Optical measurement device and scanning microscope

Abstract

To provide a technique capable of inversely transforming a detection signal of a photodetector by using an optimal transformation matrix.SOLUTION: An optical measurement device includes: an optical sensor unit for detecting pulse-like signal light so as to output a detection signal composed of exponential responses; an A / D conversion unit for converting the detection signal into a digital signal; a setting unit for setting a transformation matrix for inversely transforming the digital signal; and a calculation unit for inversely transforming the digital signal by the transformation matrix set by the setting unit so as to calculate an estimated pulse of the signal light.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The disclosure herein relates to an optical measurement device and a scanning microscope. [Background technology]

[0002] In a laser scanning microscope, a laser beam is scanned two-dimensionally over a sample, the fluorescence generated by the sample is detected by a photodetector, and an image is reconstructed and displayed based on the detection signal.

[0003] However, depending on the type of photodetector used, crosstalk may occur in the detection signal, making it impossible to correctly detect the light incident on the photodetector. For example, a semiconductor photosensor is inexpensive and highly sensitive, but has the characteristic of having a long sensor response time. Therefore, when this is used as a photodetector, crosstalk may occur in the detection signal.

[0004] To solve such problems, the scanning microscope described in Patent Document 1 estimates the light incident on the photodetector by inversely transforming the detection signal of the photodetector (optical sensor unit) using a transformation matrix (by performing convolution with an inverse matrix that cancels out the sensor response). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 058619 Summary of the Invention [Problem to be solved by the invention]

[0006] Patent Document 1 does not particularly mention aspects of a practical scanning microscope.

[0007] For example, in a practical scanning microscope, the photodetector may be replaced, a plurality of different (different characteristics) photodetectors may be provided, and the downstream circuit configuration of the photodetector (circuit configuration between the photodetector and the calculation unit that performs inverse conversion) may be different. When the photodetector is replaced, the impulse response of the photodetector differs before and after the replacement. This may occur not only when the photodetector is replaced with a different type of photodetector, but also when the photodetector is replaced with the same type of photodetector due to individual differences. In addition, when a plurality of different types of photodetectors are provided, the impulse response differs for each photodetector. In addition, when the downstream circuit configuration of the photodetector is different, the impulse response (digital signal input to the calculation unit as the impulse response) differs. In addition to the A / D conversion circuit (A / D conversion unit), the downstream circuit configuration of the photodetector may further include, for example, an AFE (Analog Front End) circuit including an analog filter circuit and an amplifier circuit and / or a digital filter circuit.

[0008] Therefore, in a practical scanning microscope, the transformation matrix used for the inverse transformation needs to be an optimal transformation matrix according to the photodetector used and the downstream circuit configuration, otherwise the light incident on the photodetector cannot be correctly estimated.

[0009] An object according to one aspect of the present invention is to provide a technique capable of performing inverse transformation on a detection signal of a photodetector using an optimal transformation matrix. [Means for solving the problem]

[0010] An optical measurement device according to one embodiment of the present invention includes an optical sensor unit that detects pulsed signal light and outputs a detection signal having an exponential response, an A / D conversion unit that converts the detection signal into a digital signal, a setting unit that sets a transformation matrix for inversely transforming the digital signal, and a calculation unit that inversely transforms the digital signal using the transformation matrix set by the setting unit to calculate an estimated pulse of the signal light.

[0011] A scanning microscope according to one aspect of the present invention includes a scanning unit that scans illumination light emitted from a light source over a sample, an optical system that focuses the illumination light scanned by the scanning unit onto the sample and focuses signal light generated at each scanning position on the sample of the illumination light, an optical measurement device according to the above-described one aspect that calculates an estimated pulse of the signal light focused by the optical system, and an image reconstruction unit that reconstructs an image of the sample based on the estimated pulse calculated by the optical measurement device. Effect of the Invention

[0012] According to the above aspect, it is possible to perform inverse transformation on the detection signal of the photodetector using an optimal transformation matrix. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 illustrates an example of a configuration of a scanning microscope according to an embodiment. [Diagram 2] 13 is a diagram illustrating an example of the configuration of a scanning microscope according to Modification 2. FIG. [Diagram 3] FIG. 1 is a diagram illustrating a plurality of transformation matrices. [Figure 4] FIG. 13 is a diagram illustrating the configuration of a scanning microscope according to a fourth modified example. [Diagram 5] FIG. 13 is a diagram illustrating the configuration of a scanning microscope according to a sixth modified example. [Figure 6] FIG. 13 is a diagram illustrating the configuration of a scanning microscope according to Modification 8. [Figure 7] FIG. 23 is a diagram illustrating the configuration of a scanning microscope according to a tenth modification. [Figure 8] 4A to 4C are diagrams illustrating examples of signals in the optical measurement device. [Figure 9] 4A to 4C are diagrams illustrating examples of signals in the optical measurement device. [Figure 10] FIG. 23 is a diagram illustrating the configuration of a scanning microscope according to a twelfth modification example. [Figure 11] 4A to 4C are diagrams illustrating examples of signals in the optical measurement device. [Figure 12] FIG. 2 is a diagram illustrating an example of a hardware configuration of a computer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0015] FIG. 1 is a diagram illustrating a configuration of a scanning microscope 1 according to an embodiment.

[0016] The scanning microscope 1 illustrated in FIG. 1 includes a light source unit 2, a scanner 3, a dichroic mirror 4, an optical system 5, an optical measurement device 6, an image reconstruction unit 7, and a display unit 8.

[0017] The light source unit 2 is an example of a light source, and generates an extremely short pulsed laser light (illumination light). The scanner 3 is an example of a scanning unit, and two-dimensionally scans the laser light emitted from the light source unit 2. The scanner 3 is, for example, a proximity galvanometer mirror in which two galvanometer mirrors are arranged in close proximity. The dichroic mirror 4 transmits the laser light from the light source unit 2 and reflects the fluorescence from the sample X. The optical system 5 focuses the laser light scanned by the scanner 3 on the sample X, and focuses the fluorescence generated in the sample X.

[0018] The optical measurement device 6 detects the fluorescence (signal light) collected by the optical system 5 and calculates an estimated pulse. The image reconstruction unit 7 generates an image of the sample X based on the estimated pulse calculated by the optical measurement device 6. More specifically, the image reconstruction unit 7 generates an image showing the amount of detected fluorescence by arranging the estimated pulse of fluorescence detected and calculated for each scanning position of the laser light by the scanner 3 at the coordinates of each scanning position. The display unit 8 displays the image generated by the image reconstruction unit 7.

[0019] The optical measurement device 6 includes an optical sensor unit 11, an A / D conversion unit 12, a setting unit 13, and a calculation unit .

[0020] The optical sensor unit 11 is a photodetector that detects pulsed fluorescence generated by irradiating a fluorescent substance present in the sample X with an extremely short pulsed laser light, and outputs a detection signal consisting of an exponential response with a certain time constant. The optical sensor unit 11 is, for example, a semiconductor optical sensor consisting of an array of multiple avalanche photodiodes operating in Geiger mode.

[0021] The A / D conversion unit 12 converts the detection signal output from the optical sensor unit 11 into a digital signal and outputs it. The A / D conversion unit 12 performs sampling at a sampling period shorter than the longest time constant of the exponential response of the optical sensor unit 11. The A / D conversion unit 12 is, for example, an A / D conversion circuit.

[0022] The setting unit 13 sets in the calculation unit 14 a transformation matrix for inversely transforming the digital signal output from the A / D conversion unit 12 .

[0023] The calculation unit 14 inversely converts the digital signal output from the A / D conversion unit 12 using a conversion matrix set by the setting unit 13 to calculate an estimated pulse of the signal light. More specifically, the calculation unit 14 calculates an estimated pulse of the signal light by performing convolution, i.e., inverse conversion, with an inverse matrix that cancels the sensor response of the optical sensor unit 11, on the detection signal output from the optical sensor unit 11. The conversion matrix set by the setting unit 13 is a conversion matrix (inverse matrix) for inverse conversion. The conversion matrix for inverse conversion is created based on data obtained by A / D converting the impulse response of the optical sensor unit 11. Since the impulse response of the optical sensor unit 11 is known, the conversion matrix for inverse conversion can be obtained in advance.

[0024] In the optical measurement device 6, if the type (characteristic) of the optical sensor unit 11 is different, the impulse response is naturally different. Furthermore, even if the type of the optical sensor unit 11 is the same, the impulse response is different due to individual differences. Therefore, the transformation matrix set in the calculation unit 14 by the setting unit 13 is obtained in advance according to the type and individual differences of the optical sensor unit 11 included in the optical measurement device 6. For example, by calculating the transformation matrix by the method described in Patent Document 1 based on the digital signal output from the A / D conversion unit 12, it is possible to obtain an optimal transformation matrix according to the type and individual differences of the optical sensor unit 11 included in the optical measurement device 6. The calculation unit 14 can correctly calculate the estimated pulse of the signal light by performing inverse transformation using such a transformation matrix.

[0025] According to this embodiment, the setting unit 13 sets in the calculation unit 14 an optimal transformation matrix according to the type and individual differences of the optical sensor unit 11 provided in the optical measurement device 6, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix.

[0026] This embodiment can be modified in various ways.

[0027] For example, as a first modification, in the scanning microscope 1 illustrated in FIG. 1, the optical measurement device 6 may further include an AFE unit and / or a digital filter unit. The AFE unit is provided between the optical sensor unit 11 and the A / D conversion unit 12, and includes, for example, an analog filter unit and an amplifier unit. The analog filter unit reduces noise in the detection signal output from the optical sensor unit 11. The amplifier unit adjusts the signal level of the output signal of the analog filter unit. The AFE unit is, for example, an AFE circuit. The digital filter unit is provided between the A / D conversion unit 12 and the calculation unit 14. The digital filter unit reduces noise in the digital signal output from the A / D conversion unit 12. The digital filter unit is, for example, a digital filter circuit.

[0028] In the first modification, the impulse response (digital signal input to the calculation unit 14 as the impulse response) differs depending on the type and individual difference of the AFE unit and / or the digital filter unit. Therefore, in the first modification, the transformation matrix set in the calculation unit 14 by the setting unit 13 is obtained in advance according to the types and individual differences of the optical sensor unit 11 and the AFE unit and / or the digital filter unit included in the optical measurement device 6. For example, when the optical measurement device 6 further includes an AFE unit and a digital filter unit, the transformation matrix is ​​calculated based on the digital signal output from the digital filter unit by the method described in Patent Document 1, so that the optimal transformation matrix according to the types and individual differences of the optical sensor unit 11, the AFE unit, and the digital filter unit included in the optical measurement device 6 can be obtained. The calculation unit 14 can correctly calculate the estimated pulse of the signal light by performing an inverse transformation using such a transformation matrix.

[0029] According to the first variant example, an optimal transformation matrix according to the types and individual differences of the optical sensor unit 11, AFE unit, and / or digital filter unit included in the optical measurement device 6 is set in the calculation unit 14 by the setting unit 13, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix.

[0030] As Modification 2, which is another modification, the scanning microscope 1 illustrated in Fig. 1 may be configured as illustrated in Fig. 2. Fig. 2 is a diagram illustrating the configuration of the scanning microscope 1 according to Modification 2. Fig. 3 is a diagram illustrating a plurality of transformation matrices.

[0031] As illustrated in FIG. 2, the optical measurement device 6 may further include a memory unit 15 that stores one or more transformation matrices, and the setting unit 13 may select the transformation matrix to be set in the calculation unit 14 from among the one or more transformation matrices stored in the memory unit 15.

[0032] The one or more transformation matrices stored in the storage unit 15 may be, for example, a transformation matrix for each optical sensor unit 11 (optical sensor units 11 with different types or individual differences) that can be included in the optical measurement device 6. In this way, for example, assuming a case where the optical sensor unit 11 is replaced, a plurality of transformation matrices that take into account the differences in type and individual differences can be stored in the storage unit 15.

[0033] The multiple transformation matrices stored in the storage unit 15 are, for example, multiple transformation matrices of m rows and n columns (transformation matrix A, transformation matrix B, and transformation matrix C) as illustrated in FIG.

[0034] According to variant example 2, even if the optical sensor unit 11 is replaced, the setting unit 13 can select an optimal transformation matrix according to the type and individual differences of the replaced optical sensor unit 11 from among the multiple transformation matrices stored in the memory unit 15 and set it in the calculation unit 14, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix.

[0035] In addition, in the second modification, the setting unit 13 may select the transformation matrix to be set in the calculation unit 14 from one or more transformation matrices stored in the storage unit 15 based on a selection input by the user. In this case, the selection input by the user may be performed, for example, by the user selecting a desired transformation matrix from one or more transformation matrices stored in the storage unit 15 via an input unit (not shown) included in the scanning microscope 1, or may be performed by the user selecting a desired optical sensor unit 11. In the latter case, the setting unit 13 may select a transformation matrix corresponding to the optical sensor unit 11 selected by the user from one or more transformation matrices stored in the storage unit 15. This allows the transformation matrix corresponding to the selection input by the user to be set in the calculation unit 14.

[0036] As another modified example, modified example 3, in the scanning microscope 1 illustrated in Fig. 2, the optical measurement device 6 may further include an AFE unit and / or a digital filter unit, similar to modified example 1. In this case, one or more transformation matrices stored in the storage unit 15 may be, for example, transformation matrices for each combination of the optical sensor unit 11, the AFE unit, and / or the digital filter unit (the optical sensor unit 11 having different types and individual differences, and the AFE unit and / or the digital filter unit having different types and individual differences) that the optical measurement device 6 may include. As a result, for example, assuming a case in which one or more of the optical sensor unit 11, the AFE unit, and / or the digital filter unit are replaced, a plurality of transformation matrices that take into account the differences in the types and individual differences can be stored in the storage unit 15.

[0037] According to variant example 3, even if one or more of the optical sensor unit 11 and the AFE unit and / or the digital filter unit are replaced, the setting unit 13 can select an optimal transformation matrix corresponding to the configuration after the replacement from among the multiple transformation matrices stored in the memory unit 15 and set it in the calculation unit 14, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix.

[0038] In addition, in the third modification, the setting unit 13 may select the transformation matrix to be set in the calculation unit 14 from one or more transformation matrices stored in the storage unit 15 based on the combination of the optical sensor unit 11 and the AFE unit and / or the digital filter unit specified by the user. The user may specify the combination of the optical sensor unit 11 and the AFE unit and / or the digital filter unit, for example, via an input unit (not shown) included in the scanning microscope 1. In this case, the setting unit 13 may select a transformation matrix corresponding to the combination specified by the user from one or more transformation matrices stored in the storage unit 15. This allows the calculation unit 14 to set an optimal transformation matrix according to the combination specified by the user.

[0039] Alternatively, the setting unit 13 may select the transformation matrix to be set in the calculation unit 14 from one or more transformation matrices stored in the storage unit 15 based on a selection input by the user. In this case, the selection input by the user may be performed, for example, by the user selecting a desired transformation matrix from one or more transformation matrices stored in the storage unit 15 via an input unit (not shown) included in the scanning microscope 1. This makes it possible to set in the calculation unit 14 a transformation matrix according to the selection input by the user.

[0040] As another modified example, Modified Example 4, the scanning microscope 1 illustrated in Fig. 1 may be configured as illustrated in Fig. 4. Fig. 4 is a diagram illustrating the configuration of the scanning microscope 1 according to Modified Example 4.

[0041] 4, the scanning microscope 1 may further include a plurality of optical elements 9 each having a different wavelength range for transmitting and reflecting fluorescence from the sample X, and the optical measurement device 6 may include a plurality of sets of optical sensor units 11 and A / D conversion units 12, and the setting unit 13 may set an optimal transformation matrix for each set in the calculation unit 14. Setting an optimal transformation matrix for each set means, for example, setting an optimal transformation matrix for each set according to the type and individual differences of the optical sensor units 11 included in the set. This allows the calculation unit 14 to correctly calculate an estimated pulse of the signal light incident on the optical sensor units 11 for each set.

[0042] In Fig. 4 (as well as Figs. 5 and 6 described later), in order to clarify the correspondence, the same alphabet as that added to the reference numeral of the optical element 9 that transmits or reflects the light incident on the optical sensor unit 11 is added to the reference numeral of the optical sensor unit 11 and the A / D conversion unit 12 of each set (for example, optical element 9a, optical sensor unit 11a, and A / D conversion unit 12a). Also, a set may be called a channel.

[0043] According to variant example 4, the setting unit 13 sets in the calculation unit 14 an optimal transformation matrix for each set according to the type and individual differences of the optical sensor units 11 included in the set, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix for each set.

[0044] As another modified example, in the scanning microscope 1 illustrated in FIG. 4, each set included in the optical measurement device 6 may further include an AFE unit and / or a digital filter unit, as in the modified example 1. More specifically, the optical measurement device 6 may include a plurality of sets of the optical sensor unit 11, the A / D conversion unit 12, and the AFE unit and / or the digital filter unit, and the setting unit 13 may set the optimal transformation matrix for each set in the calculation unit 14. Setting the optimal transformation matrix for each set means, for example, setting the optimal transformation matrix for each set according to the type and individual difference of the optical sensor unit 11 and the AFE unit and / or the digital filter unit included in the set. This allows the calculation unit 14 to correctly calculate the estimated pulse of the signal light incident on the optical sensor unit 11 for each set.

[0045] According to variant example 5, an optimal transformation matrix according to the types and individual differences of the optical sensor unit 11 and AFE unit and / or digital filter unit included in each set is set in the calculation unit 14 by the setting unit 13, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix for each set.

[0046] As a sixth modification, which is another modification, the scanning microscope 1 illustrated in Fig. 4 may be configured as illustrated in Fig. 5. Fig. 5 is a diagram illustrating the configuration of the scanning microscope 1 according to the sixth modification.

[0047] As illustrated in Fig. 5, the optical measurement device 6 may further include a storage unit 15 that stores a plurality of transformation matrices, and the setting unit 13 may select the transformation matrix to be set in the calculation unit 14 for each set from the plurality of transformation matrices stored in the storage unit 15. The plurality of transformation matrices stored in the storage unit 15 may be, for example, transformation matrices for each optical sensor unit 11 (optical sensor units 11 having different types and individual differences) that may be included in the set provided in the optical measurement device 6. This allows, for example, the storage unit 15 to store a plurality of transformation matrices that take into account the differences in type and individual differences, assuming a case in which the optical sensor unit 11 included in the set is replaced.

[0048] According to variant example 6, even if an optical sensor unit 11 included in a set is replaced, the setting unit 13 can select an optimal transformation matrix for that set according to the type and individual differences of the replaced optical sensor unit 11 from among the multiple transformation matrices stored in the memory unit 15 and set it in the calculation unit 14, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix.

[0049] As another modified example, modified example 7, in the scanning microscope 1 illustrated in FIG. 5, each set included in the optical measurement device 6 may further include an AFE unit and / or a digital filter unit, similar to modified example 5. In this case, the multiple transformation matrices stored in the storage unit 15 may be, for example, transformation matrices for each combination of the optical sensor unit 11, the AFE unit, and / or the digital filter unit (the optical sensor unit 11 having different types and individual differences, and the AFE unit and / or the digital filter unit having different types and individual differences) that may be included in the set included in the optical measurement device 6. As a result, for example, assuming a case in which one or more of the optical sensor unit 11, the AFE unit, and / or the digital filter unit included in the set are replaced, multiple transformation matrices that take into account the differences in the types and individual differences can be stored in the storage unit 15.

[0050] According to variant example 7, even if one or more of the optical sensor unit 11, the AFE unit, and / or the digital filter unit included in a set are replaced, the setting unit 13 can select an optimal transformation matrix for the set according to the post-replacement configuration from among the multiple transformation matrices stored in the memory unit 15 and set it in the calculation unit 14, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix.

[0051] As an eighth modification, which is another modification, the scanning microscope 1 illustrated in Fig. 5 may be configured as illustrated in Fig. 6. Fig. 6 is a diagram illustrating the configuration of the scanning microscope 1 according to the eighth modification.

[0052] 6, each set of optical sensor units 11 included in the optical measurement device 6 is configured to further output an identification signal of the optical sensor unit 11, and the setting unit 13 may select a transformation matrix to be set in the calculation unit 14 for each set from among a plurality of transformation matrices stored in the storage unit 15 based on the identification signal output from the optical sensor unit 11 of each set. In this case, each of the plurality of transformation matrices stored in the storage unit 15 is linked to the identification signal of the corresponding optical sensor unit 11, and becomes an optimal transformation matrix according to the type and individual differences of the optical sensor unit 11.

[0053] According to variant example 8, for each set, an optimal transformation matrix according to the type and individual differences of the optical sensor units 11 included in the set is set in the calculation unit 14 by the setting unit 13 based on the identification signal output from the optical sensor units 11 included in the set, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix for each set.

[0054] As another modified example, in the scanning microscope 1 illustrated in FIG. 6, each set included in the optical measurement device 6 may further include an AFE unit and / or a digital filter unit, as in the modified example 7. In this case, the multiple transformation matrices stored in the storage unit 15 may be, for example, transformation matrices for each combination of the optical sensor unit 11, the AFE unit, and / or the digital filter unit (the optical sensor unit 11 having different types and individual differences, and the AFE unit and / or the digital filter unit having different types and individual differences) that may be included in the set included in the optical measurement device 6. The setting unit 13 may select the transformation matrix to be set in the calculation unit 14 for each set from the multiple transformation matrices stored in the storage unit 15 based on the identification signal output from the optical sensor unit 11 of each set. In this case, each of the multiple transformation matrices stored in the storage unit 15 is linked to the identification signal of the corresponding optical sensor unit 11, and is an optimal transformation matrix according to the type and individual difference of the optical sensor unit 11 and the AFE unit and / or the digital filter unit included in the set including the optical sensor unit 11.

[0055] According to the ninth variant, for each set, an optimal transformation matrix according to the types and individual differences of the optical sensor unit 11 and the AFE unit and / or the digital filter unit included in the set is set in the calculation unit 14 by the setting unit 13 based on the identification signal output from the optical sensor unit 11 included in the set, so that the calculation unit 14 can perform inverse transformation using the optimal transformation matrix for each set.

[0056] As Modification 10, which is another modification, the scanning microscope 1 illustrated in Fig. 1 may be configured as illustrated in Fig. 7. Fig. 7 is a diagram illustrating the configuration of the scanning microscope 1 according to Modification 10. Fig. 8 is a diagram illustrating each signal in the optical measurement device 6.

[0057] As illustrated in FIG. 7, the optical measurement device 6 further includes a metering unit 16 that measures (photon counts) the magnitude of the estimated pulse (estimated pulse calculated by the arithmetic unit 14) output from the arithmetic unit 14 by threshold processing, and the image reconstruction unit 7 may generate an image having the number of detected photons detected by the optical sensor unit 11 instead of generating an image having the signal intensity of the detection signal of the optical sensor unit 11 corresponding to the amount of fluorescence detected at each pixel (each scanning position). In this case, the metering unit 16 can measure the magnitude of the estimated pulse by, for example, determining that the magnitude of the estimated pulse calculated by the arithmetic unit 14 is one photon of fluorescence when it is equal to or greater than a predetermined threshold and smaller than twice the predetermined threshold, and determining that the magnitude of the estimated pulse is two photons of fluorescence when it is equal to or greater than twice the predetermined threshold and smaller than three times the predetermined threshold. Specifically, the information on the magnitude of the estimated pulse can be measured by dividing the integral signal of the estimated pulse by the area of ​​the unit pulse. The image reconstruction unit 7 can generate an image showing the fluorescence intensity by arranging the information on the magnitude of the estimated pulse measured by the metering unit 16 in correspondence with each scanning position.

[0058] In the tenth modification, when pulsed signal light is input to the optical measurement device 6 (optical sensor section 11), each signal in the optical measurement device 6 becomes, for example, as shown in FIG.

[0059] 8, for example, when a pulsed signal light a is input to the optical sensor unit 11, the output of the optical sensor unit 11 becomes a detection signal b, the output of the A / D conversion unit 12 becomes a digital signal c, and the output of the calculation unit 14 becomes an estimated pulse d. Then, when the estimated pulse d is input to the measurement unit 16, the measurement unit 16 measures the magnitude of the estimated pulse d by threshold processing. For example, when the magnitude of the estimated pulse d is equal to or greater than a predetermined threshold value ("1.0") and smaller than twice the predetermined threshold value ("2.0"), it is determined that the magnitude of the estimated pulse d is one photon of fluorescence, and when the magnitude of the estimated pulse d is equal to or greater than twice the predetermined threshold value ("2.0") and smaller than three times the predetermined threshold value ("3.0"), it is determined that the magnitude of the estimated pulse d is two photons of fluorescence.

[0060] According to the tenth modification, the image reconstruction section 7 can generate an image having the number of detected photons detected by the optical sensor section 11.

[0061] In addition, in the scanning microscopes 1 relating to each of the modified examples 1 to 9, similar to modified example 10, the optical measurement device 6 may further include a measuring section 16, and the image reconstruction section 7 may generate an image having the number of detected photons detected by the optical sensor section 11.

[0062] 7, the measuring unit 16 may integrate the estimated pulse output from the calculation unit 14 and measure the integrated magnitude of the estimated pulse by threshold processing, instead of measuring the magnitude of the estimated pulse output from the calculation unit 14 by threshold processing. For example, the estimated pulse output from the calculation unit 14 may be integrated over a predetermined time, and the number of photons within the integration time may be totaled and measured.

[0063] In the eleventh modification, when a pulsed signal light is input to the optical measurement device 6 (optical sensor unit 11), each signal in the optical measurement device 6 is, for example, as shown in Fig. 9. Fig. 9 is a diagram illustrating each signal in the optical measurement device 6.

[0064] 9, for example, when a pulsed signal light a is input to the optical sensor unit 11, the output of the optical sensor unit 11 becomes like the detection signal b, the output of the A / D conversion unit 12 becomes like the digital signal c, and the output of the calculation unit 14 becomes like the estimated pulse d, as in the example shown in FIG. 8. Then, when the estimated pulse d is input to the measurement unit 16, the measurement unit 16 integrates the estimated pulse d and measures the magnitude of the estimated pulse e, which is the integration result, by threshold processing. For example, when the magnitude of the estimated pulse e is equal to or greater than a predetermined threshold value ("1.0") and smaller than twice the predetermined threshold value ("2.0"), it is determined that the estimated pulse e is one photon of fluorescence, and when the magnitude of the estimated pulse e is equal to or greater than twice the predetermined threshold value ("2.0") and smaller than three times the predetermined threshold value ("3.0"), it is determined that the estimated pulse e is two photons of fluorescence, and the magnitude of the estimated pulse e is measured.

[0065] According to the eleventh modification, it is possible to obtain a high degree of quantitativeness in the measurement of the number of photons, reduce the influence of noise, and perform measurement with high accuracy. The integration time may correspond to the exposure time.

[0066] In the eleventh modification, when integrating the estimated pulse output from the calculation unit 14, the measurement unit 16 may integrate for the same time period as the time period corresponding to the number of multiplexes of the multidiagonal matrix, which is the transformation matrix set in the calculation unit 14. This makes it possible to obtain high quantitativeness in addition to high time resolution.

[0067] In addition, in the eleventh modification, when integrating the estimated pulse output from the calculation unit 14, the measurement unit 16 may detect the rising and falling edges of the estimated pulse by threshold processing to determine a detection region, and integrate the estimated pulse in the determined detection region. This dynamically changes the integration timing and time, suppresses the detection signal from crossing the exposure time, and provides higher measurement performance.

[0068] In addition, in the scanning microscopes 1 relating to each of the modified examples 1 to 9, similar to modified example 11, the optical measurement device 6 may further include a measuring unit 16, and the measuring unit 16 may integrate the estimated pulse output from the calculation unit 14 and measure the magnitude of the integrated estimated pulse by threshold processing.

[0069] As Modification 12, which is another modification, the scanning microscope 1 illustrated in Fig. 7 may be configured as illustrated in Fig. 10. Fig. 10 is a diagram illustrating the configuration of the scanning microscope 1 according to Modification 12. Fig. 11 is a diagram illustrating each signal in the optical measurement device 6.

[0070] 10, the optical measurement device 6 may further include a gain adjustment unit 17 that adjusts the gain of a signal between the A / D conversion unit 12 and the calculation unit 14. The gain adjustment unit 17 is, for example, a gain adjustment circuit.

[0071] The gain adjustment unit 17 adjusts the gain of the digital signal output from the A / D conversion unit 12 so that the magnitude of the estimated pulse (estimated pulse calculated by the calculation unit 14) output from the calculation unit 14 is normalized to a constant value. In detail, when any of a plurality of optical sensor units 11 having different types or individual differences is provided in the optical measurement device 6, the gain adjustment unit 17 adjusts the gain of the digital signal so that the magnitude of the estimated pulse output from the calculation unit 14 becomes the same (i.e., normalized to a constant value) if the optical signal input to the optical sensor unit 11 is the same, regardless of which optical sensor unit 11 is provided in the optical measurement device 6.

[0072] For example, when two optical sensor units 11 (say, optical sensor unit A and optical sensor unit B) having different types or individual differences are provided in an optical measurement device 6, the signals in the optical measurement device 6 when each optical sensor unit 11 is provided will be, for example, as shown in FIG. 11.

[0073] 11, for example, when a pulsed signal light a is input to the optical sensor unit 11, if the optical sensor unit 11 is the optical sensor unit A, the detection signal b which is the output of the optical sensor unit A, the digital signal c which is the output of the A / D conversion unit 12, the estimated pulse d which is the output of the calculation unit 14 when the gain adjustment unit 17 is not present, and the estimated pulse g which is the output of the calculation unit 14 when the gain adjustment unit 17 is present (standardized case) are as shown in the left side of Fig. 11. On the other hand, if the optical sensor unit 11 is the optical sensor unit B, the detection signal b which is the output of the optical sensor unit B, the digital signal c which is the output of the A / D conversion unit 12, the estimated pulse d which is the output of the calculation unit 14 when the gain adjustment unit 17 is not present, and the estimated pulse g which is the output of the calculation unit 14 when the gain adjustment unit 17 is present (standardized case) are as shown in the right side of Fig. 11. In this way, even if the optical sensor unit 11 provided in the optical measurement device 6 is different, if the optical signal input to the optical sensor unit 11 is the same, the estimated pulse g output from the calculation unit 14 will be the same due to normalization by gain adjustment by the gain adjustment unit 17.

[0074] According to the modification 12, even if the optical sensor unit 11 provided in the optical measurement device 6 is different, if the optical signal input to the optical sensor unit 11 is the same, the estimated pulse output from the calculation unit 14 will be the same. Therefore, it is not necessary to change the threshold value used by the weighing unit 16 when performing threshold processing for each optical sensor unit 11 provided in the optical measurement device 6, and the weighing unit 16 needs to have only one threshold value to use for threshold processing.

[0075] In addition, in the modification 12, the gain adjustment unit 17 included in the optical measurement device 6 may be provided between the optical sensor unit 11 and the A / D conversion unit 12. In this case, the gain adjustment unit 17 adjusts the gain of the detection signal output from the optical sensor unit 11 so that the magnitude of the estimated pulse output from the calculation unit 14 is normalized to a constant value.

[0076] As another modification, modification 13, in modification 11, the optical measurement device 6 may further include a gain adjustment unit 17 between the A / D conversion unit 12 and the calculation unit 14, as in modification 12. In this case, the gain adjustment unit 17 adjusts the gain of the digital signal output from the A / D conversion unit 12 so that the estimated pulse (estimated pulse calculated by the calculation unit 14) output from the calculation unit 14 is integrated by the measurement unit 16, and the magnitude of the integrated estimated pulse is normalized to a constant value. In detail, when any of a plurality of optical sensor units 11 having different types and individual differences is provided in the optical measurement device 6, even if any of the optical sensor units 11 is provided in the optical measurement device 6, if the optical signal input to the optical sensor unit 11 is the same, the estimated pulse output from the calculation unit 14 is integrated, and the gain adjustment unit 17 adjusts the gain of the digital signal so that the magnitude of the integrated estimated pulse is the same (i.e., normalized to a constant value).

[0077] According to variant 13, as in variant 12, even if the optical sensor unit 11 provided in the optical measurement device 6 is different, as long as the optical signal input to the optical sensor unit 11 is the same, the estimated pulse output from the calculation unit 14 is integrated by the measuring unit 16 and the magnitude of the integrated estimated pulse becomes the same. Therefore, it is not necessary to change the threshold value used by the measuring unit 16 when performing threshold processing for each optical sensor unit 11 provided in the optical measurement device 6, and the measuring unit 16 is required to have only one threshold value to use for threshold processing.

[0078] In addition, in the modification 13, the gain adjustment unit 17 included in the optical measurement device 6 may be provided between the optical sensor unit 11 and the A / D conversion unit 12. In this case, the gain adjustment unit 17 adjusts the gain of the detection signal output from the optical sensor unit 11 so that the estimated pulse output from the calculation unit 14 is integrated by the measurement unit 16 and the magnitude of the integrated estimated pulse is normalized to a constant value.

[0079] In the above-described embodiment and modified examples, the scanning microscope 1 may include a light source section that generates continuous laser light (for example, a CW (Continuous wave) laser) instead of the light source section 2 that generates extremely short pulsed laser light.

[0080] Furthermore, the scanning microscope 1 performs imaging of the fluorescence intensity, but may also perform imaging of the fluorescence lifetime.

[0081] A part of the scanning microscope 1 may be realized by a computer 100 illustrated in Fig. 12. Fig. 12 is a diagram illustrating an example of the hardware configuration of the computer 100.

[0082] As illustrated in FIG. 12, a computer 100 includes a processor 101, a memory 102, an input device 103, an output device 104, a storage device 105, a portable storage medium drive device 106, a communication interface 107, and an input / output interface 108, each of which is connected to a bus 109 and capable of transmitting and receiving data to and from each other.

[0083] The processor 101 may be, for example, a single processor, or a multi-processor or multi-core. The processor 101 executes programs such as an OS (Operating System) and applications to perform various processes. The memory 102 includes a RAM (Random Access Memory) and a ROM (Read Only Memory). The RAM temporarily stores a part of the program executed by the processor 101. The RAM is also used as a working memory area for the processor 101. The ROM stores the programs executed by the processor 101 and various data required for executing the programs.

[0084] The input device 103 is a keyboard, a mouse, a joystick, a touch panel, etc. The output device 104 is a display device such as a liquid crystal display, a printer, etc.

[0085] The storage device 105 is a device for storing data, and is an HDD (Hard Disk Drive), an SSD (Solid State Drive), etc. The portable storage medium driving device 106 drives a portable storage medium 106a, and accesses the stored contents thereof to read and write data. The portable storage medium 106a is a memory device, a flexible disk, an optical disk, a magneto-optical disk, etc. This portable storage medium 106a also includes a CD-ROM (Compact Disc Read Only Memory), a DVD (Digital Versatile Disc), a Blu-ray Disc, a USB (Universal Serial Bus) memory, an SD card memory, etc.

[0086] The communication interface 107 is connected to a network by wire or wirelessly, and is an interface for communicating with an external device connected to the network. The input / output interface 108 is connected to an external device, and is an interface for inputting and outputting data to and from the external device.

[0087] In such a computer 100, the programs executed by the processor 101 and various data required for executing the programs may be stored in the storage device 105 or the portable storage medium 106a, not limited to the memory 102. Furthermore, the programs executed by the processor 101 and various data required for executing the programs may be stored in the storage device 105 or the portable storage medium 106a from an external device via a network or a communication interface 107.

[0088] Furthermore, computer 100 is not limited to the example shown in FIG. 12, and may be configured to include multiple components that are part of the example shown in FIG. 12, or may be configured to omit some of the components.

[0089] Furthermore, the computer 100 may be configured to include hardware such as a microprocessor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a programmable logic device (PLD), a field-programmable gate array (FPGA), etc. For example, the processor 101 may be implemented using at least one of these pieces of hardware.

[0090] 12 , the A / D conversion unit 12, the digital filter unit, or the gain adjustment unit 17 are connected to the input / output interface 108, and the output signal of the A / D conversion unit 12, the digital filter unit, or the gain adjustment unit 17 is input to the input / output interface 108. In addition, the functions of the setting unit 13, the calculation unit 14, the measurement unit 16, and the image reconstruction unit 7 are realized by the processor 101, the function of the storage unit 15 is realized by one or more of the memory 102, the storage device 105, and the portable storage medium 106a, the function of the display unit 8 is realized by the output device 104, and the function of the input unit (not shown) included in the scanning microscope 1 is realized by the input device 103.

[0091] Although the embodiments of the present invention (including modified examples) have been described above, the present invention is not limited to the above-described embodiments, and various improvements and modifications can be made without departing from the gist of the present invention. [Explanation of symbols]

[0092] 1. Scanning microscope 2 Light source section 3. Scanner 4 Dichroic mirror 5 Optical system 6. Optical measurement equipment 7 Image reconstruction unit 8 Display 9 Optical Elements 11 Optical sensor section 12 A / D conversion section 13 Setting section 14 Arithmetic section 15 Storage section 16 Measuring section 17 Gain adjustment section 100 Computers 101 Processor 102 Memory 103 Input Device 104 Output Device 105 Storage device 106 Portable storage medium drive device 106a Portable storage media 107 Communication Interface 108 Input / Output Interface 109 Bus X Sample

Claims

1. an optical sensor unit that detects a pulsed signal light and outputs a detection signal having an exponential response; an A / D converter for converting the detection signal into a digital signal; A setting unit that sets a transformation matrix for inversely transforming the digital signal; a calculation unit that calculates an estimated pulse of the signal light by inversely transforming the digital signal using the transformation matrix set by the setting unit; An optical measurement device comprising:

2. A storage unit for storing one or more transformation matrices; Further comprising: The setting unit selects the transformation matrix to be set from the one or more transformation matrices stored in the storage unit.

2. The optical measurement device according to claim 1 .

3. The setting unit selects the transformation matrix to be set from the one or more transformation matrices stored in the storage unit based on a selection input by a user.

3. The optical measurement device according to claim 2.

4. a plurality of sets each including the optical sensor unit and the A / D conversion unit; The setting unit sets the transformation matrix for each of the sets.

2. The optical measurement device according to claim 1 .

5. a storage unit for storing a plurality of transformation matrices; Further comprising: The setting unit selects the transformation matrix to be set for each set from among the plurality of transformation matrices stored in the storage unit.

5. The optical measurement device according to claim 4.

6. The optical sensor unit further outputs an identification signal of the optical sensor unit, the setting unit selects the transformation matrix to be set for each set from among the plurality of transformation matrices stored in the storage unit based on the identification signal.

6. The optical measurement device according to claim 5.

7. a measuring unit that measures the magnitude of the estimated pulse calculated by the calculation unit through threshold processing; 2. The optical measurement device according to claim 1, further comprising:

8. a measuring unit that integrates the estimated pulse calculated by the calculation unit and measures the magnitude of the integrated estimated pulse by threshold processing; 2. The optical measurement device according to claim 1, further comprising:

9. a gain adjustment unit that adjusts a gain of the detection signal or the digital signal so that the magnitude of the estimated pulse calculated by the calculation unit is normalized to a constant value; 8. The optical measurement device according to claim 7, further comprising:

10. a gain adjustment unit that integrates the estimated pulse calculated by the calculation unit and adjusts a gain of the detection signal or the digital signal so that the magnitude of the integrated estimated pulse is normalized to a constant value; 9. The optical measurement device according to claim 8, further comprising:

11. The optical sensor unit is a semiconductor optical sensor having a plurality of avalanche photodiodes operating in Geiger mode arranged therein.

11. The optical measurement device according to claim 1 , wherein the optical measurement device is a casing.

12. a scanning unit that scans the sample with illumination light emitted from the light source; an optical system that focuses the illumination light scanned by the scanning unit on the sample and focuses signal light generated at each scanning position on the sample of the illumination light; an optical measurement device according to claim 1 , which calculates an estimated pulse of the signal light collected by the optical system; an image reconstruction unit that reconstructs an image of the sample based on the estimated pulse calculated by the optical measurement device; A scanning microscope comprising:

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