Fluorescence microscope system and method

Abstract

To be able to acquire high quality images in a fluorescence microscopy system and method for imaging a sample containing at least two different fluorophores.SOLUTION: A fluorescence microscope system includes: an illumination system 104 configured to emit illumination light for exciting fluorophores; and an optical detection system 112 configured to produce an image of a sample 102 based on fluorescence emitted by the excited fluorophores. Further the system includes a control unit 124 configured to determine based on at least one property of each of the fluorophores and based on at least one parameter of the optical detection system and / or at least one parameter of the illumination system, whether to image the sample in a simultaneous imaging mode or in a sequential imaging mode. In the simultaneous imaging mode, the fluorophores are imaged simultaneously. In the sequential imaging mode, the fluorophores are divided into a first group and at least one second group, and these fluorophores are imaged one behind the other.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a fluorescence microscope system for imaging a sample. The present invention further relates to a method for imaging a sample comprising at least two different fluorophores using a fluorescence microscope system. [Background technology]

[0002] When a sample is prepared with two or more fluorophores, each fluorophore is often recorded separately. Because imaging each different fluorophore may require changing filters, changing light sources, or switching between them, a significant amount of time elapses between different image acquisitions. However, this is undesirable for many applications. Therefore, known microscope systems use multiple excitation sources and detectors to simultaneously excite and detect two or more fluorophores. However, nonlinear effects such as cross-excitation of fluorophores and, especially, bleaching due to high-intensity illumination, can severely affect image quality. Summary of the Invention [Problem to be solved by the invention]

[0003] SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a fluorescence microscope system and method for imaging a sample containing at least two different fluorophores, which allows for the acquisition of high quality images. [Means for solving the problem]

[0004] The above-mentioned object is achieved by the subject matter of the independent claims. Advantageous embodiments are set out in the dependent claims and the following description.

[0005] A proposed fluorescence microscope system for imaging a sample containing at least two different fluorophores includes an illumination system configured to emit illumination light for exciting the fluorophores and an optical detection system configured to generate an image of the sample based on fluorescence emitted by the excited fluorophores. The fluorescence microscope system further includes a control unit configured to determine whether to image the sample in a simultaneous imaging mode or a sequential imaging mode based on at least one property of each of the different fluorophores and based on at least one parameter of the optical detection system and / or at least one parameter of the illumination system. In the simultaneous imaging mode, the different fluorophores are imaged simultaneously. In the sequential imaging mode, the different fluorophores are divided into a first group and at least one second group, and the fluorophores in the first group and the fluorophores in the second group are imaged one after the other.

[0006] The properties of each of the different fluorophores may in particular be one of the following: excitation spectrum, emission spectrum, brightness and concentration of the fluorophore inside the sample. The parameters of the optical detection system may in particular be one of the following: magnification, numerical aperture, detector gain, detector exposure time, scan speed, pinhole size, detector averaging, detector directionality, detection spectrum, detection filter settings and imaging modality, such as confocal or wide-field imaging. The parameters of the illumination system may in particular be one of the following: light source power, light source spectrum and excitation filter settings.

[0007] The control unit assists the user of the fluorescence microscope system in imaging a sample by considering any number of these parameters to determine whether to image the sample in simultaneous or sequential imaging mode. In particular, the control unit checks for common sources of error that occur when simultaneously exciting and imaging fluorophores, such as cross-excitation, over-excitation, and overlapping emission spectra. The control unit also ensures that an imaging mode is selected that allows the acquired image data to be automatically processed, for example, by spectral separation. Furthermore, sample preparation and staining, fluorophore exposure time, or even the expression of specific proteins in the sample all affect the behavior of fluorophores. Therefore, by adapting the imaging mode to the specific sample and current imaging parameters of the microscope system, high-quality images can be obtained without requiring special know-how on the part of the user.

[0008] According to one preferred embodiment, the control unit is configured to separate the different fluorophores into a first group and at least one second group based on at least one characteristic of each of the different fluorophores. According to this embodiment, the different fluorophores are separated into different groups. The fluorophores within the different groups are excited simultaneously, whereas the different groups are excited sequentially. By separating the fluorophores based on at least one characteristic, this embodiment prevents undesirable effects that adversely affect image quality, such as cross-excitation and over-excitation. For example, by grouping the fluorophores based on their excitation and emission spectra, undesirable cross-excitation is prevented. Similarly, if the fluorophores are grouped by their brightness, cancellation of less bright fluorophores can be prevented. According to this embodiment, as many fluorophores as possible are imaged together, thereby shortening the image acquisition period while ensuring high image quality.

[0009] According to another preferred embodiment, the control unit is configured to separate the different fluorophores into a first group and at least one second group such that the emission spectra of the different fluorophores in each group have minimal overlap. According to this embodiment, the different fluorophores in each group can be easily separated by spectral separation, thereby minimizing crosstalk between the different fluorophores. In this way, the sample can be imaged with high spectral quality.

[0010] According to another preferred embodiment, the control unit is configured to determine minimum and / or maximum values ​​for the output power of at least one light source of the illumination system based on at least one characteristic of each of the different fluorophores and / or at least one characteristic of the sample, and to decide whether to image the sample in sequential or simultaneous imaging mode by comparing the operating range of the output power of the light source with these minimum and / or maximum values. The minimum value can be determined, for example, from the minimum power required to excite each of the different fluorophores. The maximum value can be determined based on the power required to bleach one or more of the different fluorophores.

[0011] For example, a sample contains two fluorophores, A and B, each excitable by a different light source. Due to an error in the staining, fluorophore A is very weakly stained and requires a lot of energy for excitation, while fluorophore B is very strongly stained and responds very well to excitation. The user selects confocal imaging and uses the automatic illumination setting, which, due to the very different dynamics of the fluorophores, results in one light source being set to maximum and the other to minimum. As a result of cross-excitation, neither fluorophore A nor B is optimally excited, and the control unit suggests sequential imaging mode.

[0012] According to this embodiment, the control unit determines whether the power required to excite but not bleach the different fluorophores is within the operating range of the light source or light sources used, in other words, the control unit is configured to decide whether to image the sample in sequential or simultaneous imaging mode based on the dynamic range of at least one light source of the illumination system.

[0013] According to another preferred embodiment, the control unit is configured to determine whether to image the sample in sequential or simultaneous imaging mode based on the excitation spectra of the different fluorophores. For example, if the excitation spectra of the different fluorophores overlap significantly, the different fluorophores can be excited by the same excitation light and thus simultaneously. If the excitation spectra of the different fluorophores are significantly different, two or more light sources may be required. However, excitation of multiple fluorophores may be undesirable, especially if the emission spectra of the fluorophores overlap significantly. This is undesirable because it would make it difficult to separate the different fluorophores during spectral separation or using filter settings. Therefore, in this embodiment, the control unit can also check whether strong overlap is likely to occur by comparing the spectra of the different fluorophores, for example by calculating the overlap integral, Euclidean distance, or spectral angle, and comparing the result with a predetermined threshold. This embodiment thus facilitates spectral separation, thereby further improving image quality.

[0014] According to another preferred embodiment, the control unit is configured to determine whether to image the sample in sequential or simultaneous imaging mode based on the spectrum of the excitation light emitted by the illumination system. The spectrum of the excitation light is determined, inter alia, by the light source and / or excitation filter settings. The spectrum of the excitation light determines which of the different fluorophores can be excited. If there is not enough overlap between the spectrum of the excitation light and the excitation spectrum of a particular fluorophore, that particular fluorophore cannot be excited by the excitation light. Therefore, it is necessary to select the appropriate excitation light source and / or excitation filter settings for each fluorophore. Therefore, it may be necessary to image some of the different fluorophores in succession in order to change the excitation light source and / or excitation filter settings between simultaneous image acquisitions.

[0015] According to another preferred embodiment, the control unit is configured to decide whether to image the sample in sequential or simultaneous imaging mode by comparing the excitation spectra of the different fluorophores with the spectrum of the excitation light emitted by the illumination system. In this embodiment, the control unit checks whether one or more light sources are "over-exciting" one or more of the fluorophores. This "over-excitation" can cause bleaching of the sample due to non-linear effects and detector saturation, which, if not taken into account, can rapidly degrade image quality.

[0016] According to another preferred embodiment, the control unit is configured to determine whether to image the sample in sequential or simultaneous imaging mode based on the emission spectra of the different fluorophores. Large overlaps in the emission spectra of some fluorophores can make their separation difficult, especially when these specific fluorophores are located close to each other. By imaging these specific fluorophores in succession, the fluorophores can be clearly distinguished, thereby improving image quality.

[0017] According to another preferred embodiment, the control unit is configured to determine whether to image the sample in sequential or simultaneous imaging mode by comparing the overlap of the emission spectra of the different fluorophores with the detection spectrum of the optical detection system. The detection spectrum is determined, inter alia, by the type of detector used and / or the detection filter settings. Cross-excitation occurs when the emission spectra of two or more different fluorophores overlap significantly within the detection spectrum of the optical detection system. This may require imaging some of the different fluorophores in succession to facilitate spectral separation and improve image quality.

[0018] According to another preferred embodiment, the control unit is configured to determine whether to image the sample in sequential or simultaneous imaging mode by comparing the brightness values ​​of at least two of the different fluorophores. The brightness values ​​can be, for example, maximum values ​​per fluorophore channel, percentile scores per fluorophore channel, or average values ​​per fluorophore channel. A less bright fluorophore can easily be overwhelmed by a more bright fluorophore. In this embodiment, the control unit checks whether one or more fluorophores have a significantly lower brightness than the other fluorophores and determines the imaging mode accordingly.

[0019] In particular, the control unit is configured to determine the brightness value based on the measured brightness value measured by the optical detection system. In other words, in this embodiment, the brightness of a particular fluorophore is determined in situ. The brightness of some fluorophores varies significantly depending on factors such as sample preparation and fluorophore concentration. Therefore, determining brightness in situ is significantly more reliable than using a predetermined value.

[0020] According to another preferred embodiment, the control unit is configured to determine whether to image the sample in sequential or simultaneous imaging mode based on the dynamic range of at least one detector of the optical detection system. The dynamic range is determined by the type of detector and, in part, by the detection filter settings. The dynamic range thus determines which of the different fluorophores the optical detection system can detect with sufficient signal brightness and signal-to-noise ratio for all fluorophores without saturating any of the detectors of the optical detection system. This requires selecting appropriate detector and / or detection filter settings for each fluorophore or group of fluorophores. It may therefore be necessary to image some of the different fluorophores in succession in order to change the detector and / or detector filter settings between simultaneous image acquisitions.

[0021] According to another preferred embodiment, in the sequential imaging mode, the illumination system emits a first illumination light for exciting a fluorophore of the first group and at least one second illumination light for exciting a fluorophore of the second group in succession, and in the simultaneous imaging mode, the illumination system emits a third illumination light for simultaneously exciting different fluorophores. In this embodiment, the different imaging modes are characterized by different illuminations, i.e., different combinations of illumination wavelengths or illumination intensities. In the sequential imaging mode, different illumination lights are used to excite the fluorophores of the first group and the fluorophore of the second group. In the simultaneous imaging mode, the fluorophores of the first group and the fluorophore of the second group are excited by the same illumination light.

[0022] According to another preferred embodiment, the control unit is configured to decide whether to image the sample in sequential or simultaneous imaging mode based on at least one characteristic of the sample. The characteristic of the sample can in particular be one of the following: the type of sample and the concentration of fluorophores within the sample. These characteristics, among others, affect the behavior of the fluorophores, in particular their brightness, excitation spectrum and emission spectrum. Taking these factors into account makes it possible to make a more reliable decision on which imaging mode to use.

[0023] According to another preferred embodiment, the control unit is configured to set at least one parameter of the optical detection system and / or at least one parameter of the illumination system based on at least one property of each of the different fluorophores and / or at least one property of the sample. In this embodiment, the control unit is configured to assist the user in setting the parameters so that the different fluorophores can be imaged simultaneously, if possible. If this is not possible, the control unit will set the parameters so that the different fluorophores can be imaged as little as possible in succession. By assisting the user, the microscope system ensures high image quality even for non-expert users.

[0024] According to another preferred embodiment, the control unit is configured to provide feedback to the user if the control unit determines, for example based on imaging settings entered by the user, such as target values ​​for brightness or signal-to-noise ratio, that the sample should be imaged in sequential imaging mode. In this embodiment, the user is informed that the imaging settings, i.e., the parameters of the optical detection system and / or the illumination system, do not allow the sample to be imaged in simultaneous imaging mode. The user can then continue imaging in simultaneous imaging mode, switch to imaging in sequential imaging mode, or change the imaging settings.

[0025] The present invention further relates to a method for imaging a sample comprising at least two different fluorophores using a fluorescence microscope system, the method comprising: determining whether to image the sample in a simultaneous imaging mode or a sequential imaging mode based on at least one property of each of the different fluorophores and based on at least one parameter of an optical detection system of the fluorescence microscope system and / or at least one parameter of an illumination system of the fluorescence microscope system; if it is determined that the sample should be imaged in the simultaneous imaging mode, simultaneously imaging the different fluorophores; if it is determined that the sample should be imaged in the sequential imaging mode, separating the different fluorophores into a first group and at least one second group and imaging the fluorophores of the first group and the fluorophores of the second group in succession.

[0026] This method has the same advantages as the fluorescence microscope system and can be supplemented with the features of the dependent claims relating to the sample carrier and the imaging system.

[0027] Specific embodiments are described below with reference to the drawings. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram of a fluorescence microscope system for imaging a sample, according to one embodiment. [Figure 2] 2 is a flowchart of a method for determining whether to image a sample in simultaneous or sequential imaging mode using the fluorescence microscope system according to FIG. 1 . [Figure 3] 1 is a flowchart of a first sub-process of the above method. [Figure 4] 10 is a flowchart of a second sub-process of the above method. [Figure 5] 10 is a flowchart of a third sub-process of the above method. [Figure 6] 1 is a graph showing the emission spectra of different phosphors. DETAILED DESCRIPTION OF THE INVENTION

[0029] FIG. 1 is a schematic diagram of a fluorescence microscope system 100 for imaging a sample 102, according to one embodiment.

[0030] The illumination system 104 of the fluorescence microscope system 100 is configured to generate different excitation lights for exciting different fluorophores disposed within the sample 102. The illumination system 104 according to this embodiment includes a white light source 106 and a first interchangeable filter unit 108. The white light source 106 emits white light toward the first interchangeable filter unit 108 into an illumination beam path 110. The first interchangeable filter unit 108 includes two or more filters that block all wavelengths of the white light except for a single wavelength or a predetermined range of wavelengths in order to generate the different excitation lights. One of the filters is moved into the illumination beam path 110 by the first interchangeable filter unit 108 at a time, thereby generating specific excitation lights that are further guided along the illumination beam path 110. The excitation filter setting defines which filter is currently moved into the illumination beam path 110, and thus which specific excitation lights are generated.

[0031] According to an alternative embodiment, the illumination system 104 can include two or more different light sources, each configured to generate a specific excitation light and direct the specific excitation light into the illumination beam path 110. In this alternative embodiment, the first interchangeable filter unit 108 can be omitted.

[0032] The optical detection system 112 of the fluorescence microscope system 100 is configured to generate an image of the sample 102 based on the fluorescence emitted by the excited fluorophores. The optical detection system 112 according to this embodiment includes an objective lens 114 directed toward the sample 102, a second interchangeable filter unit 116, and a detector element 118. The objective lens 114 receives the fluorescence emitted by the excited fluorophores and guides the fluorescence toward a detection beam path 120. The second interchangeable filter unit 116 includes two or more filters that block all wavelengths of the fluorescence except for a single wavelength or a predetermined wavelength range, each for generating a different detection light. One of the filters is moved into the detection beam path 120 by the second interchangeable filter unit 116 at a time, thereby generating a specific detection light that is further guided along the detection beam path 120 toward the detector element 118. A detection filter setting defines which filter is currently moved into the detection beam path 120. By blocking undesired wavelengths of fluorescence, it can be ensured that only fluorescence emitted by a single fluorophore or group of fluorophores is detected by detector element 118 .

[0033] According to an alternative embodiment, the optical detection system 112 can include two or more different detector elements, each sensitive to a single wavelength or range of wavelengths for detecting fluorescence emitted by a single fluorophore or group of fluorophores. According to this alternative embodiment, the second interchangeable filter unit 116 can be omitted and / or replaced by different filters, each positioned in front of a different detector element.

[0034] According to this embodiment, a beam splitter 122 is arranged at the intersection of the illumination beam path 110 and the detection beam path 120, which according to this embodiment are perpendicular to each other. The beam splitter 122 is configured so that the excitation light is guided to the sample 102 via the objective lens 114. The beam splitter 122 is further configured so that the fluorescence light received by the objective lens 114 is guided towards the second interchangeable filter unit 116.

[0035] The fluorescence microscope system 100 further includes a control unit 124, an input unit 126, and an output unit 128. The control unit 124 is connected to the illumination system 104, the optical detection system 112, the input unit 126, and the output unit 128. The control unit 124 is configured to set parameters of the illumination system 104 and the optical detection system 112, for example, based on user input via the input unit 126. The control unit 124 is also configured to output, via the output unit 128, one or more images of the sample 102 generated based on the fluorescence emitted by the excited fluorophores. The control unit 124 is further configured to determine whether the sample 102 is better imaged in a simultaneous or sequential imaging mode, and output the determination result via the output unit 128. In the simultaneous imaging mode, different fluorophores are imaged simultaneously. In sequential imaging mode, the different fluorophores are separated into a first group and at least one second group, and the fluorophores in the first group are imaged in succession with the fluorophores in the second group. Exemplary embodiments of methods for determining whether to image the sample 102 in simultaneous or sequential imaging mode are described below with reference to Figures 2-6.

[0036] FIG. 2 is a flowchart of a method for determining whether to image a sample 102 in a simultaneous or sequential imaging mode using the fluorescence microscope system 100 described above.

[0037] The process starts in step S200. In step S202, the control unit 124 receives a set of parameters on which to base the decision. This set of parameters may include at least one characteristic of each of the different fluorophores disposed within the sample 102, at least one characteristic of the sample 102 itself, at least one parameter of the optical detection system 112, and / or at least one parameter of the illumination system 104. These parameters may be entered into the control unit 124 by a user or may be received by the control unit 124 from, for example, the illumination system 104 and / or the optical detection system 112.

[0038] A first sub-process is performed in step 204. In the first sub-process, the control unit 124 determines whether to image the sample 102 in a simultaneous imaging mode or a sequential imaging mode based on at least one parameter of the illumination system 104. The first sub-process is described in more detail below with reference to FIG.

[0039] A second sub-process is performed in step 206. In the second sub-process, the control unit 124 determines whether to image the sample 102 in a simultaneous imaging mode or a sequential imaging mode based on at least one parameter of the optical detection system 112. The second sub-process is described in more detail below with reference to FIG.

[0040] In step 208, a third sub-process is performed in which the control unit 124 determines whether to image the sample 102 in a simultaneous imaging mode or a sequential imaging mode based on at least one characteristic of each of the different fluorophores. The third sub-process is described in more detail below with reference to FIG. 5.

[0041] Steps S204, S206, and S208 can be performed in any order or simultaneously. If the control unit 124 determines in any of steps S204, S206, or S208 that the sample 102 is best imaged in sequential imaging mode, the control unit 124 outputs this result via the output unit 128 in step S210 for notifying the user. Otherwise, the control unit 124 outputs via the output unit 128 that the sample 102 can be imaged in simultaneous imaging mode and / or continues imaging the sample 102 in simultaneous imaging mode in step S212. The process ends in step S214.

[0042] FIG. 3 is a flowchart of the first sub-process of the above method.

[0043] In step 300, the first subprocess starts. In step 302, the control unit 124 determines minimum and maximum values ​​for the output of the light source 106 of the illumination system 104 based on at least one characteristic of each of the different fluorophores and / or at least one characteristic of the sample 102. In step 304, the control unit 124 compares these minimum and maximum values ​​with the operating range of the light source 106. If either the maximum or minimum value is outside the operating range of the light source 106, the control unit 124 decides to image the sample 102 in sequential imaging mode. In other words, the decision on which imaging mode to use is based on the dynamic range of the light source 106. If the fluorescence microscope system 100 includes more than one light source 106, step 304 is repeated for each light source 106.

[0044] In step 306, the control unit 124 determines whether all of the different fluorophores can be excited by the excitation light currently being emitted by the illumination system 104. If all of the different fluorophores cannot be excited by the same excitation light, meaning different excitation filter settings or light sources 106 must be used, the control unit 124 determines that the sample 102 must be imaged in a sequential imaging mode.

[0045] Steps 302, 304 and 306 can be performed in any order or simultaneously. The first sub-process ends in step S308.

[0046] FIG. 4 is a flowchart of the second sub-process of the above method.

[0047] The second sub-process starts in step 400. In step 402, the control unit 124 determines how much of the dynamic range of one or more detector elements 118 is to be used based on the emission spectra of each different fluorophore. If the used dynamic range exceeds a threshold, the control unit 124 determines that the sample 102 should be imaged in sequential imaging mode.

[0048] In step 404, the control unit 124 determines whether cross-excitation occurs based on the wavelength spectrum of the one or more excitation lights used and the excitation spectra of the different fluorophores, and if significant cross-excitation occurs, the control unit 124 determines that the sample 102 should be imaged in sequential imaging mode.

[0049] If more than one light source is used, the control unit 124 determines whether the light sources used over-excite one or more of the different fluorophores in step 406. If over-excitation occurs, the control unit 124 determines that the sample 102 should be imaged in sequential imaging mode.

[0050] Steps 402, 404 and 406 can be performed in any order or simultaneously. The second sub-process ends in step S408.

[0051] FIG. 5 is a flowchart of the third sub-process of the above method.

[0052] The third sub-process starts in step 500. In step 502, the control unit 124 determines whether the emission spectra of two or more of the different fluorophores overlap in the detection spectra of the detector elements 118 being used. If there is a significant overlap in the detection spectra and the fluorophores can be excited separately, the control unit 124 determines that the sample 102 should be imaged in sequential imaging mode. Step 502 is described in more detail below with reference to FIG. 6.

[0053] In step 504, the control unit 124 determines a brightness histogram for each of the different fluorophores. The control unit 124 then compares the brightness histograms, and if the difference in brightness for two or more of the different fluorophores exceeds a threshold, the control unit 124 determines that the sample 102 should be imaged in sequential imaging mode.

[0054] Steps 502 and 504 can be performed in any order or simultaneously. The third sub-process ends in step S506.

[0055] FIG. 6 is a graph 600 showing the emission spectra of different phosphors.

[0056] The abscissa 602 of graph 600 (not shown) represents wavelength in nm. The ordinate 604 of graph 600 (not shown) represents relative intensity in percent. Emission spectra 606, 608, and 610 of three different exemplary fluorophores are shown as solid lines. Three detection wavelength ranges 612, 614, and 616 of three exemplary detectors are shown as filled rectangles. As can be seen from FIG. 6 , an emission maximum 618 of a first fluorophore falls within the detection wavelength range 612 of the first detector, an emission maximum 620 of a second fluorophore falls within the detection wavelength range 614 of the second detector, and an emission maximum 622 of a third fluorophore falls within the detection wavelength range 616 of the third detector. However, there is significant overlap between the emission spectrum 608 of the second fluorophore and the emission spectrum 610 of the third fluorophore within the detection wavelength range 616 of the third detector. In particular, if the signal of the second fluorophore is weaker than the signal of the third fluorophore, it may be difficult to further process, for example by spectral separation. In this case, the control unit 124 will decide to image the sample 102 in sequential imaging by imaging the first fluorophore and the third fluorophore in a first image, and then imaging the second fluorophore in a second image. In this embodiment, the first fluorophore and the third fluorophore belong to a first group, while the second fluorophore belongs to a second group.

[0057] Identical or similarly acting elements are designated by the same reference numerals in all figures. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0058] While some aspects have been described in the context of an apparatus, it will be apparent that these aspects also represent a description of a corresponding method, where a block or apparatus corresponds to a step or feature of a step, and similarly, aspects described in the context of a step also represent a description of a corresponding block or item or feature of a corresponding apparatus. [Explanation of symbols]

[0059] 100 Microscope System 102 samples 104 Irradiation System 106 Light source 108 Replaceable filter unit 110 Irradiation beam path 112 Optical Detection System 114 Objective Lens 116 Second replaceable filter unit 118 detector elements 120 detection beam path 122 Beam Splitter 124 Control Unit 126 input units 128 output units 600 graphs 602 Abscissa 604 Ordinate 606, 608, 610 Emission spectrum 612, 614, 616 Detection wavelength range 618,620,622 Maximum luminescence

Claims

1. 1. A fluorescence microscope system (100) for imaging a sample (102) containing at least two different fluorophores, the fluorescence microscope system (100) comprising: an illumination system (104) configured to emit illumination light for exciting the phosphor; an optical detection system (112) configured to generate an image of the sample (102) based on the fluorescence emitted by the excited fluorophores; a control unit (124) configured to determine whether the sample (102) is imaged in a simultaneous imaging mode or a sequential imaging mode based on at least one property of each of the different fluorophores and based on at least one parameter of the optical detection system (112) and / or at least one parameter of the illumination system (104); Including, In the simultaneous imaging mode, the different phosphors are imaged simultaneously, In the sequential imaging mode, the different phosphors are divided into a first group and at least one second group, and the phosphors in the first group and the phosphors in the second group are imaged successively. A fluorescence microscope system (100).

2. the control unit (124) is configured to separate the different phosphors into the first group and at least one second group based on at least one characteristic of each of the different phosphors. The fluorescence microscope system (100) of claim 1.

3. the control unit (124) is configured to separate the different phosphors into the first group and at least one second group such that the emission spectra of the different phosphors within each group have minimal overlap. The fluorescence microscope system (100) of claim 2.

4. The control unit (124) determining a minimum and / or a maximum value of the output of at least one light source (106) of the illumination system (104) based on at least one characteristic of each of the different phosphors and / or at least one characteristic of the sample (102); determining whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode by comparing the operating range of the output of the light source (106) to the minimum and / or maximum values; It is configured as follows: The fluorescence microscope system (100) of claim 1.

5. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode based on the excitation spectra of the different fluorophores. The fluorescence microscope system (100) of claim 1.

6. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode based on the spectrum of the excitation light emitted by the illumination system (104). The fluorescence microscope system (100) of claim 1.

7. the control unit (124) is configured to determine whether to image the sample (102) in a sequential or simultaneous imaging mode by comparing the excitation spectra of the different fluorophores with the spectrum of excitation light emitted by the illumination system (104). The fluorescence microscope system (100) of claim 1.

8. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode based on the emission spectra of the different fluorophores. The fluorescence microscope system (100) of claim 1.

9. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode by comparing an overlap of the emission spectra of the different fluorophores with the detected spectrum of the optical detection system (112). The fluorescence microscope system (100) of claim 1.

10. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode by comparing brightness values ​​of at least two of the different fluorophores. The fluorescence microscope system (100) of claim 1.

11. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode based on a dynamic range of at least one detector of the optical detection system (112). The fluorescence microscope system (100) of claim 1.

12. In the sequential imaging mode, the illumination system (104) sequentially emits a first illumination light for exciting the fluorophores of the first group and at least one second illumination light for exciting the fluorophores of the second group; In the simultaneous imaging mode, the illumination system (104) emits a third illumination light for simultaneously exciting different fluorophores. The fluorescence microscope system (100) of claim 1.

13. the control unit (124) is configured to determine whether to image the sample (102) in a sequential imaging mode or a simultaneous imaging mode based on at least one characteristic of the sample (102). The fluorescence microscope system (100) of claim 1.

14. the control unit (124) is configured to set at least one parameter of the optical detection system (112) and / or at least one parameter of the illumination system (104) based on at least one property of each of the different fluorophores and / or at least one property of the sample (102). The fluorescence microscope system (100) of claim 1.

15. the control unit (124) is configured to provide feedback to a user if the control unit (124) determines that the sample (102) should be imaged in the sequential imaging mode. The fluorescence microscope system (100) of claim 1.

16. 1. A method for imaging a sample (102) containing at least two different fluorophores using a fluorescence microscope system (100), the method comprising: determining whether to image the sample (102) in a simultaneous imaging mode or a sequential imaging mode based on at least one property of each of the different fluorophores and based on at least one parameter of an optical detection system (112) of the fluorescence microscope system (100) and / or at least one parameter of an illumination system (104) of the fluorescence microscope system (100); if it is determined that the sample (102) should be imaged in the simultaneous imaging mode, imaging each of the different fluorophores simultaneously; if it is determined that the sample (102) should be imaged in the sequential imaging mode, separating the different fluorophores into a first group and at least one second group, and imaging the fluorophores of the first group and the fluorophores of the second group in succession; A method comprising:

Images