Controller and method for imaging apparatus

Abstract

To provide a controller and a method for an imaging apparatus.SOLUTION: A controller controls a continuous light source and a pulsed light source so as to emit first and second excitation lights, and controls an optical detection unit so as to receive phosphor light and so as to separate the received phosphor light into two spectral detection channels. The first and second spectral detection channels correspond to first and second wavelength bands that include portions of emission spectra of first and second fluorophore species, respectively. The controller controls the optical detection unit so as to detect photon arrival time of the received phosphor light relative to a pulse of the second excitation light, and determines temporal correlation between the pulse of the second excitation light and the photon arrival time and, based thereon, a respective count of photons received in the respective spectral detection channel. Each photon count is obtained by counting the photons respectively emitted by each phosphor species.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a controller for an imaging device.The present invention also relates to a spectral separation method. [Background technology]

[0002] In fluorescence microscopy, spectral unmixing refers to a set of methods for decomposing a recorded fluorophore light spectrum into the spectra of various fluorophore species. The contributions of individual fluorophore species to the recorded spectrum are generally extracted based on the spectral characteristics of the fluorophore species' emission spectra. Known methods that use the spectral characteristics of fluorophore species include, for example, linear spectral unmixing, phasor unmixing, and hyperspectral unmixing. However, the more overlap there is between the emission spectra, the more difficult it becomes to extract the spectra of individual fluorophore species. Furthermore, the quality of the unmixing results depends heavily on a proper understanding of the spectral characteristics of the fluorophore species, especially how these spectral characteristics vary in a given sample.

[0003] Fluorescence lifetimes of fluorophore species can also be used to separate individual spectra. Known lifetime-based separation techniques include TauSeparation (see, e.g., MJ Roberti et al., "TauSense: a fluorescence lifetime-based tool set for everyday imaging," Nature Methods, September 2020, based on D.R. James et al., "Recovery of underlying distributions of lifetimes from fluorescence decay data," Chemical Physics Letters, Vol. 126, No. 1, 1986, pp. 7-11, and / or Merola et al., "Picosecond tryptophan fluorescence of thioredoxin: evidence for discrete species in slow exchange," Biochemistry, August 28, 1989, pp. 3383-3398), pattern fit separation, and phasor separation. Fluorescence lifetimes can be used as additional information, or separations can be entirely lifetime-based. Using fluorescence lifetimes as a distinctive feature to separate the spectra of various fluorophore species allows separation despite overlapping emission spectra, as long as the fluorophore species have different fluorescence lifetimes. There are drawbacks to using lifetime-based separation methods. Generally, lifetime-based spectral separation approaches require a large photon dose, which may not be available. Lifetime-based approaches also require sophisticated determination of the fluorescence lifetime of the fluorophore species being used. Summary of the Invention [Problem to be solved by the invention]

[0004] The challenge is therefore to provide a controller and a spectral separation method for an imaging device that allows for robust spectral separation and does not require a priori knowledge of the fluorescence lifetimes of the fluorophore species used. [Means for solving the problem]

[0005] The above-mentioned object is achieved by the subject matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

[0006] A proposed controller for an imaging device is configured to control a continuous light source of the imaging device to emit a first excitation light for exciting a first phosphor species and a pulsed light source of the imaging device to emit a second excitation light for exciting a second phosphor species. The first excitation light has a first wavelength range, and the second excitation light has a second wavelength range. The controller is also configured to control an optical detection unit of the imaging device to receive phosphor light emitted by the excited first and second phosphor species and to control the optical detection unit of the imaging device to separate the received phosphor light into at least two spectral detection channels. The first spectral detection channel corresponds to a first wavelength band that includes at least a portion of the emission spectrum of the first phosphor species. The second spectral detection channel corresponds to a second wavelength band that includes at least a portion of the emission spectrum of the second phosphor species. The controller is also configured to control the optical detection unit of the imaging device to detect photon arrival times of the received phosphor light relative to pulses of the second excitation light. The controller is further configured to determine a temporal correlation between the pulse of the second excitation light and the photon arrival time, and to determine a first number of photons and a second number of photons received in the first and / or second spectral detection channels based on the temporal correlation. The first number of photons is a count of photons emitted by the first fluorophore species, and the second number of photons is a count of photons emitted by the second fluorophore species. The temporal correlation between the pulse of the second excitation light and the photon arrival time can be determined, for example, as described in EP 3761010, the entire contents of which are incorporated herein by reference. Alternatively or additionally, detection of the photon arrival time of the received fluorophore light relative to the pulse of the second excitation light can be achieved by appropriate time gating settings of one or more detection channels. Furthermore, any fitting or non-fitting lifetime analysis approach can be used as a basis for determining lifetime correlation or lifetime decorrelation of the detected signals.

[0007] A continuous light source in the sense of this specification is a light source that emits continuous light, e.g., uninterrupted light. A pulsed light source in the sense of this specification is a light source that emits light having a pulsed shape, or a light source that emits continuous light, in which case this continuous light is converted into pulsed light, e.g., by a chopper wheel or other means of temporarily interrupting the light beam.

[0008] Preferably, the first wavelength band is selected so that the first spectral detection channel captures a majority of the phosphor light emitted by the first phosphor species. Similarly, the second wavelength band is selected so that the second spectral detection channel captures a majority of the phosphor light emitted by the second phosphor species. However, overlap between the emission spectra of the first and second phosphor species is inevitable. Therefore, the first wavelength band may include the phosphor light emitted by the second phosphor species, and / or the second spectral detection channel may include the phosphor light emitted by the first phosphor species. Spectral separation is required to separate the contributions of the first and second phosphor species to the first and second spectral detection channels, respectively. The proposed controller is configured to perform this spectral separation, as described below.

[0009] The pulsed light source is controlled to emit the second excitation light periodically, where one period includes intervals during which the pulsed light source emits the second excitation light and intervals during which the pulsed light source does not emit the second excitation light. The second phosphor species is excited only during the intervals during which the second excitation light is emitted. As a result, the emission of phosphor light by the second phosphor species exhibits a periodic pattern that is highly correlated with the pattern in which the pulsed light source emits the second excitation light. Therefore, the arrival times of the phosphor light photons are also correlated with the pattern in which the pulsed light source emits the second excitation light. The continuous light source is controlled to emit the first excitation light continuously. Therefore, the first phosphor species is continuously excited and emits phosphor light continuously. As a result, the emission of phosphor light by the first phosphor species does not exhibit a periodic pattern that is correlated with the pattern in which the pulsed light source emits the second excitation light. The emission of phosphor light by the first phosphor species is uncorrelated in the sense that noise is uncorrelated.

[0010] By determining the correlation between photon arrival times and the emission pattern of the pulsed light source, the controller can determine how many of the detected photons are likely emitted by a first fluorophore species, i.e., a first photon count, and how many of the detected photons are likely emitted by a second fluorophore species, i.e., a second photon count. The first and second photon counts can then be used to separate the contributions of the first and second fluorophore species to the first and / or second spectral detection channels. The separation performed by the proposed controller does not require a priori knowledge of the fluorescence lifetimes of the two fluorophore species used. Furthermore, the separation performed by the proposed controller is robust in the sense that it is not sensitive to changes in the fluorescence lifetimes of the two fluorophore species that may occur during the experiment.

[0011] In one preferred embodiment, the controller is configured to control at least one additional continuous light source of the imaging device to emit third excitation light for exciting a third fluorescent species. The third excitation light has a third wavelength range. According to this embodiment, the controller is also configured to control the optical detection unit of the imaging device to separate the received fluorescent light into at least three spectral detection channels. The third spectral detection channel corresponding to the third wavelength band includes at least a portion of the emission spectrum of the third fluorescent species and at least a portion of the emission spectrum of the second fluorescent species. According to this embodiment, the controller is further configured to determine a third number of photons received in the third spectral detection channel based on the temporal correlation, the third number of photons being a count of photons emitted by the third fluorescent species.

[0012] Preferably, the third wavelength band is selected so that the third spectral detection channel captures a majority of the phosphor light emitted by the third phosphor species. However, due to overlap between the emission spectra of the phosphor species, the third wavelength band may include the phosphor light emitted by the second phosphor species. Spectral separation is required to separate the phosphor species contributions to the third spectral detection channel. In this embodiment, the controller is configured to perform this spectral separation in the third spectral detection channel.

[0013] The controller determines a correlation between the photon arrival times and the emission pattern of the pulsed light source in the third spectral detection channel. The emission of phosphor light by the second phosphor species is correlated with the emission pattern of the pulsed light source, while the emission of phosphor light by the third phosphor species is uncorrelated. From this correlation, the controller can therefore determine how many photons counted in the third spectral detection channel are attributed to the second phosphor species, and therefore how many photons are attributed to the third phosphor species, i.e., a third photon count. The third photon count can then be used to separate the contributions of the second and third phosphor species to the third spectral detection channel.

[0014] The third wavelength band may also contain the phosphor light emitted by the first phosphor species. In this case, additional separation is required. However, by selecting the third wavelength band appropriately, the contributions of the first phosphor species to the third spectral detection channel can be minimized to the point where they are negligible.

[0015] In another preferred embodiment, the controller is configured to control at least one additional pulsed light source of the imaging device to emit a fourth excitation light to excite a fourth fluorophore species. The fourth excitation light has a fourth wavelength range. According to this embodiment, the controller is also configured to control the optical detection unit of the imaging device to separate the received fluorophore light into at least three spectral detection channels, preferably four spectral detection channels. The fourth spectral detection channel, corresponding to the fourth wavelength band, includes at least a portion of the emission spectrum of the fourth fluorophore species and at least a portion of the emission spectrum of the first, second, and / or third fluorophore species. According to this embodiment, the controller is also configured to control the optical detection unit of the imaging device to detect photon arrival times of the received fluorophore light relative to pulses of the fourth excitation light and / or to detect photon arrival times of the received fluorophore light relative to pulses of the second excitation light. The controller is further configured to determine a temporal correlation between the fourth pulse of excitation light and / or the second pulse of excitation light and the photon arrival time, and to determine a fourth number of photons received in the fourth spectral detection channel based on the temporal correlation, the fourth number of photons being a count of photons emitted by the fourth fluorophore species.

[0016] Preferably, the fourth wavelength band is selected so that the fourth spectral detection channel captures a majority of the phosphor light emitted by the fourth phosphor species. However, due to overlap between the emission spectra of the phosphor species, the fourth wavelength band may include phosphor light emitted by the first, second, and / or third phosphor species. Spectral separation is required to separate the phosphor species contributions to the fourth spectral detection channel. In this embodiment, the controller is configured to perform this spectral separation in the fourth spectral detection channel.

[0017] The additional pulsed light source is controlled to emit the fourth excitation light periodically, where one period includes intervals during which the pulsed light source emits the fourth excitation light and intervals during which the pulsed light source does not emit light. The fourth phosphor species is excited only during intervals during which the fourth excitation light is emitted and the phosphor species is illuminated by the fourth excitation light. As a result, the emission of phosphor light by the fourth phosphor species exhibits a periodic pattern that is highly correlated with the pattern in which the additional pulsed light source emits the fourth excitation light. Therefore, the arrival times of photons of the phosphor light emitted by the fourth phosphor species are also correlated with the pattern in which the additional pulsed light source emits the fourth excitation light. In contrast, the arrival times of photons of the phosphor light by the first and / or third phosphor species are not correlated with the pattern in which the additional pulsed light source emits the fourth excitation light.

[0018] The controller determines a correlation between the photon arrival times and the emission pattern of the additional pulsed light source in the fourth spectral detection channel. This allows the controller to determine how many photons counted in the fourth spectral detection channel are attributed to the fourth fluorophore species, i.e., the fourth photon number, and therefore how many photons are attributed to the first and / or third fluorophore species, and therefore how many photons should be assigned to the first or third photon number rather than the fourth photon number. The fourth photon number can then be used to separate the contributions of the first, third, and fourth fluorophore species to the fourth spectral detection channel.

[0019] The fourth wavelength band may also include the phosphor light emitted by the second phosphor species. By appropriately selecting the fourth wavelength band, the contribution of the second phosphor species to the fourth spectral detection channel can be minimized to a negligible level. Furthermore, the pattern, i.e., periodic length, of the additional pulsed light source can be different from that of the pulsed light source emitting the second excitation light. In other words, the pulse frequencies of the (second and fourth) pulsed light sources can be different, and / or the pulses emitted by the (second and fourth) pulsed light sources can be emitted asynchronously, or the pulses can irradiate the phosphor species asynchronously. This allows the contribution of the second phosphor species to the fourth spectral detection channel to be identified based on their correlation with the pattern of the pulsed light source emitting the second excitation light. On the other hand, the contribution of the fourth phosphor species to the fourth spectral detection channel can be identified based on their correlation with the pattern of the additional pulsed light source emitting the fourth excitation light.

[0020] The controller can be configured to control the additional light sources to emit additional excitation light. The additional light sources can be pulsed or continuous light sources. For each additional light source, the controller is configured to control the optical detection unit to separate the received fluorophore light into additional spectral detection channels corresponding to additional wavelength bands. The separation into additional spectral detection channels is performed according to the same principles as described above.

[0021] The first, second, third, and / or fourth wavelength ranges of the excitation light can have a width of at least 2 nm, for example, 10 nm, 20 nm, or up to 50 nm, but may extend up to about 300 nm. Alternatively, the first, second, third, and / or fourth wavelength ranges can include only one wavelength. Thus, the terms wavelength and wavelength range are used interchangeably herein when referring to excitation light. The first, second, third, and / or fourth wavelength bands of the spectral detection channel can have a width of at least 2 nm, preferably at least 10 nm, and up to 300 nm. Alternatively, the first, second, third, and / or fourth wavelength bands can include only one wavelength.

[0022] The wavelength bands of the spectral detection channels can be arranged so that a wavelength band containing primarily fluorophore light emitted by fluorophore species excited by a continuous light source is followed by a wavelength band containing primarily fluorophore light emitted by fluorophore species excited by a pulsed light source. With this alternating wavelength band arrangement, crosstalk between fluorophore species excited by one type of light source, i.e., a continuous light source or a pulsed light source, can be distinguished in the individual spectral detection channels, thereby significantly reducing, and in ideal cases even completely eliminating, "unwanted" crosstalk contributions detected during detection. This, in turn, makes it easier to distinguish fluorophore species based on the temporal correlation between photon arrival times and the emission pattern of the pulsed light source.

[0023] The wavelength ranges of the excitation light and the wavelength bands of the spectral detection channels can be arranged in a particularly alternating manner, although this may not be an ideal arrangement depending on the size of the Stokes shift of the fluorophore species used. In particular, for large Stokes shifts, a different arrangement of the wavelength ranges and wavelength bands may be required.

[0024] In another preferred embodiment, the controller is configured to use machine learning or deep learning to determine the first, second, third, and / or fourth photon counts. In this embodiment, the controller uses machine learning or deep learning to distinguish between various fluorescent signals. In particular, the controller uses machine learning or deep learning to determine the temporal correlation between the excitation light pulses of the pulsed light source and the photon arrival times. The use of machine learning or deep learning can significantly assist in determining the first, second, third, and / or fourth photon counts, thereby improving the reliability of the controller. Machine learning techniques include, but are not limited to, support vector machines and neural networks. Most machine learning and deep learning techniques require either supervised or unsupervised training using an appropriate training dataset. The choice of training dataset depends on the specific task of the machine learning or deep learning technique being used. Suitable training datasets for determining the first, second, third, and / or fourth photon counts can include simulated data, e.g., generated by a Monte Carlo method.

[0025] The present invention also relates to an imaging device including the controller described above. The imaging device also includes at least one continuous light source configured to emit a first excitation light and at least one pulsed light source configured to emit a second excitation light. The first excitation light has a first wavelength range and the second excitation light has a second wavelength range. The imaging device further includes an optical detection unit configured to receive fluorophore light emitted by the excited first and second fluorophore species, separate the received fluorophore light into at least two spectral detection channels, and detect photon arrival times of the received fluorophore light relative to pulses of the second excitation light.

[0026] The imaging device has the same advantages as the controller described above and can be complemented with the features of the dependent claims relating to the controller.

[0027] The optical detection unit of the imaging device is configured to detect photon arrival times, i.e. the optical detection unit is capable of time-resolved detection of the photons of the received phosphor light. The time-resolved detection of the photons of the received phosphor light is achieved, for example, by assigning an arrival time to each detected photon. Preferably, the optical detection unit includes at least one detector element capable of assigning an arrival time to each detected photon.

[0028] In one preferred embodiment, the optical detection unit includes at least one detector element capable of counting photons. In this embodiment, the optical detection unit is configured to assign an arrival time to each detected photon by the at least one detector element capable of counting photons. Counting photons can improve the temporal resolution of the detector elements. The temporal resolution significantly affects the quality of determining the temporal correlation between the pulse of excitation light and the photon arrival time, and thus the quality of determining the first, second, third, and / or fourth photon counts. Thus, improving the temporal resolution significantly improves the separation performed by the controller of the imaging device.

[0029] In another preferred embodiment, the optical detection unit comprises at least one objective, preferably a microscope objective, for receiving the fluorophore light emitted by the first and second fluorophore species. The objective can provide magnification, which allows for resolving small details that would not be visible to the naked eye. In this way, for example, smaller structures of the sample can be imaged by the imaging device.

[0030] In another preferred embodiment, the continuous light source and / or pulsed light source includes at least one laser light source. The laser light source can be, for example, a pulsed laser light source in the VIS range or IR range. Using laser light to excite the phosphor species has several advantages. For example, laser light exhibits coherence, making it easier to focus, and laser light has a higher power density than incoherent light. Alternatively or additionally, the continuous light source and / or pulsed light source can include other light sources, such as LED elements or gas discharge lamps such as argon lamps.

[0031] In another preferred embodiment, the pulsed light source includes a supercontinuum laser light source or a white-light laser source configured to emit supercontinuum laser light. Supercontinuum laser light typically includes a frequency range of one octave or more. Therefore, the supercontinuum laser light source or the white-light laser light source can be used to excite a wide range of fluorescent species, for example, by extracting various single wavelength ranges or single wavelengths from the supercontinuum laser light or the white laser light used as excitation light derived from the supercontinuum laser light, for example, by using a filter, particularly a tunable filter such as an acousto-optical tunable filter. In this embodiment, fewer light sources are used, making the imaging device more compact.

[0032] In another preferred embodiment, the optical detection unit includes at least two detector elements and a beam splitting means configured to direct received phosphor light having a wavelength range within a first wavelength band to a first detector element capable of assigning an arrival time to a detected photon, and to direct received phosphor light having a wavelength range within a second wavelength band to a second detector element capable of assigning an arrival time to a detected photon. In this embodiment, the beam splitting element and the first and second detector elements are used as a means for generating first and second spectral detection channels. Compared to other means for generating first and second spectral detection channels, the use of a beam splitting element and the first and second detector elements is easy to implement, cost-effective, and reliable. Another advantage of such an optical device is that the phosphor light received by the optical detection unit is distributed among multiple detector elements, resulting in little or no phosphor light loss.

[0033] In another preferred embodiment, the optical detection unit includes a multispectral or hyperspectral camera configured to generate at least two spectral detection channels and capable of assigning an arrival time to a detected photon. A multispectral camera is configured to capture a limited number of wavelength bands, typically less than or around 10 wavelength bands. Each of these wavelength bands can be a spectral detection channel of the optical detection unit. A hyperspectral camera is configured to capture tens or hundreds of wavelength bands per pixel. In other words, hyperspectral images have significantly higher spectral resolution. A larger number of wavelength bands allows for finer differentiation of fluorescent sources based on their emission spectra, thereby increasing the sensitivity and reliability of the imaging device.

[0034] In another preferred embodiment, the imaging device is a microscope, in particular a confocal microscope.

[0035] The present invention further relates to a method for spectral separation using an imaging device, the method comprising the steps of: exciting a first fluorophore species with a first excitation light having a first wavelength range emitted by a continuous light source; exciting a second fluorophore species with a second excitation light having a second wavelength range emitted by a pulsed light source; receiving fluorophore light emitted by the excited first and second fluorophore species; and separating the received fluorophore light into at least two spectral detection channels, the first spectral detection channel corresponding to a first wavelength band including at least a portion of the emission spectrum of the first fluorophore species and the second spectral detection channel corresponding to a second wavelength band including at least a portion of the emission spectrum of the first fluorophore species. The spectral detection channel corresponds to a second wavelength band including at least a portion of the emission spectrum of the second phosphor species; detecting photon arrival times of the received phosphor light relative to a pulse of second excitation light; determining a temporal correlation between the pulse of second excitation light and the photon arrival times; and determining a first photon number and a second photon number of the photons received in the first spectral detection channel and / or the second spectral detection channel based on the temporal correlation, wherein the first photon number is a count of photons emitted by the first phosphor species and the second photon number is a count of photons emitted by the second phosphor species.

[0036] This method has the same advantages as the controller and imaging device described above and can be complemented with the features of the dependent claims relating to the controller and / or imaging device. [Brief explanation of the drawings]

[0037] Specific embodiments will be described below with reference to the drawings. [Figure 1] FIG. 1 is a diagram illustrating a schematic of an imaging device according to one embodiment. [Figure 2] 2 shows a schematic representation of an alternative optical detection unit of the imaging device according to FIG. 1; [Figure 3] FIG. 3 shows a schematic diagram of a spectral detection channel of the imaging device according to FIGS. 1 and 2; [Figure 4] 4 is a graph that schematically illustrates photon arrival times of fluorescent photons emitted by a first fluorescent species. [Figure 5] 10 is a graph that schematically illustrates photon arrival times of fluorescence photons emitted by a second fluorophore species. [Figure 6] 1 is a graph that schematically illustrates photon arrival times of fluorescence photons received in one of the spectral detection channels. [Figure 7] 3 is a flowchart of a spectral separation method using the imaging device according to FIGS. 1 and 2; [Figure 8a] FIG. 1 shows a schematic diagram of an emission spectrum and a corresponding phasor plot. [Figure 8b] FIG. 1 shows a schematic diagram of an emission spectrum and a corresponding phasor plot. [Figure 8c] FIG. 1 shows a schematic diagram of an emission spectrum and a corresponding phasor plot. DETAILED DESCRIPTION OF THE INVENTION

[0038] FIG. 1 is a schematic diagram of an imaging device 100 according to one embodiment.

[0039] The imaging device 100 is configured to image a sample 102 containing various fluorophore species by fluorescence imaging. In this embodiment, the imaging device 100 is exemplarily formed as a microscope. However, the imaging device 100 is not limited to a microscope.

[0040] The imaging device 100 illustratively includes four continuous light sources and three pulsed light sources. Only the first continuous light source 104 and the first pulsed light source 106 are shown in FIG. 1 . The remaining light sources are represented by small black dots. The continuous light source 104 is configured to emit first, third, fifth, and seventh excitation light beams 302a, 302c, 302e, and 302g (see FIG. 3 ), respectively. The pulsed light source 106 is configured to emit second, fourth, and sixth excitation light beams 302b, 302d, and 302f (see FIG. 3 ), respectively. Each excitation light beam excites a different fluorophore species disposed within the sample 102.

[0041] To capture an image of the sample 102, the imaging device 100 includes an optical detection unit 108. The optical detection unit 108 includes an objective lens 110 directed toward the sample 102, a beam splitting unit 112, and a detector element 114. The objective lens 110 receives fluorophore light emitted by excited fluorophore species disposed in the sample 102 and directs the received detection light toward the detector element 114. The beam splitting unit 112 is configured to direct excitation light into the sample 102 through the objective lens 110 and direct the received fluorophore light toward the detector element 114. In this embodiment, the detector element 114 is configured as a multispectral camera capable of time-resolved detection of photons of the received fluorophore light. The multispectral camera is configured to capture seven different wavelength bands 300a, 300b, 300c, 300d, 300e, 300f, and 300g (see FIG. 3). Each of the seven wavelength bands 300a, 300b, 300c, 300d, 300e, 300f, and 300g corresponds to a spectral detection channel of the optical detection unit 108. An alternative optical detection unit 200 is described below with reference to FIG.

[0042] The imaging device 100 further includes a controller 116. The controller 116 is connected to the optical detection unit 108 and the light sources 104 and 106 and is configured to control the imaging device 100. Specifically, the controller 116 is configured to implement a spectral separation method, which will be described later with reference to Figures 4 to 7.

[0043] FIG. 2 shows a schematic diagram of an alternative optical detection unit 200 of the imaging device 100 according to FIG.

[0044] The optical detection unit 200 according to Fig. 2 differs from the optical detection unit 108 according to Fig. 1 in how the spectral detection channels are realized: in this embodiment, the spectral detection channels are realized by individual detector elements 202a, 202b rather than by a multispectral camera.

[0045] According to this embodiment, the optical detection unit 200 includes seven beam-splitting elements 206a, 206b, such as dichroic elements or acousto-optic tunable filters (AOTFs), arranged along the detection beam path 204. Only the first two beam-splitting elements 206a, 206b are shown in FIG. 2 . The remaining beam-splitting elements and additional detector elements are represented by small black dots in FIG. 2 . Each of the beam-splitting elements 206a, 206b is configured to direct a specific wavelength band of the received fluorophore light to one of the detector elements 202a, 202b and direct the remaining fluorophore light toward the next beam-splitting element 206a, 206b along the detection beam path. Each detector element 202a, 202b receives fluorophore light of a predetermined wavelength band, thereby realizing a spectral detection channel.

[0046] FIG. 3 is a schematic diagram of a spectral detection channel of the imaging device 100 according to FIGS.

[0047] Figure 3 is a wavelength graph showing wavelengths decreasing to the left and wavelengths increasing to the right. Wavelength bands 300a, 300b, 300c, 300d, 300e, 300f, and 300g corresponding to the seven spectral sensing channels are shown as rectangles. As can be seen from Figure 3, there is no overlap between the spectral sensing channels.

[0048] The wavelength ranges of the excitation lights are shown as vertical lines. The first excitation light, designated "cw" in FIG. 3, is emitted by one of the continuous light sources 104 and has a first wavelength 302a of 405 nm. The second excitation light, designated "wll" in FIG. 3, is emitted by one of the pulsed light sources 106, specifically a white light laser, and has a second wavelength 302b of 440 nm. The third excitation light, designated "wll" in FIG. 3, is emitted by one of the continuous light sources 104 and has a third wavelength 302c of 458 nm. The fourth excitation light, designated "wll" in FIG. 3, is emitted by one of the pulsed light sources 106, specifically a white light laser, and has a fourth wavelength 302d of 535 nm. The fifth excitation light, designated "wll" in FIG. 3, is emitted by one of the continuous light sources 104 and has a fifth wavelength 302e of 594 nm. The sixth excitation light is emitted by one of the pulsed light sources 106, in particular by a white light laser, and has a sixth wavelength 302f of 640 nm. The seventh excitation light is emitted by one of the continuous light sources 104 and has a seventh wavelength 302g of 730 nm. According to this embodiment, the emission wavelengths of the continuous light source 104 and the pulsed light source 106 are alternated.

[0049] FIG. 3 also shows emission spectra 306a, 306b, 306c, 306d, 306e, 306f, and 306g of various phosphor species. Each of the various phosphor species is excited by a different excitation light. Due to the Stokes shift, the emission spectra 306a, 306b, 306c, 306d, 306e, 306f, and 306g of the phosphor species contain wavelengths longer than the excitation light that excites that phosphor species. Therefore, the emission spectra 306a, 306b, 306c, 306d, 306e, 306f, and 306g of the phosphor species in FIG. 3 are always to the right of the wavelengths 302a, 302b, 302c, 302d, 302e, 302f, and 302g of the excitation light that excites that phosphor species. The emission spectra 306a, 306c, 306e, 306g of phosphor species excited by continuous light source 104 are shown as solid lines, while the emission spectra 306b, 306d, 306f of phosphor species excited by pulsed light source 106 are shown as dashed lines.

[0050] The first fluorophore species is excited by a first excitation light. The emission spectrum 306a of the first fluorophore species falls almost entirely within a first wavelength band 300a of a first spectral detection channel. However, the emission spectrum 306a of the first fluorophore species extends into a second wavelength band 300b of a second spectral detection channel. The second fluorophore species is excited by a second excitation light. The emission spectrum 306b of the second fluorophore species falls almost entirely within a second wavelength band 300b of the second spectral detection channel and extends into a third wavelength band 300c of a third spectral detection channel. The third fluorophore species is excited by a third excitation light. The emission spectrum 306c of the third fluorophore species falls almost entirely within a third wavelength band 300c of the third spectral detection channel and extends into a fourth spectral detection channel. The fourth fluorophore species is excited by a fourth excitation light. The emission spectrum 306d of the fourth phosphor species falls almost entirely within the fourth wavelength band 300d of the fourth spectral detection channel and extends into the fifth wavelength band 300e of the fifth spectral detection channel. The fifth phosphor species is excited by the fifth excitation light. The emission spectrum 306e of the fifth phosphor species falls almost entirely within the fifth wavelength band 300e of the fifth spectral detection channel and extends into the sixth wavelength band 300f of the sixth spectral detection channel. The sixth phosphor species is excited by the sixth excitation light. The emission spectrum 306f of the sixth phosphor species falls almost entirely within the sixth wavelength band 300f of the sixth spectral detection channel and extends into the seventh wavelength band 300g of the seventh spectral detection channel. The seventh phosphor species is excited by the seventh wavelength band 300g of the seventh excitation light. The emission spectrum 306g of the seventh fluorophore species falls almost entirely within the seventh spectral detection channel.

[0051] Crosstalk exists in all spectral detection channels except the first. However, because the emission wavelengths 302a, 302c, 302e, and 302g of the continuous light source 104 and the emission wavelengths 302b, 302d, and 302f of the pulsed light source 106 are alternated, only crosstalk between fluorophore species excited by different types of light sources 104 and 106, i.e., the continuous light source and the pulsed light source, is always present. This can be used to separate the contributions of different fluorophore species to a single spectral detection channel. Spectral separation methods are described below with reference to Figures 4-7.

[0052] It should be understood that the wavelength arrangement of excitation light in wavelength bands 300a, 300b, 300c, 300d, 300e, 300f, and 300g of spectral detection channels and 302a, 302b, 302c, 302d, 302e, 302f, and 302g shown in FIG. 3 is exemplary. The shortest wavelength excitation light may be emitted by pulsed light source 106 instead of continuous light source 104 as shown in FIG. 3. It is also not necessary to alternate continuous light source 104 and pulsed light source 106 with respect to their emission wavelengths or wavelength ranges as shown in FIG. 3, as long as the two overlapping emission spectra in each spectral detection channel are emission spectra of two fluorophore species excited by different types of light sources, i.e., one of pulsed light source 106 and one of continuous light source 104. For example, if one of the fluorophore species has a large Stokes shift, the order of emission wavelengths of the continuous light source 104 and the pulsed light source 106 may be different from the alternating arrangement shown in FIG.

[0053] FIG. 4 is a graph 400 that schematically illustrates photon arrival times for fluorescent photons emitted by a first fluorophore species.

[0054] The horizontal axis 404 of the graph 400 represents time in nanoseconds. The vertical axis 402 of the graph 400 represents the number of photons counted over a short interval on a logarithmic scale.

[0055] The continuous light source 104 continuously emits the first excitation light, thereby continuously exciting the first phosphor species. The phosphor light emission by the first phosphor species is uncorrelated in the sense that the emission of the fluorescent photons, and therefore the arrival times, are randomly distributed in time.

[0056] As can be seen from Figure 4, the first fluorophore species emits photons continuously. The exact number of photons emitted during each short interval will vary slightly. However, the number of photons emitted does not show a pattern that correlates with the emission of excitation light by any of the pulsed light sources 106.

[0057] FIG. 5 is a graph 500 that schematically illustrates photon arrival times for fluorescent photons emitted by a second fluorophore species.

[0058] The horizontal axis 504 of graph 500 represents time in nanoseconds. The vertical axis 502 of graph 500 represents the number of photons counted over a short interval on a logarithmic scale. Graph 500 thus resembles a fluorescence decay histogram.

[0059] The second excitation light is emitted in short pulses by one of the pulsed light sources 106, followed by a short period during which the pulsed light source 106 does not emit excitation light. The second phosphor species is excited only during the period during which the pulsed light source 106 emits the second excitation light. The emission of phosphor light by the second phosphor species will exhibit a pattern that correlates with the pattern in which the pulsed light source 106 emits the second excitation light.

[0060] 5, the second fluorophore species does not initially emit photons, and what is seen to the left of step edge 506 is essentially noise, containing no detected fluorescence photons. After pulsed light source 106 emits a pulse of second excitation light, the second fluorophore species immediately begins emitting fluorescent light. The number of photons of emitted fluorescent light is high immediately following the pulse, resulting in step edge 506 in the photon count, which then decreases to its initial level linearly, as opposed to exponentially, over a short period called the fluorescence lifetime decay.

[0061] FIG. 6 is a graph 600 that schematically illustrates photon arrival times for fluorescence photons received in the second spectral detection channel.

[0062] The horizontal axis 604 of the graph 600 represents time in nanoseconds. The vertical axis 602 of the graph 600 represents the number of photons counted over a short interval on a logarithmic scale.

[0063] The second spectral detection channel receives the phosphor light emitted by the first phosphor species and the phosphor light emitted by the second phosphor species. Therefore, the photon counts shown in graph 600 of Figure 6 can be considered a combination of the photon counts shown in graphs 400 and 500 of Figures 4 and 5. In the case of graph 600 of Figure 6, the baseline photon count (e.g., shown to the left of side edge 606) is higher than in graph 500 of Figure 5. This relatively high baseline signal (which may include some noise level) includes uncorrelated photons emitted by the first phosphor species excited by continuous light source 104.

[0064] All of the fluorophore light initially received in the second spectral detection channel is due to the first fluorophore species being excited by the continuous light source 104. After the pulsed light source 106 emits a pulse of second excitation light, the second spectral detection channel also receives fluorophore light emitted by the second fluorophore species, as seen by the step edge 606 in the photon counts.

[0065] By determining the temporal correlation between the photon arrival time and the pulse of second excitation light, the number of first photons emitted by the first fluorophore species and received in the second spectral detection channel can be determined, and the number of second photons emitted by the second fluorophore species and received in the second spectral detection channel can be determined. For example, because the photon arrival times of photons emitted by the first fluorophore species are uncorrelated, the photons can be treated as random background noise. Therefore, to determine the number of photons emitted by the second fluorophore species, the number of photons emitted by the first fluorophore species can be subtracted from the total photon count in the second spectral detection channel. This separates the contributions of the first fluorophore species and the second fluorophore species to the second spectral detection channel.

[0066] So far, separation has only been described in the context of the second spectral sensing channel, however everything that has been described so far with reference to Figures 4 to 6 also applies to the third to seventh spectral sensing channels.

[0067] FIG. 7 is a flowchart of a spectral separation method using the imaging device 100 according to FIGS.

[0068] The process starts in step S700. In step S702, the controller 116 controls the continuous light source 104 to emit first, third, fifth, and seventh excitation lights for exciting the first, third, fifth, and seventh fluorophore species. In step S704, the controller 116 controls the pulsed light source 106 to emit second, fourth, and sixth excitation lights for exciting the second, fourth, and sixth fluorophore species. Steps S702 and S704 can be performed simultaneously or sequentially in any order. In step S706, the controller 116 controls the optical detection unit 108, 200 to receive fluorophore light emitted by the excited fluorophore species. For example, in step S706, the controller 116 can control the objective lens 110 of the optical detection unit 108, 200 to focus the fluorophore light on the sample 102. In step S708, the controller 116 controls the optical detection units 108, 200 to separate the received fluorophore light into spectral detection channels. The controller 116 can control the detection unit, e.g., a multispectral camera, to record photons within wavelength bands 300a, 300b, 300c, 300d, 300e, 300f, and 300g corresponding to the spectral detection channels. Alternatively, the controller 116 can control the beam splitting elements 206a, 206b to direct the wavelength bands 300a, 300b, 300c, 300d, 300e, 300f, and 300g corresponding to the spectral detection channels to the detector elements 202a, 202b. For example, the controller 116 can select a particular filter from a filter wheel or control an AOTF. In step S710, the controller 116 controls the optical detection units 108, 200 to detect photon arrival times of the received phosphor light relative to the second, fourth, and sixth pulses of excitation light emitted by the pulsed light source 106. In step S712, the controller 116 determines a temporal correlation between the second, fourth, and sixth pulses of excitation light and the photon arrival times.In step S714, controller 116 determines the number of received photons corresponding to each fluorophore species for each spectral detection channel based on the temporal correlation. The separation performed in step S714 is based on the principles described above with reference to Figures 4-6. The process ends in step S716.

[0069] FIG. 8a is a schematic diagram of the emission spectrum and the corresponding phasor plot.

[0070] The top row of Figure 8a shows three spectral channels corresponding to three different wavelength bands 300a, 300b, and 300c of the spectral channels shown in Figure 3. The leftmost and center wavelength bands 300a and 300b contain an emission spectrum 306a of one of the phosphor species. Because the phosphor species corresponding to emission spectrum 306a is excited by a continuous light source, emission spectrum 306a is shown as a solid line. Below each spectral channel in Figure 8a, corresponding phasor plots 808a, 808b, and 808c are shown.

[0071] Phasor plots and related analytical approaches can be used to analyze fluorescence lifetimes. Phasor FLIM (Fluorescence Lifetime Imaging Microscopy) provides a 2D graphical view of the lifetime distribution. This graphical view allows for rapid differentiation and separation of various lifetime distributions within a FLIM image. Because every species has a specific phasor, multiple molecular species are resolved within a single pixel. When data are acquired using a time-resolved single-photon counting (TCSPC) system, the phasor FLIM distribution is derived from a Fourier transform, as described, for example, by Digman et al., "The Phasor Approach to Fluorescence Lifetime Imaging Analysis," Biophys. J. (2008) 94, 2nd ed., pp. L14-L16. Each pixel in the image corresponds to a point in the phasor plot. The information contained in the phasor approach can be readily interpreted, resulting in a lifetime distribution. The most important rules for interpreting phasor plots are explained, for example, by E. Gratton in "The Phasor approach: Application to FRET analysis and Tissue Autofluorescence," 13th LFD workshops 2018, October 22–26, 2018, Laboratory for Fluorescence Dynamics (LFD), University of California, Irvine, USA. Lanzano et al. (2015) in their publication "Encoding and decoding spatio-temporal information for super-resolution microscopy," Nature Communications, 6:6701, 10.1038 / ncomms7701, describe a method called SPLIT that can preserve the signal of early photons by applying the phasor approach. Here, the lifetime decay of each pixel is converted into a polar coordinate plot called a phasor plot.In this plot, the coordinate of each pixel, called the phasor, is a linear combination of the coordinates of three components: the high-resolution signal, the low-resolution signal, and the background. The weight of the high-resolution component is used as the intensity of the resulting image, which contains information from all detected photons.

[0072] The fluorophore species corresponding to the emission spectrum 306a is continuously excited. The detected fluorophore light emissions emitted by the fluorophore species in the two spectral channels corresponding to the leftmost and middle wavelength bands 300a and 300b are uncorrelated in the sense that the fluorescence photon emissions, and therefore arrival times, are randomly distributed in time. This can be observed by the distribution 806a shown by the black dots in the lower right corner of the leftmost phasor plot 808a corresponding to the leftmost wavelength band 300a. The same is true for the second part of the emission spectrum 306a in the middle wavelength band 300b and the distribution 806b in the corresponding phasor plot 808b. The rightmost wavelength band 300c does not detect fluorescence, and therefore the corresponding phasor plot 808c has no entries.

[0073] FIG. 8b is a schematic diagram of the emission spectrum and the corresponding phasor plot.

[0074] The top row of Figure 8b shows three spectral channels corresponding to three different wavelength bands 300b, 300c, and 300d from those shown in Figure 3. The central and rightmost wavelength bands 300c and 300d contain emission spectrum 306c of a different phosphor species than that shown in Figure 8a. This phosphor species is excited by a pulsed light source, and thus emission spectrum 306c is shown as a dashed line. Below each of wavelength bands 300b, 300c, and 300d, corresponding phasor plots 808b, 808c, and 808d are shown.

[0075] The phosphor species corresponding to emission spectrum 306c is excited by the pulsed light source 106, e.g., by one of the pulsed light sources, with a short pulse, followed by a short period during which the pulsed light source does not emit excitation light. The phosphor species corresponding to emission spectrum 306c is excited only during the period during which the pulsed light source emits an individual excitation light. The emission of phosphor light by this phosphor species will exhibit a pattern that correlates with the pattern of the pulsed light source emitting the second excitation light, as described above with reference to FIG. 5 . This can be observed by the representation of distribution 806c, shown as a black circle with few dots, in the central phasor plot 808c. The same is true for the second portion of emission spectrum 306c in the rightmost wavelength band 300d and distribution 806d in the corresponding phasor plot 808d. The leftmost wavelength band 300b does not detect fluorescence, and therefore the corresponding phasor plot 808b has no entries.

[0076] FIG. 8c is a schematic diagram of the emission spectrum and the corresponding phasor plot.

[0077] The situation shown in Figure 8c is a combination of the situations shown in Figures 8a and 8b. Phasor plots 808a, 808b, and 808c in Figure 8c show first-type distributions 806a, 806b and second-type distributions 806c, 806d, which are clearly separable from one another. The first-type distributions 806a, 806b, indicated by black dots in the leftmost and central phasor plots 808a, 808b, correspond to uncorrelated photon arrival times (because they correspond to fluorophore species excited by a continuous light source). The second-type distributions 806c, 806d, indicated by white dots in the central and rightmost phasor plots 808b, correspond to correlated photon arrival times resulting from fluorophore species excited by a pulsed light source. Because the various fluorophore species can be clearly distinguished by the phasor plots, the detected individual contributions of the various fluorophore species can be separated. This is possible even without a priori knowledge of the fluorescence lifetime of the fluorophore species being used.

[0078] 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 " / ". All individual features of the embodiments and all combinations of the individual features of the embodiments with each other, as well as all combinations of those individual features in combination with individual features or features in the description and / or claims, are considered to be disclosed.

[0079] Although some aspects have been described in the context of an apparatus, it will be appreciated that these aspects also represent descriptions of corresponding methods, where blocks or apparatus correspond to steps or features of steps.

[0080] Analogously, 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]

[0081] 100 Imaging Device 102 samples 104 Continuous Light Source 106 Pulsed Light Source 108 Optical Detection Unit 110 Objective Lens 112 Beam Splitting Unit 114 detector elements 116 Controller 200 Optical Detection Unit 202a, 202b detector elements 204 Beam Path 206a, 206b Beam splitting element 300a, 300b, 300c, 300d, 300e, 300f, 300g wavelength band 302a, 302b, 302c, 302d, 302e, 302f, 302g Wavelength range 306a, 306b, 306c, 306d, 306e, 306f, 306g Emission spectrum 400 graphs 402 Vertical Axis 404 Horizontal axis 500 graphs 502 vertical axis 504 Horizontal Axis 506 Side edge 600 graphs 602 Vertical Axis 604 Horizontal axis 606 Side edge 808a, 808b, 808c Phasor plots 806a,806b,806c,806d distribution

Claims

1. A controller (116) for an imaging device (100), The controller (116) is configured to control the continuous light source (104) of the imaging apparatus (100) to emit first excitation light having a first wavelength range (302a) in order to excite a first phosphor species. The controller (116) is configured to control the pulse light source (106) of the imaging apparatus (100) to emit a second excitation light having a second wavelength range (302b) in order to excite a second phosphor species. The controller (116) is configured to control the optical detection units (108, 200) of the imaging apparatus (100) to receive phosphor light emitted by the excited first phosphor species and the second phosphor species. The controller (116) is configured to control the optical detection units (108, 200) of the imaging apparatus (100) to separate the received phosphor light into at least two spectral detection channels, wherein the first spectral detection channel corresponds to a first wavelength band (300a) including at least a portion of the emission spectrum (306a) of the first phosphor species, and the second spectral detection channel corresponds to a second wavelength band (300b) including at least a portion of the emission spectrum (306b) of the second phosphor species. The controller (116) is configured to control the optical detection units (108, 200) of the imaging device (100) to detect the photon arrival time of the received phosphor light relative to the pulse of the second excitation light. It is configured to determine the temporal correlation between the pulse of the second excitation light and the photon arrival time, The controller (116) is configured to determine the number of first and second photons received in the first spectral detection channel and / or the second spectral detection channel based on the temporal correlation, wherein the number of first photons is the number of photons emitted by the first phosphor species, and the number of second photons is the number of photons emitted by the second phosphor species. Controller (116).

2. The controller (116) is configured to control at least one additional continuous light source (104) of the imaging apparatus (100) to emit a third excitation light having a third wavelength range (302c) in order to excite a third phosphor species. The controller (116) is configured to control the optical detection units (108, 200) of the imaging apparatus (100) to separate the received phosphor light into at least three spectral detection channels, wherein the third spectral detection channel corresponds to a third wavelength band (300c) that includes at least a portion of the emission spectrum (306c) of the third phosphor species and a portion of the emission spectrum (306b) of the second phosphor species. The controller (116) is configured to determine the number of third photons received in the third spectral detection channel based on the temporal correlation, and the number of third photons is the number of photons emitted by the third phosphor species. The controller (116) according to claim 1.

3. The controller (116) is configured to control at least one additional pulsed light source (106) of the imaging apparatus (100) to emit a fourth excitation light having a fourth wavelength range (302d) in order to excite a fourth phosphor species. The controller (116) is configured to control the optical detection units (108, 200) of the imaging apparatus (100) to separate the received phosphor light into at least three spectral detection channels, wherein the fourth spectral detection channel corresponds to a fourth wavelength band (300d) that includes at least a portion of the emission spectrum (306d) of the fourth phosphor species and a portion of the emission spectra (306a, 306c) of the first, second, and / or third phosphor species. The controller (116) is configured to control the optical detection units (108, 200) of the imaging device (100) to detect the photon arrival time of the received phosphor light relative to the fourth excitation light pulse. The controller (116) is configured to determine the temporal correlation between the pulse of the fourth excitation light and the photon arrival time. The controller (116) is configured to determine the number of fourth photons received in the fourth spectral detection channel based on the temporal correlation, and the number of fourth photons is the number of photons emitted by the fourth phosphor species. A controller (116) according to claim 1 or 2.

4. The controller (116) is configured to determine the first number of photons, the second number of photons and / or the third number of photons and / or the fourth number of photons using machine learning or deep learning. A controller (116) according to claim 1 or 2.

5. An imaging device (100), wherein the imaging device (100) is A controller (116) according to claim 1 or 2, A continuous light source (104) configured to emit first excitation light having a first wavelength range (302a), A pulsed light source (106) configured to emit a second excitation light having a second wavelength range (302b), An optical detection unit (108, 200) is configured to receive phosphor light emitted by the excited first phosphor species and the second phosphor species, separate the received phosphor light into at least two spectral detection channels, and detect the photon arrival time of the received phosphor light relative to the pulse of the second excitation light, An imaging apparatus (100) including [a specific component].

6. The optical detection unit (108, 200) includes at least one detector element (114, 202a, 202b) which can assign one arrival time to one detected photon. The imaging apparatus (100) according to claim 5.

7. The optical detection unit (108, 200) includes at least one detector element (114, 202a, 202b) capable of counting photons. The imaging apparatus (100) according to claim 5.

8. The optical detection unit (108, 200) includes at least one objective lens (110) for receiving the phosphor light emitted by the first phosphor species and the second phosphor species. The imaging apparatus (100) according to claim 5.

9. The continuous light source (104) and / or the pulsed light source (106) include at least one laser light source. The imaging apparatus (100) according to claim 5.

10. The pulsed light source (106) includes a supercontinuous laser light source or a white light laser source configured to emit supercontinuous laser light. The imaging apparatus (100) according to claim 9.

11. The optical detection unit (200) includes at least two detector elements (202a, 202b) and beam splitting means, wherein the beam splitting means is configured to orient the received phosphor light having a wavelength range within a first wavelength band (300a) to a first detector element (202a) that can assign it to a photon with a detected arrival time, and is configured to orient the received phosphor light having a wavelength range within a second wavelength band (300b) to a second detector element (202b) that can assign it to a photon with a detected arrival time. The imaging apparatus (100) according to claim 5.

12. The optical detection unit (108) includes a multispectral camera or hyperspectral camera configured to generate at least two spectral detection channels and assign one arrival time to one photon, The imaging apparatus (100) according to claim 5.

13. The imaging device (100) is a microscope, and more particularly a confocal microscope. The imaging apparatus (100) according to claim 5.

14. A method for separating spectra using an imaging device (100), the method comprising the following steps, namely, a) A step of exciting a first phosphor species with first excitation light having a first wavelength range (302a) emitted from a continuous light source (104), b) A step of exciting a second phosphor species with a second excitation light having a second wavelength range (302b) emitted by a pulsed light source (106), c) Receiving phosphor light emitted by the excited first phosphor species and the second phosphor species, d) A step of separating the received phosphor light into at least two spectral detection channels, wherein the first spectral detection channel corresponds to a first wavelength band (300a) including at least a portion of the emission spectrum (306a) of the first phosphor species, and the second spectral detection channel corresponds to a second wavelength band (300b) including at least a portion of the emission spectrum (306b) of the second phosphor species, e) A step of detecting the photon arrival time of the received phosphor light relative to the pulse of the second excitation light, f) A step of determining the temporal correlation between the pulse of the second excitation light and the photon arrival time, g) A step of determining the number of first and second photons of photons received in the first spectral detection channel and / or the second spectral detection channel based on the temporal correlation, wherein the number of first photons is the number of photons emitted by the first phosphor species and the number of second photons is the number of photons emitted by the second phosphor species. A method that includes this.

Images