Control system and method for determining illumination intensity in fluorescence microscope as well as corresponding microscope system

Abstract

To provide a control system (150) for automatically determining illumination intensity (Pk) of a light source (120k) of a fluorescence microscope (100).SOLUTION: A light source is used for stimulating fluorophore (130) in a sample (110), and configured to vary illumination intensity, and a microscope has a detector (140) for detecting image intensity (B) of the sample. A control system is configured to: automatically determine (304) a control value of the illumination intensity after a change (302) in a light path (164 and 168) for achieving a desired value of an observation parameter (B) characterizing a sample observation. Determining is based on a value of the illumination intensity being set before the change, a value of the observation parameter being used before the change, and a physical model (M) of the light path. The physical model is configured to associate the illumination intensity with the observation parameter, taking into account an imaging characteristic of an optical component (124, 132, 160m and 160m+1) in the light path.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention substantially relates to a light source for a fluorescence microscope, a control system for automatically determining the illumination intensity of a light source used to stimulate a phosphor in a sample, a fluorescence microscope, a microscope system comprising such a control system, and a corresponding method. [Background technology]

[0002] In particular, with fluorescence microscopy, when the objective lens and / or other components in the optical path are changed, and illumination parameters such as the field of view and intensity of the intermediate image are kept constant, the illumination intensity incident on the objective field of view (and the sample) changes. In addition, the intensity detected on the detector changes differently.

[0003] However, when examining or imaging a sample using fluorescence microscopy, a constant illumination intensity or image intensity is typically required in the sample even after changing the objective lens. Therefore, it is necessary to readjust the illumination intensity or power of the light source. [Overview of the project] [Means for solving the problem]

[0004] In light of the aforementioned shortcomings and problems, an improved method for adjusting illumination intensity in fluorescence microscopy is needed. According to embodiments of the present invention, a microscopy system and method comprising a control system, a fluorescence microscope, and a method having the features of the independent claims are proposed. Advantageous further developments form the subject of the dependent claims and subsequent descriptions.

[0005] Embodiments of the present invention generally relate to a microscope system comprising a fluorescence microscope, particularly an epifluorescence microscope, and a control system for such a fluorescence microscope. The control system may comprise one or more processors, which may be a separate computer (system) or a processor integrated into the microscope. The microscope comprises at least one light source used to stimulate at least one phosphor (also called a fluorescent dye or pigment, which is a fluorescent compound that can re-emit light upon photoexcitation) in a sample to be examined and / or imaged by the microscope. The at least one light source is configured to vary its illumination intensity (also called illumination power or illumination luminance). The fluorescence microscope further comprises at least one detector (which may be part of a detection unit comprising multiple detectors) used and configured to detect image intensity, wherein the image intensity is the fluorescence light emitted from the excited fluorescent dye in the sample. The image intensity also depends on the illumination intensity of the light source.

[0006] In this regard, the term “light source” includes any light-emitting device suitable for exciting a phosphor, preferably a solid-state light source. For the excitation of a phosphor, a predetermined excitation wavelength must be present in the spectrum of the light source. Therefore, such a light source can be a broadband light source containing the excitation wavelength, a narrowband light source containing the excitation wavelength, or a light source equipped with a downstream filter that filters the spectrum containing the excitation wavelength from the light source spectrum. The same applies to the term “detector.” Such a detector or detection unit must be able to detect the wavelength of the fluorescence emission emitted by the phosphor under discussion. For this purpose, a corresponding broadband detector or a corresponding narrowband detector with sufficient sensitivity to the corresponding wavelength of fluorescence emission can be used. Furthermore, a broadband detector equipped with an upstream filter may also be used, which filters the relevant wavelength of fluorescence emission, making it accessible to the detector’s sensor. The aforementioned filters, connected downstream of the light source or upstream of the detector, can be designed as filter wheels or filter slides, as spectral splitting layers, spectrometers or monochromators, or, for example, in the form of acousto-optics or liquid crystal-based systems.

[0007] Each light source in this application is intended to directly excite phosphors assumed by the user to be present in the imaged sample. Each of these light sources can have its illumination intensity or brightness individually adjusted. In this way, the light beam incident on the directly assigned phosphor, and consequently the light beam obtained from the fluorescence emission ultimately incident on the detector, can be adjusted. When a single light source excites two or more phosphors, it is preferable to consider so-called cross-excitation in the illumination control method.

[0008] Depending on the specific type of inspection or imaging performed by the user, this may require changes to the optical path from at least one light source to the sample (also called the illumination path) and / or the optical path from the sample to at least one detector (also called the imaging path). Such changes typically involve changing the microscope's objective lens currently positioned in the optical path. In addition, other components, such as filters, positioned in the optical path may be inserted, removed, or replaced, which can also lead to changes in the optical path regarding illumination intensity. If those illumination parameters, such as the field of view and intensity of the intermediate image, remain unchanged, the illumination intensity incident on the objective field of view will consequently change due to the change in the objective lens. In addition to the illumination intensity incident on the sample and phosphor, the imaging intensity, i.e., the intensity of light emitted from the phosphor and reaching at least one detector, may also change.

[0009] Therefore, the illumination intensity and / or imaging intensity of the light source must be adjusted to maintain a constant level of illumination and / or imaging intensity on the sample, or to reset it to a desired value (such a desired value may be, for example, a suitable value for achieving appropriate illumination for a particular inspection). For example, manual resetting is quite time-consuming. Therefore, such adjustments should preferably be performed by an automated method. Automated illumination control methods may work based on databases and lookup tables that can adapt filter settings to arc lamps and filters. However, creating data for lookup tables and databases is time-consuming and needs to be adapted to changes in hardware such as light sources. Furthermore, such methods cannot fully utilize the possibility of fine wavelength-specific adjustments of modern solid-state fluorescent light sources (floodlights).

[0010] The preferred method for achieving automatic lighting control is the use of iterative methods. However, for such iterative methods to converge quickly, i.e., to rapidly reach the final setting of the lighting intensity, a good starting point for the iteration is essential.

[0011] To overcome such problems, a control system according to one embodiment of the present invention is configured to automatically determine a control value for the illumination intensity of at least one light source after a change in the optical path in order to achieve a desired value for observation parameters that characterize sample observation. The control system is preferably configured to automatically detect the change in the optical path by one or more sensors, for example, located in an objective turret. The observation parameters are, in particular, based on or include a desired illumination intensity in the sample. The observation parameters may also be based on or include a desired imaging intensity of the sample detected by at least one detector. The desired value for the observation parameters is, in particular, a value desired by the user for observation of the sample, for example, the same value that was used or set before the change. However, any other value may also be selected or used. The desired value for the imaging intensity of the sample detected in the detection unit may be defined for at least one phosphor based on a value derived from photon statistics detected in the detector, for example, based on the signal-to-noise ratio in at least one detector. Preferably, the control system also automatically adjusts the illumination intensity of at least one light source according to the determined control value for illumination intensity. This is done, in particular, by matching the power supplied to at least one light source.

[0012] Furthermore, determining the control value of the illumination intensity of the at least one light source is based on the illumination intensity value set before the change in the optical path, the value of the observation parameter used before the change in the optical path, and a physical model of the optical path. This physical model associates or correlates the illumination intensity of the at least one light source with the observation parameter, taking into account the imaging characteristics of the optical components in the optical path.

[0013] In physical models, different types of such imaging characteristics can be considered. These preferably include at least one of the group consisting of the magnification of the objective lens, the transmittance of the objective lens, the numerical aperture of the objective lens, the refractive index of the immersion medium, the area of ​​the illumination field diaphragm in the intermediate image, the ratio of the illuminated pupil area to the entire pupil area in the objective lens, the magnification of the system optical components, and / or the focal length. The latter may include, for example, a magnification device, a camera adapter, a focal length illumination module, and the like.

[0014] Embodiments of the present invention utilize an implementation of such a physical model based on first-principles optical data to calculate the expected image intensity and / or illumination intensity after a change such as an objective lens change, from data known before the change, i.e., current illumination intensity and image luminance, as well as basic optical data. This can be used, in particular, to generate a suitable starting point for automatic (iterative) illumination adjustment after a change in the objective lens, thus leading to faster convergence.

[0015] The aforementioned physical model will be briefly explained below, followed by a more detailed explanation of the figures. In the case of an epifluorescence wide-field microscope, for example, the illumination power from light incident on the objective pupil, which functions as a condenser pupil in the case of epifluorescence, is diffused into the illuminated pupil region. The power incident on the sample is the transmittance of the objective lens multiplied by the ratio of the pupil region of the objective lens to the illuminated pupil region. The objective lens can be assumed to have a specific magnification and can be designed for an immersion medium with a specific refractive index. This allows for the determination of the intensity in the sample.

[0016] If the optical stress on the specimen should be kept constant when the objective lens is changed, the illumination power of the light source should be appropriately adjusted. Assuming that only the (power-linearized) drive current of the light source can be changed and the light source spectrum cannot be changed, the new drive current can be easily derived.

[0017] If the intensity detected (image intensity) in at least one detector should be kept constant, the detected solid angle and the amount of fluorescence collected (due to the isotropy of fluorescence emission) can be determined. Assuming that the reduction ratio of the pixel area of ​​the array detector relative to the sample changes in a manner similar to the change in the reduction ratio of the illumination aperture when the objective lens is changed, these two area effects cancel each other out, leaving only vignetting and solid angle as factors affecting the fluorescence intensity detected per pixel. Also, as with the case of a magnification device, if the pixel area reduction ratio factor changes independently of the illumination aperture reduction ratio, this factor can be easily incorporated into the model in the form of each area magnification.

[0018] While constant factors and transmission factors can be ignored, transmission effects can be easily included by recalculating the overlap integral of cross-excitation and cross-emission from the spectral data of the phosphor and the transmission, irradiation, and sensitivity spectra of the optical system.

[0019] This allows for the determination of a new (and very good) value of illumination intensity or light source immediately after or even during a change of the objective lens. As described above, such a value is preferably used as a starting value or starting point in an iterative method to further fine-tune the illumination intensity to achieve a desired value of the inspection parameter. If the starting value already satisfies the convergence criteria of the iterative method, the iteration may be omitted entirely.

[0020] In such an iterative method, the illumination intensity value is preferably changed sequentially until a specified target value is reached (or a convergence criterion is met) for illumination or imaging intensity (e.g., by the signal-to-noise ratio for each phosphor) in the sample. This change in illumination intensity in this iterative method includes increasing or decreasing the illumination intensity.

[0021] In typical iterative methods, the initial or starting value of the illumination intensity is set to, for example, zero or the value used before the objective lens was changed. However, in the proposed physical model, it is possible to generate a starting value very close to the final value, and thus the iterative method converges very quickly. This reduces the number of iteration steps required. When imaging intensity is used as the observation parameter, the image intensity can be detected directly, in particular, in at least one detector. When illumination intensity in the sample is used as the observation parameter, the image intensity in at least one detector can be detected and used (after recalculation as necessary) to determine the appropriate value of illumination intensity. In the case of cross-emission, the imaging intensity for each phosphor can be reconstructed by demixing from the acquired data (see also below for cross-emission).

[0022] In a preferred embodiment, a fluorescence microscope is used having at least two light sources used to stimulate different phosphors among at least two phosphors in a sample, the at least two light sources configured to vary their illumination intensity independently. A corresponding control system is configured to automatically determine, individually, the control values ​​for the illumination intensity of each of the at least two light sources after changes in the optical path. The physical model can be used for each light source, each having individual parameters such as illumination intensity. A preferred method for individually determining the control values ​​for the illumination intensity of each of the at least two light sources is by determining the cross-excitation and / or cross-emission of the at least two phosphors in the sample to account for crosstalk effects in excitation and / or detection. Such crosstalk effects in the detector allow for very precise determination of the required intensity.

[0023] In this regard, it should be noted that when at least two detectors are used, each detector appropriately assigned to a particular phosphor will, by its sensitivity spectrum, also detect to some extent the fluorescence emission from other phosphors. Such cross-emitting emission can be taken into account by determining the degree of crosstalk while setting the illumination intensity of the light source. Similarly, a particular excitation wavelength typically excites not only the assigned phosphor but also other phosphors to some extent; this is called cross-excitation.

[0024] The cross-excitation and cross-emission of a system comprising detectors having respective detection spectra, light sources having respective light source spectra, and phosphors having respective excitation and emission spectra can be empirically determined from known spectral data, such as system design data and calibration measurements, as well as subsequent calculations of appropriate overlap integrals of the light source spectrum and excitation spectrum to obtain the matrix elements of the cross-excitation matrix, and subsequent calculations of appropriate overlap integrals of the detection spectrum and emission spectrum to obtain the matrix elements of the cross-emission matrix. Alternatively, control measurements can be performed using individually labeled specimens on the system, and the respective matrix elements can be derived from these controls (measurements).

[0025] As described above, one embodiment of the present invention also relates to a method for automatically determining the illumination intensity of a fluorescence microscope. Any further details of the method, preferred embodiments and advantages are to be seen with reference to the above, which are also applied accordingly.

[0026] The present invention also relates to a computer program having program code for performing a method according to the present invention when executed in a control system comprising one or more processors or an embodiment of the present invention.

[0027] Further advantages and embodiments of the present invention will become apparent from the description and accompanying figures.

[0028] It should be noted that the features described above and those further described below can be used in further combinations or individually, without departing from the scope of the present invention, not only in the combinations shown, but also in other ways. [Brief explanation of the drawing]

[0029] [Figure 1] This figure schematically illustrates a microscope system according to a preferred embodiment of the present invention. [Figure 2] This figure schematically illustrates a microscope system according to a further preferred embodiment of the present invention. [Figure 3] This is a flowchart of a method according to a preferred embodiment of the present invention. [Modes for carrying out the invention]

[0030] Figure 1 shows, as an example, a light source 120 for the excitation wavelength. k A microscope system 102 comprising a fluorescence microscope 100 according to a preferred embodiment of the present invention is shown very schematically. The light source is advantageously an LED or laser having a corresponding spectrum, and a filter can be connected downstream as needed. In principle, a broadband light source can also be used, from which a desired excitation wavelength can be filtered by a filter wheel or filter slide.

[0031] As shown in Figure 1, the light source 120 k It emits illumination light along the illumination beam path 164, and the illumination light is directed to the objective lens 160 of the microscope 100. m You will be guided there. Objective lens 160 m On the way there, the illumination beam path 164 is connected to the collector lens 122, area A LF Illumination field aperture 124, pupil illumination diameter 2r LFIt passes through the condenser lens 126 having [it], and is reflected by the spectral splitting element 166. This spectral splitting element 166 is preferably a dichroic element, which deflects the corresponding excitation wavelength and is transparent to the corresponding fluorescence emission. Since the details of the optical elements of the fluorescence microscope are well known in sufficient detail from the prior art, they are shown only very schematically in FIG. 1.

[0032] Objective lens 160 m has a radius f ref ·NA m / β m (reference focal length f ref , numerical aperture NA m and magnification β m ), transmittance T m and immersion refractive index n m of the pupil 132. The illumination beam path 164 is directed towards the sample 110 through the objective lens 160 m and stimulates the phosphor 130 disposed thereon to emit fluorescence emission. The illuminated objective field 134 of the illumination beam path has an area A I . The emitted fluorescence emission is imaged onto the detector 140 through the objective lens 160 m , the tube lens 136 and any other optical imaging elements as necessary. The detection beam path hitting the detector is indicated by 168.

[0033] The illumination intensity or luminance P k (λ) (“illumination power”), thus, the irradiation power of the light source 120 k can be set individually. A control system 150 is provided which is configured to set the illumination intensity. In addition, the control system 150 is in communication with, or operably connected to, means for setting the illumination intensity of the light source. The detector 140 detects the distribution of each phosphor 130 in the recorded image of the sample 110, thus the image intensity B. The control system 150 is in communication with, or operably connected to, the detector 140 to enable recording of a signal corresponding to the image intensity.

[0034] When using the fluorescence microscope 100, the illumination intensity P of the light sourcek (λ) can be set to a desired value by the control system 150. If there are two or more light sources (see also Figure 2), for example, this can be done in such a way that a predetermined set value for the signal-to-noise ratio is set for each distinguishable phosphor. Crosstalk from the detector due to the emission spectra of other phosphors not directly assigned, and cross-excitation of phosphors considered by the irradiation spectra of other light sources not directly assigned can also be taken into account.

[0035] The fluorescence microscope 100 includes an objective lens 160 positioned in the optical path (including the illumination beam path 164 and the imaging beam path 168). m In addition, for example, the objective lens 160 can be used in the turret 162 or other objective lens changing mechanism. m A further objective lens 160 is positioned alongside it. m+1 A 160 objective lens is provided. m+1 is radius f ref ·NA m+1 / β m+1 (Reference focal length f ref , numerical aperture NA m+1 , magnification β m+1 ), transmittance T m+1 and immersion refractive index n m+1 It has eyes that have eyes.

[0036] Depending on the inspection and / or imaging requirements, the user may change the objective lens during the inspection, i.e., objective lens 160 m Remove from the optical path, objective lens 160 m+1 This is moved into the optical path. As described above, changing the objective lens typically requires adjusting the illumination intensity to achieve the desired illumination intensity in the sample 110 and / or the desired imaging intensity in the detector 140.

[0037] It should be noted that a typical user requirement is that the illumination intensity of the light source be equal to (or as close as possible to) the value used before the objective lens change, so that the illumination intensity or imaging intensity in the sample is equal to (or as close as possible to) the value used before the objective lens change. However, different desired values ​​may be used, such as a specific range of the value before the change, e.g., ±10% or ±5% of the value, or a completely different value. The determination of the required illumination intensity and the adjustment of the illumination intensity (or power) of the light source are performed automatically by the control system 150. The adaptation process can be initiated, for example, by inputting a desired value (via a user interface, etc.), or can be initiated automatically with / after the objective lens change (especially when the desired value of the imaging intensity is equal to the value used previously).

[0038] In the following, the physical model used in the concept of the present invention will be described based on the fluorescence microscope 100 and its parameters shown in Figure 1, as well as the imaging characteristics of each optical component in the optical path described above. In the case of an epifluorescence wide-field microscope, for example, the light source 120 k (or k) From this, the illumination power P of wavelength λ incident on the objective pupil, which functions as a condenser pupil in the case of incident illumination. k (λ) is the illuminated pupil region A I It will be spread.

[0039] The incident power on sample 110 is equal to the objective lens 160. m Transmittance T of (or m) m (λ) is multiplied by the ratio of the pupil region to the illuminated pupil region, where, without loss of generality, we can assume that the objective pupil 132 is uniformly illuminated; otherwise, the ratio should be replaced by a superposition integral. The objective lens has a reference focal length f ref Magnification β m and numerical aperture NA m It has a refractive index n m It is assumed that it is designed for immersion media.

[0040] radius r LF (diameter 2r LF ) Uniform pupil illumination and area A in the intermediate image LFAssuming the illumination field diaphragm and , the intensity I due to the objective lens m in the sample m It will be as follows:

[0041]

number

[0042] In that formula, the first term is the reduction ratio of the illumination field aperture, the second term is vignetting, and the third term is the transmittance of the objective lens. When changing the objective lens, if you try to keep the light stress on the sample (i.e., the illumination intensity on the sample) constant, for example, you can change the illumination intensity or power of the light source k, I m =I m+1 It must be adapted so that it becomes so. Here, I m+1 The objective lens is 160 m+1 This is the intensity in the sample due to (or m+1). Therefore, in the above formula, the illumination intensity I m+1 To determine, objective lens 160 m The value of objective lens 160 m+1 Each of these values ​​must be replaced.

[0043] Assuming that the spectrum cannot be changed and only the (power-linearized) drive current of the light source can be changed, the objective lens 160 corresponds to the new illumination intensity. m+1 Light source 120 that uses k New drive current P k,m+1 The (control values) are as follows:

[0044] P k,m+1 =P k,m ·F m / F m+1 Here, current P k,m Light source 120 k and objective lens 160 m This is the drive current (control value) of the light source P. k,mAlternatively, the corresponding illumination intensity typically yields an illumination intensity on the sample that is very close to the desired value, such as the value before changing the objective lens, and it should be noted that this value can be used as a control value for setting the light source. Nevertheless, slight deviations may exist.

[0045] Therefore, in a preferred embodiment, as described above, the value P determined in this manner is k,m This can be used as a starting point or starting value in an iterative method to determine a final control value that enables achieving a better illumination intensity in the sample. In such an iterative method, a starting value for illumination intensity or power can be set, as determined by a physical model, and each illumination intensity or each imaging intensity (in the detector) in the sample is determined. Depending on the difference between this value and the desired value, the illumination intensity can be further adjusted.

[0046] These steps can be repeated until a desired value of illumination intensity or imaging intensity in the sample is achieved, or until the deviation of the achieved value from the desired value falls below a predetermined threshold.

[0047] A particular advantage of using such physical models is that they allow for the very effective consideration of many parameters of optical components that affect the illumination intensity in the sample. Values ​​obtained from such determinations or calculations are a much better starting point for iterative methods than, for example, the values ​​used before changing the objective lens (i.e., the starting point is much closer to the final value). As a result, iterative methods for determining the final control value converge much faster than when such physical models are not used. Since iterative methods typically require repeated changes in the illumination intensity of the light source and subsequent detections, the use of physical models results in less energy consumption and less unwanted irradiation of the sample.

[0048] In another embodiment, the intensity of the image detected in the detector (image intensity) should have a desired value after the change of the objective lens (e.g., kept constant). Therefore, the signal-to-noise ratio must also have a specific value, for example, it must be kept constant (due to changes in the field of view, this can only be considered on a statistical basis, such as histogram data). The detected solid angle and the isotropy of the fluorescence emission of the objective lens 160 m The amount of fluorescence light collected and the following equation are given by:

[0049]

number

[0050] The formula excludes supercritical angle fluorescence by restricting the solid angle to 2π. Assuming that the reduction ratio of the pixel area of ​​the array detector relative to the sample changes in a manner similar to the change in the reduction ratio of the illumination field aperture when the objective lens is changed, these two area effects cancel each other out, and the fluorescence intensity B detected for each pixel is... m The only remaining factors influencing this are vignetting and solid angle, which are as follows:

[0051]

number

[0052] Note that constant factors and transmission factors are ignored here. Transmission effects can be easily included by recalculating the overlap integral of cross-excitation and cross-emission from the spectral data of the phosphor and the transmission, irradiation, and sensitivity spectra of the optical system. As with the illumination intensity in the sample, the objective lens 160 corresponds to the new imaging intensity. m+1 Light source 120 that uses k New drive current P k,m+1 The following applies:

[0053] P k,m+1 =P k,m ·B m / Bm+1 Here, current P k,m is the drive current of the light source 120 k and the objective lens 160 m . Similar to the illumination intensity situation in the sample described above, the value thus achieved is typically very accurate. Nevertheless, for an even better final control value, an iterative method can be used.

[0054] FIG. 2 very schematically shows a microscope system 202 including a fluorescence microscope 200 according to a further preferred embodiment of the present invention. The fluorescence microscope 200 basically corresponds to the fluorescence microscope 100 of FIG. 1. However, the fluorescence microscope 200 includes, for example, two light sources 120 k , 120 k+1 for two different excitation wavelengths, which are within a common light housing 120. That is, in addition to the light source of the fluorescence microscope 100, there is a further light source 120 k+1 . In addition, the light source 120 k+1 can vary the illumination intensity and is, in particular, independent of the light source 120 k . The optical paths 164, 168 are common to all light sources. By using a plurality of light sources, their illumination intensities can be set simultaneously, so that sequential operations or sequential settings are not required.

[0055] Furthermore, the sample 110 includes two phosphors denoted as 130 j , 130 j+1 (whereas in FIG. 1 only the reference numeral 130 was used). Each phosphor will be stimulated by a different excitation wavelength. Furthermore, two (individual) detectors 140 i , B i+1 for detecting the imaging intensities B i , 140 i+1is provided, for example, within a common housing 142. In this example, two light sources, two phosphors and two detectors are shown, but depending on the particular needs or wishes of the user or inspection, three or more of each of them can also be used. It should be noted that the numbers of light sources, phosphors and detectors do not necessarily have to match.

[0056] For each light source that emits an excitation wavelength, a corresponding phosphor can be assigned, the phosphor emits fluorescent radiation, and the fluorescent radiation is detected by a corresponding detector. Thus, a detector is assigned to each distinguishable phosphor.

[0057] For each of the light sources, the respective illumination intensity after changing the objective lens (or another change in the optical path) can be determined individually by a physical model as described above for a single light source. In particular, the crosstalk effect in excitation and / or detection can be taken into account as described above.

[0058] FIG. 3 shows, by way of a flow chart, a method according to a preferred embodiment of the invention, in which the illumination intensity of the light source of the fluorescence microscope 100 as shown in FIG. 1 is adjusted. In step 300, an inspection of the sample is carried out using the objective lens m and a specific value P as the control value (drive current) of the light source, which corresponds to a specific value of the illumination intensity P k,m of the light source. Thus, the illumination intensity I (inspection parameter) in the sample should have a value of I = I k of that value. m

[0059] In step 302, the objective lens m (160 m ) is replaced by the objective lens m + 1 (160 m+1 ). After or during that replacement, in step 304, a new control value P k,m+1 (drive current) of the light source is determined, and the value I mThis yields an illumination intensity I value in the sample that is very close to the given value. This is done using the physical model M described above with respect to Figure 1. In particular, the imaging characteristics of optical components present in the optical path (which may change with changes in the objective lens) are taken into consideration.

[0060] According to the formula P k,m+1 =P k,m ·F m / F m+1 Here I m =F m P k,m New control value P k,m+1 This is determined and set. As mentioned above, this new control value is the value I before the objective lens change. m This produces an illumination intensity I in the sample that is close to this.

[0061] To better adjust the control value, and also the value I m Alternatively, to achieve an illumination intensity I in the sample that is even closer to any other desired value, an iterative method is performed in step 306. Such an iterative method involves slightly changing the control value and observing the illumination intensity I obtained in the sample. Examples of such iterative methods can be found in literature referring to numerical optimization, and for example, the Newton method or quasi-Newton method is particularly easy to adapt to the current model. These steps are performed until the illumination intensity I in the sample is the desired value I = I m+1 The process is repeated until the desired value is reached, and depending on the situation, such a desired value can be considered achieved if, for example, a small range of ±5% of the value is achieved. Then, the corresponding final control value P' is provided to provide the corresponding illumination intensity of the light source. k,m+1 This setting allows for further inspection while the illumination intensity on the sample remains (almost) constant, even when the objective lens is changed.

[0062] As used herein, the term "and / or" includes all possible combinations of one or more of the related items and may be abbreviated as " / ".

[0063] While several embodiments have been described in the context of the apparatus, it is clear that these embodiments also represent descriptions of the corresponding methods, where blocks or apparatus correspond to steps or features of steps. Similarly, embodiments described in the context of steps also represent descriptions of the corresponding blocks, items, or features of the corresponding apparatus.

[0064] Some embodiments relate to a microscope that includes a system such as those described in relation to one or more of Figures 1 to 3. Alternatively, the microscope may be part of a system such as those described in relation to one or more of Figures 1 to 3, or may be connected to a system such as those described in relation to one or more of Figures 1 to 3. Figure 1 shows a schematic diagram of a system 102 configured to carry out the method described herein. The system 102 includes a microscope 100 and a computer system 150 (or control system). The microscope 100 is configured to take images and is connected to the computer system 150. The computer system 150 is configured to carry out at least a portion of the method described herein. The computer system 150 may be configured to run machine learning algorithms. The computer system 150 and the microscope 100 may be separate entities, or they may be integrated within a single common housing. The computer system 150 may be part of the central processing system of the microscope 100, and / or the computer system 150 may be part of the dependent components of the microscope 100, such as sensors, actors, cameras, or lighting units.

[0065] The computer system 150 may be a local computer device (e.g., a personal computer, laptop, tablet computer, or mobile phone) comprising one or more processors and one or more storage devices, or it may be a distributed computer system (e.g., a cloud computing system comprising one or more processors and one or more storage devices distributed to various locations such as local clients and / or one or more remote server farms and / or data centers). The computer system 150 may include any circuit or combination of circuits. In one embodiment, the computer system 150 may include one or more processors, which can be of any kind. As used herein, the processor may be intended to be any kind of computing circuit, such as a microprocessor for a microscope or microscopic component (e.g., a camera), a microcontroller, a composite instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a graphics processor, a digital signal processor (DSP), a multicore processor, a field-programmable gate array (FPGA), or any other kind of processor or processing circuit. Other types of circuits that may be included in the computer system 150 may be custom circuits, application-specific integrated circuits (ASICs), etc., such as one or more circuits (communication circuits, etc.) used in wireless devices such as mobile phones, tablet computers, laptop computers, two-way radios, and similar electronic systems. The computer system 150 may also include one or more storage devices that may include one or more memory elements suitable for a particular application, such as main memory in the form of random access memory (RAM), one or more hard drives and / or one or more drives that handle removable media such as compact discs (CDs), flash memory cards, digital video discs (DVDs), etc.The computer system 150 may also include a display device, one or more speakers and a controller which may include a keyboard and / or mouse, trackball, touchscreen, voice recognition device, or any other device which enables a user of the system to input information into and receive information from the computer system 150.

[0066] Some or all of the steps may be performed by a hardware device (or by using a hardware device), such as a processor, microprocessor, programmable computer, or electronic circuit. In some embodiments, one or more of the most critical steps may be performed by such a device.

[0067] Depending on certain implementation requirements, embodiments of the present invention may be implemented in hardware or software. This implementation is feasible using a non-transient recording medium, which is a digital recording medium, etc., that stores electronically readable control signals and cooperates (or can cooperate) with a programmable computer system to carry out each method. Examples include floppy disks, DVDs, Blu-rays, CDs, ROMs, PROMs and EPROMs, EEPROMs, or FLASH memory. Thus, the digital recording medium may be computer-readable.

[0068] Some embodiments of the present invention include a data carrier having electronically readable control signals that can cooperate with a programmable computer system so as to carry out any of the methods described herein.

[0069] Generally, embodiments of the present invention can be implemented as a computer program product comprising program code, which operates to perform one of the methods when the computer program product is executed on a computer. This program code may be stored, for example, on a machine-readable carrier.

[0070] Another embodiment includes a computer program stored in a machine-readable carrier for carrying out any of the methods described herein.

[0071] Therefore, in other words, embodiments of the present invention are computer programs having program code for carrying out any of the methods described herein when the computer program is executed on a computer.

[0072] Accordingly, another embodiment of the present invention is a recording medium (or data carrier or computer-readable medium) containing a stored computer program for carrying out any of the methods described herein when executed by a processor. The data carrier, digital recording medium, or recording medium is typically tangible and / or non-transient. Another embodiment of the present invention is an apparatus, such as those described herein, comprising a processor and a recording medium.

[0073] Therefore, another embodiment of the present invention is a data stream or signal sequence representing a computer program for carrying out any of the methods described herein. The data stream or signal sequence may be configured to be transmitted, for example, over a data communication connection, such as the Internet.

[0074] Another embodiment includes processing means, for example, a computer or programmable logic device configured or adapted to carry out any of the methods described herein.

[0075] Another embodiment includes a computer having an installed computer program for carrying out any of the methods described herein.

[0076] Another embodiment of the present invention includes an apparatus or system configured to transfer (e.g., electronically or optically) a computer program for carrying out any of the methods described herein to a receiver. The receiver may be, for example, a computer, a mobile device, a storage device, etc. The apparatus or system may include, for example, a file server for transferring the computer program to the receiver.

[0077] In some embodiments, a programmable logic device (e.g., a field-programmable gate array) may be used to perform some or all of the functions of the methods described herein. In some embodiments, the field-programmable gate array may cooperate with a microprocessor to carry out any of the methods described herein. Generally, the methods are advantageously carried out by any hardware device. [Explanation of symbols]

[0078] 100,200 fluorescence microscopes 102,202 Microscope Systems 110 samples 120 Light source housing 120 k ,120 k+1 light source 122 Collector Lens 124 Illumination field aperture 126 Condenser Lens 130,130 j ,130 j+1 Phosphors 132 The pupil of the objective lens 134 Illuminated objective field of view 136 Tube Lenses 140 Detector Housing 140,140i ,140 i+1 detector 150 control systems 160 m ,160 m+1 Objective lens 162 Objective lens changing mechanism 164 Illumination beam path 168 Imaging beam path A LF Area of ​​the field of view aperture A I Area of ​​the illuminated objective field of view 2r LF Pupil illumination diameter T m Transmittance β m magnification NA m Number of mouth openings f ref focal length n m Immersion refractive index P k (λ), P k+1 (λ) Illumination intensity B, B i ,B i+1 image intensity 300-308 Method Steps

Claims

1. A control system (150) for automatically determining the illumination intensity (P ｋ , 120 ｋ＋１ ), P ｋ , P ｋ＋１ ) of at least one light source (120 The at least one light source (120 ｋ , 120 ｋ＋１ ) is used to stimulate at least one phosphor (130, 130 ｊ , 130 ｊ＋１ ) in the sample (110), The at least one light source (120 ｋ , 120 ｋ＋１ ) is configured to vary the illumination intensity (P ｋ , P ｋ＋１ ). The fluorescence microscope (100) detects the image intensity (B, B ｉ , B ｉ＋１ ) of the sample (110) and has at least one detector (140, 140 ｉ , 140 ｉ＋１ ) used for this purpose. The control system (150) automatically determines (304) a control value (P ｋ，ｍ＋１ ) of the illumination intensity (P ｋ , P ｋ＋１ ) of the at least one light source (120 ｋ , 120 ｋ＋１ ) after a change (302) in the optical path (164, 168) in order to achieve a desired value (I ｍ＋１ ) of the observation parameters (I, B, B ｉ , B ｉ＋１ ) characterizing the sample observation, whereby the optical path includes at least one of an illumination path (164) from the at least one light source (120 ｋ , 120 ｋ＋１ ) to the sample (110) and an imaging path (168) from the sample (110) to the at least one detector (140, 140 ｉ , 140 ｉ＋１ ).​​​​​​​​​​​​​​​​​​​​​​​​ Said at least one light source (120 ｋ , 120 ｋ＋１ ) of said illumination intensity (P ｋ , P ｋ＋１ ) of said control value (P ｋ，ｍ＋１ ) to determine (304) is the illumination intensity (P ｋ , P ｋ＋１ ) value (P ｋ，ｍ ) set before said change (302) in said optical path (164, 168), and the observation parameter (I, B, B ｉ , B ｉ＋１ ) value (I ｍ ) used before said change (302) in said optical path, and the physical model (M) of said optical path (164, 168), and is based on, The physical model (M) takes into account the imaging characteristics of the optical components (124, 132, 160 ｍ , 160 ｍ＋１ ) in the optical paths (164, 168) and associates the illumination intensity (P ｋ , 120 ｋ＋１ ) of the at least one light source (120 ｋ , P ｋ＋１ ) with the observation parameters (I, B, B ｉ , B ｉ＋１ ). A control system (150).

2. The control system (150) is further configured to determine a final control value (P') of the illumination intensity (P, P) based on an iterative method (306) such that a desired value (I) of the observation parameters (I, B, B, B) is achieved. ｉ ,B ｉ＋１ ), ｍ＋１ ), ｋ P ｋ＋１ ), ｋ，ｍ＋１ ). The control value (P) determined based on the physical model (M) ｋ，ｍ＋１ is used as the starting value of the iterative method (306). The control system (150) according to Claim 1.

3. The imaging characteristics of the optical components (124, 132, 160 ｍ , 160 ｍ＋１ ) in the optical path considered in the physical model are the magnification (β ｍ , 160 ｍ＋１ ) of the objective lens (160 ｍ ), the transmittance (T ｍ , 160 ｍ＋１ ) of the objective lens (160 ｍ ), the numerical aperture (NA ｍ , 160 ｍ＋１ ) of the objective lens (160 ｍ ), the refractive index (n ｍ ) of the immersion medium, the area (A ＬＦ ) of the illumination field stop (124) in the intermediate image, the ratio of the total pupil area to the illuminated pupil area in the objective lens (160 ｍ , 160 ｍ＋１ ), and at least one of the group consisting of the magnification of the system optical components and the focal length of the system optical components, The control system (150) according to Claim 1.

4. The observation parameter is based on or includes a desired illumination intensity (I) in the sample (110). The control system (150) according to Claim 1.

5. The observation parameter is based on a desired imaging intensity (B, B ｉ , 140 ｉ＋１ ), 140 ｉ , B ｉ＋１ ) of the sample (110) detected in the at least one detector (140, 140 ｉ , B ｉ＋１ ), or includes a desired imaging intensity (B, B The control system (150) according to Claim 1.

6. said at least one detector (140, 140 ｉ , 140 ｉ＋１ ), the desired value of the imaging intensity (B, B ｉ , B ｉ＋１ ) of the sample (110) detected therein is defined based on a value derived from photon statistics detected by the detector (140) for said at least one phosphor (130, 130 ｊ , 130 ｊ＋１ ), The control system (150) according to Claim 5.

7. The desired value (I ｍ＋１ ) of the observation parameter is deviated from the value (I ｍ ) of the observation parameter used before the change in the illumination path (164, 168) by 10% or less, preferably 5% or less, and more preferably, the desired value of the observation parameter is equal to the value of the observation parameter used before the change in the illumination path (164, 168). The control system (150) according to Claim 1.

8. The modification (302) in the optical path (164, 168) is a modification of the objective lens (160 ｍ , 160 ｍ＋１ ) of the fluorescence microscope (100) currently disposed in the optical path (164, 168). The control system (150) according to Claim 1.

9. At least two light sources (120 ｋ , 120 ｋ＋１ ) of the fluorescence microscope (100) are used to automatically determine the illumination intensities (P ｋ , P ｋ＋１ ). To this end, the at least two light sources (120 ｋ , 120 ｋ＋１ ) are used to stimulate different phosphors among at least two phosphors (130 ｊ , 130 ｊ＋１ ) in the sample (110). The at least two light sources (120 ｋ , 120 ｋ＋１ ) are configured to individually vary the illumination intensity (P ｋ , P ｋ＋１ ). After the change in the optical path (164, 168), the control system (150) is further configured to automatically determine individually the control values of the illumination intensity (P ｋ , 120 ｋ＋１ ) of each of the at least two light sources (120 ｋ , P ｋ＋１ ). The control system (150) according to Claim 1.

10. each of the at least two light sources (120 ｋ , 120 ｋ＋１ ) to individually determine the control value of the illumination intensity (P ｋ , P ｋ＋１ ) further includes determining at least one of cross-excitation and cross-emission of the at least two phosphors (130 ｊ , 130 ｊ＋１ ) in the sample (110) in order to take into account the crosstalk effect in at least one of excitation and detection, The control system (150) according to Claim 9.

11. The control system (150) is further configured to automatically detect the change (302) in the optical path (164, 168). The control system (150) according to Claim 1.

12. The control system (150) is particularly configured to adapt the lighting intensity (P ｋ , P ｋ＋１ ) of the at least one light source (120 ｋ , 120 ｋ＋１ ) by adapting the power supplied to the at least one light source (120 ｋ , 120 ｋ＋１ ) automatically according to the determined control value (P ｋ，ｍ＋１ ) of the lighting intensity. The control system (150) according to Claim 1.

13. The control system (150) adapts the power supplied to the at least one light source (120 ｋ , 120 ｋ＋１ ) to automatically adapt the illumination intensity (P ｋ , P ｋ＋１ ) of the at least one light source (120 ｋ , 120 ｋ＋１ ) according to the determined control value (P ｋ，ｍ＋１ ) of the illumination intensity. The control system (150) according to Claim 12.

14. A microscope system (102) comprising a fluorescence microscope (100) and the control system (150) according to any one of Claims 1 to 13, wherein the fluorescence microscope (100) is At least one phosphor (130, 130 ｊ , 130 ｊ＋１ ) configured to be used to stimulate and configured to vary said illumination intensity (P ｋ , P ｋ＋１ ), and at least one light source (120 ｋ , 120 ｋ＋１ ). At least one detector (140, 140 ｉ , 140 ｉ＋１ ) configured to be used to detect the image intensity of the sample (110) the control system (150) according to any one of Claims 1 to 13, and a microscope system (102).

15. The fluorescence microscope (100) is configured as an epi-fluorescence microscope. The microscope system (102) according to Claim 14.

16. A method for automatically determining the illumination intensity (P ｋ , 120 ｋ＋１ ), P ｋ , P ｋ＋１ ) of at least one light source (120 The at least one light source (120 ｋ , 120 ｋ＋１ ) is used to stimulate at least one phosphor (130, 130 ｊ , 130 ｊ＋１ ) in the sample, and the at least one light source (120 ｋ , 120 ｋ＋１ ) is configured to vary the illumination intensity (P ｋ , P ｋ＋１ ). The fluorescence microscope (100) detects the image intensity (B, B ｉ , B ｉ＋１ ) of the sample (110) using at least one detector (140, 140 ｉ , 140 ｉ＋１ ). The method includes determining (304) a control value (P ｋ，ｍ＋１ ) of the illumination intensity (P ｋ , P ｋ＋１ ) of the at least one light source (120 ｍ＋１ , 120 ｋ , 120 ｋ＋１ ) after a change (302) in the optical path (164, 168) in order to achieve a desired value (I) of the observation parameter (I) characterizing the sample observation, whereby the optical path includes at least one of an illumination path (164) from the at least one light source (120 ｋ , 120 ｋ＋１ ) to the sample (110) and an imaging path (168) from the sample (110) to the at least one detector (140, 140 ｉ , 140 ｉ＋１ ).​​​​​​​​​​​​​​​​​​​​ Said at least one light source (120 ｋ , 120 ｋ＋１ ), said control value (P ｋ , P ｋ＋１ ) of said illumination intensity (P ｋ，ｍ＋１ ) is determined (304) based on the value (P ｋ，ｍ ) of the illumination intensity set before said change (302) in said illumination path (164, 168), the value (I ｍ ) of said observation parameter used before said change in said optical path (164, 168), and the physical model (M) of said optical path (164, 168). The physical model (M) associates the illumination intensity (P, P) of the at least one light source (120, 120) with the observation parameters in consideration of the imaging characteristics of the optical components (124, 132, 160, 160) in the optical paths (164, 168). ｍ , 160 ｍ＋１ ), considering the imaging characteristics of the optical components (124, 132, 160 ｋ , 120 ｋ＋１ ) in the optical paths (164, 168), ｋ , P ｋ＋１ ) with the observation parameters. A method.

17. The method further includes determining, based on an iterative method, a final control value (P' ｋ , P ｋ＋１ ) of the illumination intensity (P ｋ，ｍ＋１ ) at which the desired value of the observation parameter is achieved. The control value (P) determined based on the physical model is used as the starting value of the iterative method. ｋ，ｍ＋１ ​ The method according to Claim 16.

18. The change in the optical path (164, 168) is a change in the objective lens (160 ｍ , 160 ｍ＋１ ) of the fluorescence microscope (100) that is currently disposed in the optical path (164, 168). The method according to Claim 16.

19. The method uses an epi-fluorescence microscope. The method according to Claim 16.

20. A computer program having program code for performing the method according to any one of Claims 16 to 19 when executed on one or more processors or the control system (150) according to any one of Claims 1 to 13.

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