity of the fluorescence radiation generated in the reference calibration sample in the fluorescence channel of the reference fluorescence microscope is determined from the acquired reference fluorescence image. This information is stored in memory, for example, in tabular form. The same steps are performed on the fluorescence microscope to be adapted: an adaptation shading correction is generated using the fluorescence microscope to be adapted, with the aid of an already used shading reference sample or another shading reference sample. Subsequently, under a predetermined system configuration and with the previously generated adaptation shading correction applied, an adaptation fluorescence image of the artificial reference calibration sample is acquired through the detector of the fluorescence microscope to be adapted, and the average adaptation fluorescence image intensity of the fluorescence radiation generated in the reference calibration sample in the fluorescence channel of the fluorescence microscope to be adapted is determined. And store them in memory, for example, in tabular form. Adaptation shading correction or adaptation fluorescence image linguistically distinguishes the fluorescence microscope to be adapted from other microscopes, such as a reference fluorescence microscope. Finally, according to The average reference fluorescence image intensity and average fitted fluorescence image intensity Calculate the correction factor K for the fluorescence channel under consideration. FI And store it in the internal memory of the microscope to be adapted, wherein, with the help of a correction factor K FI It can correct each adjustable system parameter of the fluorescence microscope to be adapted, which is linearly related to the fluorescence image intensity, to match the fluorescence image intensity. Average reference fluorescence image intensity. and average fitted fluorescence image intensity The fluorescence intensity of the corresponding detector across the entire detected fluorescence image was characterized. This two-part calibration method is only possible using a manual reference calibration sample. This reference calibration sample is characterized by its long-term stability. Advantageously, the reference calibration sample is formed from a uniformly fluorescent glass substrate with high photostability, meaning that the properties and structure of the sample remain stable and constant under electromagnetic radiation. Furthermore, the reference calibration sample has an emission band from 400 nm to 850 nm, allowing the excitation radiation from the microscope to excite the fluorescent components of the sample, which are emitted within the wavelength range of interest in fluorescence microscopy. Advantageously, the reference calibration sample is made of lanthanum phosphate glass, characterized by good chemical resistance and thermal stability, and possesses fluorescence properties advantageous for and usable in fluorescence microscopy.

[0023] Using artificial, long-term stable reference calibration samples ensures that the difference in fluorescence image intensity between two consecutive image acquisitions does not exceed 3% or 1%, thus ensuring good long-term stability.

[0024] Another advantage of the method according to the invention is that, although the reference calibration sample already possesses constant homogeneity across its entire area, the acquisition of the fitted fluorescence image of the artificial reference calibration sample is always performed at the same location on the reference calibration sample. This compensates for any residual fluctuations that may occur. Alignment of the reference calibration sample can be achieved, for example, through crosshairs or other clearly detectable markings on the reference calibration sample.

[0025] In one embodiment of the method according to the invention, the method is performed for each fluorescence channel of the fluorescence microscope to be adapted. Accordingly, a correction factor K is determined for each fluorescence channel x. FI,x , where x is the operating variable for different fluorescence channels x of the reference fluorescence microscope and the corresponding fluorescence microscope to be adapted. The same reference calibration sample can be used for this purpose, as it is capable of emitting fluorescence radiation in all fluorescence regions of interest.

[0026] The advantage of this adaptation method is that if the corresponding correction factor K is taken into account in the imaging settings of the adapted fluorescence microscope... FI,x This allows for calibration / fitting of fluorescence image intensity for any sample. For fitting accuracy, considering the average fluorescence image intensity across the entire fluorescence image acquisition is sufficient. Using the average fluorescence image intensity (mean) across the entire detector field or a portion thereof yields robust and reproducible measurements. Furthermore, pre-performed shading correction ensures that the average fluorescence image intensity is not distorted by darker pixels at the edges of the fluorescence image acquisition. Correction factor K FIThis can be multiplied by adjustable, controllable, or configurable parameters of the microscope system, which result in a linear increase in the intensity of the fluorescence image.

[0027] In a preferred embodiment of the method according to the present invention, when using a fluorescence microscope to examine the sample, a correction factor K is used. FI According to I S,Abgleich =K FI ·I S,orginal The intensity I of the radiation source for a fluorescence microscope S Correction is performed. The optical power of the fluorescent illumination source relative to the control variable I. S It increases linearly. Furthermore, it is advantageous that the radiation source used in the fluorescence microscope is automatically adjusted to an internal standard to, for example, compensate for time drift. In the context of this invention, automatic adjustment of the radiation source to an internal standard is understood as the radiation source having its own internal regulation, through which drift effects, radiation source aging, temperature fluctuations, etc., are automatically adjusted to target values, thereby minimizing their impact on image acquisition. This ensures that the radiation source always emits the same radiation power. In a special design, the radiation source can be an LED light source. The LED intensity I of the fluorescence microscope light source... LED Then according to I LED,Abgleich = K FI ·I LED,orginal Perform corrections.

[0028] The above design scheme of this method is limited by the range of values ​​for the control variables, which are typically from 0% to 100%, with a deviation of 0.1%. Because I S,Abgleich It cannot exceed 100%, therefore I S,orginal It must be significantly smaller to provide sufficient buffer for correction. When selecting I... S,orginal When setting the initial value, the expected range of field fluctuations must be considered. Therefore, to adapt to the fluorescence image intensity, the radiation source intensity I should be... S,orginal Set to a maximum of 50%. Discretization limits the possible accuracy of the fit because, for I... S,Abgleich The calculated value must be rounded. Especially in I S,orginal Smaller and correction factor K FI In larger cases, this can lead to significant intensity deviations. Corrective measures, such as replacing the radiation source, can compensate for this.

[0029] In another preferred embodiment of the method according to the invention, when using a fluorescence microscope to examine the sample, according to t Abgleich =K FI ·t orginal Through the correction factor K FIThe exposure time t is corrected. In general fluorescence imaging, the fluorescence image intensity increases linearly with exposure time t. Under this condition, the correction factor K can be adjusted. FI This is multiplied by the previous settings to achieve compensated fluorescence image intensity. This variation is also limited by the range of values ​​for the control variable t, which is typically from 0.1 ms to 10 s, with a walk length of 0.1 ms. When selecting t... orginal When setting the initial value, the expected range of fluctuations in the field must be considered. Therefore, to adapt to the fluorescence image intensity, the value of t should not be chosen to be less than 10 ms.

[0030] The correction factor applies only to acquisitions with the same system configuration between the reference fluorescence microscope and the fluorescence microscope to be adapted during the adaptation process. However, if system parameters, such as camera gain, are changed during sample acquisition, it is advantageous for the microscope user to correct those system parameters that are linearly related to fluorescence image intensity in an additional methodological step, or for the user to receive a prompt from the system indicating that the corresponding system parameters should still be adjusted to output the same fluorescence image intensity. By changing system parameters that have an unknown effect on fluorescence image intensity, such as changing the filter used, filter type, or radiation source, the correction factor loses its effectiveness, and the adaptation process with the reference fluorescence microscope must be repeated using the changed system configuration.

[0031] Advantageously, the adaptation process between the reference fluorescence microscope and the fluorescence microscope to be adapted is repeated at specific intervals to account for the effects of changes in system characteristics due to usage effects, contamination, or drift.

[0032] In another design of the method according to the invention, the detected predetermined system configuration and the determined correction factor K FI The storage device, such as a database, is stored in the fluorescence microscope to be adapted. When examining samples and acquiring fluorescence images of the samples using the fluorescence microscope, the stored correction factor K is configured for the corresponding system. FI The fluorescence image intensity of the sample acquisition is corrected so that the fluorescence image intensity of the sample acquisition is equivalent to the fluorescence image intensity of the sample when it is examined / acquired on a reference fluorescence microscope.

[0033] In another preferred embodiment of the method according to the invention, other qualified system reference calibration samples are generated based on artificial reference calibration samples by determining the intensity of a temporary fluorescence image for a reference fluorescence microscope and providing it in the memory of the fluorescence microscope to be adapted or in a database within the memory, in order to determine the corresponding correction factor K for the fluorescence microscope to be adapted. FITypically, fluorescence microscopes are calibrated using a manual master reference calibration sample before delivery to the customer. Since this master reference calibration sample is provided only once, other qualified system reference calibration samples must be generated, adapted to the master reference calibration sample, and provided to the customer for calibrating their fluorescence microscope. To adapt a qualified system reference calibration sample, the temporary fluorescence image intensity relative to a reference fluorescence microscope and its corresponding system configuration is determined and stored, for example, in a database of fluorescence microscopes to be adapted. The customer can then determine the correction factor K for adapting their fluorescence microscope based on the temporary fluorescence image intensity adapted to the selected system configuration using the system reference calibration sample. FI .

[0034] In another embodiment of the method according to the invention, the fluorescence image intensity is adapted on the fluorescence microscope to be adapted using a qualified system reference calibration sample. The actual existing system configuration of the fluorescence microscope to be adapted is checked to see if it matches the entries in the database. If they match, the fluorescence image intensity is measured, and the corresponding correction factor K is calculated using the temporary fluorescence image intensity stored for the system configuration. FI In cases of inconsistency, a corresponding prompt is output, and no measurement or calculation is performed. This prevents the output of erroneous correction values. The same applies to actual measurements using a fluorescence microscope. Before acquiring and measuring the fluorescence image intensity of the sample under study, check that the actual system configuration of the fluorescence microscope used matches the entries in the database. Where consistent, measure the fluorescence image intensity and apply the corresponding correction factor K. FI Calibration is performed. In case of inconsistency, a corresponding prompt is output, and the microscope user can readjust the system configuration. This prevents the output of erroneous measurement values.

[0035] The aforementioned technical problem is further solved by a fluorescence microscope that should be adapted to a reference fluorescence microscope. The fluorescence microscope according to the present invention, adapted to a reference fluorescence microscope, includes...

[0036] - An objective lens used to image an object through the imaging beam path.

[0037] - A radiation source used to simultaneously generate illumination radiation with at least two different wavelengths.

[0038] - At least one optical element for coupling illumination radiation into the imaging beam path.

[0039] - A detector device for detecting fluorescence radiation from an object through an imaging beam path, and

[0040] - A control unit, configured to collect and analyze measurement data from the detection unit, and

[0041] - A storage device for storing calibration data used to adapt the fluorescence image intensity of a fluorescence microscope to the fluorescence image intensity of a reference fluorescence microscope, wherein the control unit is configured to perform the following steps:

[0042] - Check that the configured system settings of the fluorescence microscope are consistent with the calibration data entries in the fluorescence microscope's storage device.

[0043] - Adaptive shadow correction is generated using a shadow reference sample through a fluorescence microscope to be adapted.

[0044] - Using the configured system settings and applying previously generated adaptation shading corrections, acquire adaptation fluorescence images of qualified system reference calibration samples through the detector of the fluorescence microscope to be adapted.

[0045] - Determine the average fitted fluorescence image intensity of the fluorescence radiation generated in the fluorescence channel of the fluorescence microscope to be fitted in a qualified system reference calibration sample. ,

[0046] - Based on the average temporary fluorescence image intensity from the database and average fitted fluorescence image intensity ,according to Calculate and store the correction factor K for this fluorescence channel. FI Among them, by means of correction factor K FI It can correct each adjustable system parameter that is linearly related to the fluorescence image intensity of the fluorescence microscope to be adapted, thereby adapting to the fluorescence image intensity, wherein,

[0047] - The qualified system reference sample is designed as a uniformly fluorescent glass substrate with an emission band of 400 nm to 850 nm.

[0048] The fluorescence microscope according to the invention, especially the control unit of the fluorescence microscope, can preferably be configured to perform one of the variations of the method according to the invention described herein.

[0049] Variations of the method according to the invention described herein can be performed using a fluorescence microscope according to the invention.

[0050] The term illumination beam path includes the radiation source and all, especially optical, components that guide and modify the beam, such as lenses, mirrors, prisms, gratings, filters, apertures, and beam splitters, through which excitation light is directed from the radiation source, particularly the light source, to the sample under investigation. The illumination beam path may include an illumination objective. The illumination objective and the microscope objective can each be microscope objectives of a type known per se. In principle, the illumination objective and the microscope objective can also be separate objectives. However, in a preferred embodiment, the illumination objective and the microscope objective are the same objective.

[0051] Excitation light is electromagnetic radiation, particularly in the visible spectrum and adjacent regions. Excitation light may also be referred to as illumination light; these two terms are used largely synonymously in this specification. The radiation source can be any light source capable of providing excitation light of the desired wavelength or multiple desired wavelengths and appropriate intensity. The light source can be, for example, a laser, multiple lasers, an LED, an LED module, or a combination of these components. Excitation light can be coherent, at least partially coherent, or incoherent.

[0052] The reference calibration sample, or a qualified system reference calibration sample, can in principle be any type of sample with long-term stable properties. As previously stated regarding reference calibration samples, qualified system reference calibration samples are also advantageously formed from a uniformly fluorescent glass substrate. Qualified system reference calibration samples also have an emission band from 400 nm to 850 nm, so that the excitation radiation of the microscope can excite the fluorescent components of the sample, which emit within the wavelength range of interest in fluorescence microscopy. Advantageously, qualified system reference calibration samples are also made of lanthanum phosphate glass, characterized by good chemical resistance and thermal stability, and possessing fluorescence properties advantageous for and used in fluorescence microscopy. The microscope according to the invention and the method according to the invention are particularly suitable for studying biological samples. The sample to be studied can in principle be any type of sample.

[0053] Light emitted by a reference calibration sample or a qualified system reference calibration sample due to illumination or excitation light is called emitted radiation or emitted light, and reaches the detector, such as a camera, through the detection beam path. For light to be called emitted light, it is only necessary that the light is emitted from or, in any case, from the illuminated sample. Typically, emitted light can be fluorescence, emitted or emitted by the reference calibration sample or a qualified system reference calibration sample, especially pigment molecules present therein, as a result of illumination by excitation light. Emitted light can also be reflected light, transmitted light, and scattered illumination light. The only requirement for the principle providing contrast is that the reference calibration sample or a qualified system reference calibration sample emits emitted light as a result of illumination by excitation light.

[0054] The emitted light reaches the detector, such as a camera, via a detection beam path. The term "detection beam path" here refers to the detector and all components, especially optical components, that guide and modify the beam, such as lenses, mirrors, prisms, gratings, filters, apertures, and beam splitters, through which the emitted radiation is directed from a reference calibration sample or a qualified system reference calibration sample to the detector. Advantageously, the sensor plane of the detector can be arranged in a plane optically conjugate to the focal plane of the microscope objective.

[0055] The type of detector used to detect emitted radiation typically depends on the type of microscope. In embodiments of the invention, the detector can be formed from multiple individual detectors. For example, the detector can include a two-dimensional spatially resolved photodetector, such as a camera or multiple cameras, a one-dimensional spatially resolved detector, such as a linear detector array, or individual photodetectors, such as point photodetectors. Specifically, the detector can include at least one of the following elements or components: a CCD element, a CMOS element, a SPAD element, or a PMT.

[0056] The term "control unit" is understood to refer to all hardware and software components that interact with the components of the optical device (i.e., a fluorescence microscope) according to the invention to achieve the intended function of said components. The control unit may in particular include computing devices, such as PCs, and camera controllers capable of reading out measurement signals. The detector's measurement data is the measurement data generated by the detector when it is illuminated with emitted light. The storage device can be a known type of storage device. It may, for example, have a database. In the context of this invention, a database is understood to be a memory from which values ​​related to the adaptation process can be read.

[0057] In the design of the fluorescence microscope according to the present invention, when acquiring the sample to be studied using the fluorescence microscope, a correction factor K can be used. FI According to I S,Abgleich =K FI ·I S,orginal The intensity of the radiation source I for calibrating the fluorescence microscope S .

[0058] In another design of the fluorescence microscope according to the present invention, the radiation source is an LED light source, wherein, when using the fluorescence microscope to examine the sample under study, a correction factor K can be used. FI According to I LED,Abgleich = K FI ·I LED,orginal Adjust LED intensity.

[0059] In another design of the fluorescence microscope according to the present invention, when acquiring the sample to be studied using the fluorescence microscope, a correction factor K can be used. FI According to t Abgleich =KFI ·t orginal Correct the exposure time t. Attached Figure Description

[0060] Advantageous embodiments of the microscope according to the invention and advantageous variations of the method according to the invention are described below, particularly in conjunction with the accompanying drawings. The features shown below can constitute an aspect of the invention either individually or in different combinations thereof. Wherein:

[0061] Figure 1 A first embodiment of the microscope according to the invention is shown in the schematic view; and

[0062] Figure 2 A first embodiment of the method according to the present invention is shown in the schematic diagram. Detailed Implementation

[0063] refer to Figure 1 A first embodiment of the microscope 100 according to the present invention is described.

[0064] Microscope 100 first includes an illumination beam path with a radiation source 10, such as a laser, laser module, or one or more LEDs, for emitting excitation light, wherein the illumination beam path is configured to guide or conduct the excitation light to sample region 1. In the illustrated example, the illumination beam path also includes a tube lens 20, an excitation filter 22, a main beam splitter 23, and a microscope objective 40. The excitation filter 23 is a transmission filter that allows only those spectral portions of the excitation light 12 desired for the corresponding experiment or calibration to pass through. The tube lens 20 produces an intermediate image plane 18, which is optically conjugate to a plane 11 in sample 2 within sample space 1. In the illumination beam path, the excitation light 12 passes through the intermediate image plane 18, through the tube lens 20 and the excitation filter 22, to the main beam splitter 23, where it is reflected toward the microscope objective 40. The excitation light 12 then passes through the back focal plane 42 of the microscope objective 40 and is subsequently guided by the microscope objective 40 into sample space 1. For real experiments, Sample 2 can be a biological sample, or for calibration, it can be a reference calibration sample or a qualified system reference calibration sample, and is prepared from a pigment that can be excited by excitation light 12. The wavelength and intensity of excitation light 12 can be appropriately selected for Sample 2 and the pigment used. Radiation source 10 can consist of a variety of different lasers or LEDs. The wavelength and / or intensity can be set.

[0065] Furthermore, the microscope 100 includes a detection beam path with a microscope objective 40 and a detector 54 for detecting emitted light 16 from a reference calibration sample or a qualified system reference calibration sample 2 in sample region 1 due to illumination by excitation light 12. The detection beam path is configured to guide the emitted light 16 to the detector 54. In the illustrated example, the microscope 100 is a wide-field fluorescence microscope, and the detector 50 is a camera, i.e., the field of view (FOV) of the detection beam path is imaged onto the sensor plane 51 of the camera 50. The sensor plane 51 is optically conjugate to plane 11 in sample space 1. The emitted light 16 emitted by sample 2 can typically be redshifted fluorescence, emitted by pigments in sample 2. A main beam splitter 23 is configured to transmit the redshifted emitted light 16 and reflect the excitation light 12. This prevents most of the excitation light 12 backscattered from sample space 1 from propagating toward the camera 50. In the detection beam path, the emitted light 16 emitted by the reference calibration sample or a qualified system reference calibration sample 2 is received by the microscope objective 40, passes through the main beam splitter 23 and the emission filter 24, and is then imaged onto the sensor plane 51 of the camera 50 by the tube lens 25. The emission filter 24 is a transmission filter that allows only those spectral portions of the emitted light 16 that are to be measured for the corresponding experiment / calibration to pass through.

[0066] In the illustrated embodiment, the excitation filter 22, the main beam splitter 23, and the emission filter 24 are arranged in a replaceable filter cube or filter module 26. To accommodate different filter cubes, each with a different excitation filter, main beam splitter, and / or emission filter (not shown), a replacement device 27, schematically shown, is provided in the illustrated embodiment. The replacement device may include, for example, a linear slider that allows the desired filter cube 26 to be introduced into the beam path in a direction perpendicular to the plane of the drawing. By replacing the filter cube, different system configurations for the excitation beam path and the detection beam path are achieved, respectively.

[0067] The light source 10 can also be configured to emit excitation light 12 with different spectral compositions for each system.

[0068] Of the components such as excitation filter 22, main beam splitter 23, and emission filter 24, only the main beam splitter 23 is necessary to guide the excitation light 12 toward the sample 2. Therefore, the replacement device 27 can also be a replacement device used only to introduce different main beam splitters into the beam path.

[0069] Furthermore, in the illustrated example, the microscope 100 includes, in particular, an automated mechanical actuator 44 for setting the relative lateral positions x, y between the sample 2 and the microscope objective 40 relative to the optical axis 41 of the microscope objective 40, and a control unit 90, such as a PC, for analyzing the emitted light 16 detected by the detector 54. In the illustrated example, the optical axis 41 of the microscope objective 40 extends along the z-axis. The mechanical actuator 44 may be, for example, part of a motorized sample stage, and in the illustrated example, is used to adjust the predetermined position of the sample 2, i.e., the predetermined x, y coordinates of the sample 2 relative to the optical axis 41, and to always precisely align it with a qualified system reference calibration sample. A right-handed orthogonal coordinate system x, y, z is shown below the mechanical actuator 44. Advantageously, the reference calibration sample or qualified system reference calibration sample 2 has a crosshair visible in a bright field so that the sample 2 can be precisely positioned and focused.

[0070] According to the invention, the control unit 90 has a storage device 91, in which the system configuration of the microscope is initially stored. Furthermore, temporary fluorescence image intensities are stored in the storage device 91, which are pre-measured or calculated through measurements at the reference fluorescence microscope 200 and / or various system integration systems 300. These values ​​can also be stored in and loaded from a database. Additionally, illumination settings for shading correction can be stored, for example, in a lookup table to ensure that the image of the manual reference calibration sample 250 and the qualified system reference calibration sample 350 is identical at the customer's location. Once the settings are created, parameterization can be locked to avoid process errors and erroneous data.

[0071] Settings created in advance with a specific system configuration and stored in memory are compared with the system configuration for the current imaging settings before each measurement. Customers can only use these settings if the current imaging settings match one of the stored system configurations.

[0072] According to the present invention, the control unit 90 sets a predetermined system configuration for using temporary fluorescence intensity 213 for the fluorescence microscope 400 to be adapted, and calculates a correction value K from the adapted fluorescence image acquired at the fluorescence microscope 400 to be adapted using a qualified system reference calibration sample 350. FI 413. This correction value was then used to adapt to the intensity of subsequently acquired fluorescence images of the samples under study.

[0073] Therefore, the method according to the invention or one of the variations of the method according to the invention described in the general part of the specification can be performed by microscope 100.

[0074] Figure 2A method for adapting fluorescence image intensity according to the present invention for different fluorescence microscope systems is schematically illustrated. The fluorescence microscope system outlined in the dashed box is a reference fluorescence microscope 200. In a first embodiment, the fluorescence image intensity x210 is measured at the reference fluorescence microscope against an artificial reference calibration sample 250. At the fluorescence microscope 300 to be adapted, the fluorescence image intensity Y211 is measured against the same artificial reference calibration sample 250. The system configurations of the reference fluorescence microscope 200 and the fluorescence microscope 300 to be adapted are checked beforehand to ensure consistency. Furthermore, shading correction (not shown) is performed using a shading reference sample. A correction value F is calculated from the measured fluorescence image intensity x210 and the measured fluorescence image intensity Y211. I,ref 270. To generate a qualified system reference calibration sample 350, the fluorescence image intensity Z 212 of the system reference calibration sample 350 was measured at a fluorescence microscope 300. Using the correction value F... I,ref 270, thereby calculating the temporary fluorescence image intensity W 213 and storing it in the memory 91 of the fluorescence microscopes 100 and 400. This temporary fluorescence image intensity W 213 is provided to the customer's microscope system 400 for adaptation. The customer can, for example, use a qualified system reference calibration sample 350 provided to them and the temporary fluorescence image intensity W 213 to... Calculate the new correction value K FI 413, to adapt the acquired fluorescence image intensity to the original reference fluorescence microscopes 100 and 200. The fluorescence image intensity A is generated by the fluorescence radiation emitted by a qualified system reference calibration sample 350 acquired by the detector on the customer fluorescence microscope 400 to be adapted. For the fluorescence image intensity, the average value on the detector's field of view is calculated, i.e., the determined fluorescence image intensities X, Y, Z, and A are the average values ​​on the detector's field of view.

[0075] List of reference numerals

[0076] 1 Sample Area

[0077] 2 samples

[0078] 10. Radiation sources, light sources, such as lasers or LED sources.

[0079] 12. Illumination radiation, illumination light, excitation light

[0080] 16. Emitted radiation or emitted light emitted by sample 2 in sample region 1

[0081] 20. Lens in the excitation beam path

[0082] 22 Excitation Filter

[0083] 23 Main beam splitter

[0084] 24 Emission Filters

[0085] 25. Detecting lenses in the beam path

[0086] 26 Filter Cubes

[0087] 27 Replacement device for filter cube 26

[0088] 40 Microscope Objectives

[0089] 41. Optical axis of microscope objective lens: 40°

[0090] 44 Mechanical actuator, xy displacement stage

[0091] 50 detection units

[0092] 51. Detection plane, optically conjugate with sample plane 11.

[0093] 54 detectors, cameras

[0094] 90 Control Unit

[0095] 91. Memory, storage device

[0096] 100 Microscope according to the present invention

[0097] 200 Reference Fluorescence Microscope

[0098] 210...212 Measured fluorescence image intensity

[0099] 213 Temporary fluorescence image intensity

[0100] 250 manual reference calibration samples

[0101] 270 is the correction value relative to the reference fluorescence microscope.

[0102] 300 fluorescence microscopes to be adapted

[0103] 350 qualified system reference calibration samples

[0104] 400 customers' fluorescence microscopes awaiting adaptation, customer systems

[0105] 410 Fluorescence image intensity measured on the customer system

[0106] 413 Calibration values ​​for customer systems to be adapted

Claims

1. A method for adapting fluorescence image intensity for different fluorescence microscopes (100, 300) and for different time points of image acquisition using fluorescence microscopes (100, 300), wherein, The method includes the following steps: - Obtain the predetermined system configuration for the reference fluorescence microscope (200). - Shadow correction is generated using a shadow reference sample via a reference fluorescence microscope (200). - Under the predetermined system configuration and with the application of previously generated shadow correction, reference fluorescence images of the artificial reference calibration sample (250) are acquired through the detector of the reference fluorescence microscope (200). - Determine the average reference fluorescence image intensity of the fluorescence radiation generated in the artificial reference calibration sample (250) in the fluorescence channel of the reference fluorescence microscope (200). (210), - Adaptive shadow correction is generated using the fluorescence microscope (300) to be adapted, with the aid of a shadow reference sample. - Under the predetermined system configuration and with the application of previously generated adaptation shading correction, the adaptation fluorescence image of the artificial reference calibration sample (250) is acquired through the detector of the fluorescence microscope (300) to be adapted. - Determine the average fitted fluorescence image intensity of the fluorescence radiation generated in the artificial reference calibration sample (250) in the fluorescence channel of the fluorescence microscope (300) to be fitted. (211), - Based on the average reference fluorescence image intensity (210) and average fitted fluorescence image intensity (211), according to Calculate and store the correction factor K for the fluorescence channel. FI (270), where, with the help of the correction factor K FI (270) It is capable of correcting each adjustable system parameter of the fluorescence microscope to be adapted, which is linearly related to the fluorescence image intensity, to adapt to the fluorescence image intensity, wherein, - The artificial reference sample was designed as a uniformly fluorescent glass substrate with an emission band of 400 nm to 850 nm.

2. The method according to claim 1, characterized in that, The acquisition of the adaptive fluorescence image of the artificial reference calibration sample was always performed at the same location on the reference calibration sample.

3. The method according to claim 1, characterized in that, The method is performed accordingly for each fluorescence channel of the fluorescence microscope (300, 400) to be adapted.

4. The method according to any one of claims 1 to 3, characterized in that, When samples are collected using fluorescence microscopes (100, 300, 400), the radiation source intensity I of the radiation source (10) of the fluorescence microscope (100, 300, 400) is... S In particular, the LED intensity of the light source for fluorescence microscopes (100, 300, 400) is adjusted by a correction factor K. FI (270, 413) According to I S,Abgleich =K FI ·I S,orginal Perform corrections.

5. The method according to any one of claims 1 to 3, characterized in that, When acquiring samples using fluorescence microscopes (100, 300, 400), the exposure time t is adjusted by a correction factor K. FI (270, 413) According to t Abgleich =K FI ·t orginal Perform corrections.

6. The method according to claim 4, characterized in that, To adapt to the fluorescence image intensity, the radiation source intensity I S,original Set to the maximum of 50%.

7. The method according to claim 5, characterized in that, To adapt to the fluorescence image intensity, the exposure time t original Set to at least 10ms.

8. The method according to claim 1, characterized in that, In an additional method step, system parameters that are linearly correlated with fluorescence image intensity are pre-calibrated.

9. The method according to claim 1, characterized in that, The obtained predetermined system configuration and the determined correction factor K FI (270, 413) are stored in the storage device (91) of the fluorescence microscope (100, 300, 400) to be adapted.

10. The method according to claim 1, characterized in that, Other qualified system reference calibration samples (350) are generated based on the manual reference calibration sample (250) by determining the temporary fluorescence image intensity (213) for the reference fluorescence microscope (200) and providing it to the storage device (91) of the fluorescence microscope (100, 300, 400) to be adapted, so as to determine the corresponding correction factor K for the fluorescence microscope (100, 300, 400) to be adapted. FI (270, 413).

11. The method according to claim 1 or 10, characterized in that, Before performing fluorescence image intensity adaptation on fluorescence microscopes (100, 300, 400) using a qualified system reference calibration sample (350), it is checked whether the actual existing system configuration of the fluorescence microscope (100, 300, 400) to be adapted is consistent with the entry from the storage device (91). If consistent, the fluorescence image intensity is measured, and the corresponding correction factor K is calculated using the temporary fluorescence image intensity (213) stored for the system configuration. FI (413) In case of inconsistency, output the corresponding prompt and do not perform measurement and calculation.

12. A fluorescence microscope (300, 400) adapted to a reference fluorescence microscope (200), comprising: - An objective lens (40) used to image an object through the imaging beam path. - A radiation source (10) for simultaneously generating illumination radiation (12) having at least two different wavelengths, - At least one optical element (23) for coupling illumination radiation into the imaging beam path, - A detector unit (50) for detecting fluorescence radiation from the object (2) via the imaging beam path, and - A control unit (90) configured to collect and analyze measurement data from the detector (54) of the detector unit (50), and - A storage device (91) for storing calibration data used to adapt the fluorescence image intensity of a fluorescence microscope (300, 400) to the fluorescence image intensity of a reference fluorescence microscope (200). Its features are, The control unit (90) is configured to perform the following steps: - Check that the configured system settings of the fluorescence microscopes (300, 400) are consistent with the entries of the calibration data in the storage device (91) of the fluorescence microscopes (300, 400). - Adaptive shadow correction is generated using the fluorescence microscope (300, 400) to be adapted, with the help of a shadow reference sample. - With the system configuration set up and the previously generated adaptation shadow correction applied, the adaptation fluorescence image of a qualified system reference calibration sample (350) is acquired through the detector of the fluorescence microscope (300, 400) to be adapted. - Determine the average fitted fluorescence image intensity of the fluorescence radiation generated in the fluorescence channel of the fluorescence microscope to be fitted (400) in a qualified system reference calibration sample (350). (211, 410), - Average temporary fluorescence image intensity from storage device (91) (213) and average fitted fluorescence image intensity (410), according to Calculate and store the correction factor K for the fluorescence channel. FI (413), where, with the help of correction factor K FI (413) It is capable of correcting each adjustable system parameter of the fluorescence microscope (400) to be adapted, which is linearly related to the fluorescence image intensity, to adapt to the fluorescence image intensity, wherein, - A qualified system reference sample is designed as a uniformly fluorescent glass substrate with an emission band of 400 nm to 850 nm.

13. The fluorescence microscope according to claim 12, characterized in that, When collecting the samples to be studied (2) using fluorescence microscopy (100, 300, 400), it is possible to use the correction factor K FI (270, 413) With the help of I S,Abgleich =K FI ·I S,orginal Radiation source intensity I of radiation source (10) for calibrating fluorescence microscopes (100, 300, 400) S .

14. The fluorescence microscope according to claim 12, characterized in that, The radiation source is an LED light source, wherein, when collecting the sample to be studied (2) using a fluorescence microscope, it is possible to pass through the correction factor K. FI (270, 413) With the help of I LED,Abgleich =K FI ·I LED,orginal Adjust LED intensity.

15. The fluorescence microscope according to claim 12, characterized in that, When collecting the samples to be studied (2) using fluorescence microscopy (100, 300, 400), it is possible to use the correction factor K FI With the help of t Abgleich =K FI ·t orginal Correct the exposure time t.

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