JP4065249B2 - Fluorescence lifetime measuring device - Google Patents

Description

  The present invention relates to a fluorescence lifetime measuring apparatus that counts fluorescence photons emitted from a sample excited by irradiating a sample with pulse excitation light and measures the fluorescence lifetime based on the counted number of fluorescence photons.

  Conventionally, there is a known method of irradiating a sample with excitation light to bring the sample into an excited state, measuring fluorescence photons emitted during the transition of the sample to the ground state, calculating the fluorescence lifetime, and examining the excited state of the sample. Yes. As a method for calculating the fluorescence lifetime, there is a time gate method in which a sample is irradiated with pulsed excitation light, the number of fluorescence photons emitted from the sample is measured in a plurality of time zones, and the fluorescence lifetime is calculated from the measured number of fluorescence photons. It is known (see Non-Patent Document 1).

"Multiple Time-Gate Module for Fluorescence Lifetime Imaging" C.J.DE GRAUW and H.C.GERRITSEN, APPLIED SPECTROSCOPY, Volume 55, Number 6, 2001.

  By the way, when acquiring a fluorescence lifetime distribution image, a scanning fluorescence microscope apparatus is often used, and the speed at which excitation light scans a sample is constant. In order to measure the fluorescence lifetime with a small error, it is necessary to count a predetermined number of fluorescent photons. Since the emission probability of fluorescent photons increases and decreases in accordance with the irradiation intensity of excitation light, it is common to match the irradiation intensity of excitation light with reference to a site with a low emission probability of fluorescent photons. That is, the excitation light irradiation time is increased or the number of times of excitation light irradiation is increased for a portion where the emission probability of the fluorescent photon is small. However, if a part having a low emission probability of fluorescent photons is used as a reference, the irradiation time is increased or the number of irradiations is increased to scan the excitation light, resulting in obtaining a fluorescence lifetime distribution image. There is a problem that it takes a lot of time.

  In addition, when the excitation light is irradiated on the basis of the part where the emission probability of the fluorescence photon is low, the part where the emission probability of the fluorescence photon is high is too strong and the discoloration proceeds and the sample is damaged. There was a problem of doing.

  The present invention has been made in view of the above, and an object of the present invention is to provide a fluorescence lifetime measuring apparatus that can acquire a fluorescence lifetime distribution image with a uniform error in a short time with a simple configuration.

  In order to achieve the above object, a fluorescence lifetime measuring apparatus according to claim 1 scans a sample while irradiating it with pulsed excitation light, and counts the fluorescence photons emitted from the sample excited by the pulsed excitation light. In the fluorescence lifetime measuring apparatus that measures the fluorescence lifetime based on the counted number of fluorescent photons, every time the counted number of fluorescent photons reaches a predetermined number, the irradiation position of the pulse excitation light is changed to the next irradiation position. A scanning control means for scanning is provided.

  According to a second aspect of the present invention, in the fluorescence lifetime measuring apparatus according to the present invention, the irradiation position corresponds to a predetermined region in one pixel.

  Further, the fluorescence lifetime measuring apparatus according to claim 3 is the above invention, the sample is scanned while irradiating the sample with pulse excitation light, and the fluorescence photons emitted from the sample excited by the pulse excitation light are counted, In the fluorescence lifetime measuring apparatus for measuring the fluorescence lifetime based on the counted number of fluorescence photons, the fluorescence lifetime measurement region of the sample is scanned in advance with excitation light of a predetermined light intensity, and the fluorescence intensity distribution in the fluorescence lifetime measurement region Fluorescence intensity measuring means for measuring the excitation light intensity corresponding to the intensity of the excitation light so that the number of fluorescent photons emitted from each predetermined area in the fluorescence lifetime measurement area exceeds a predetermined number based on the fluorescence intensity distribution A scanning speed calculation means for calculating the scanning speed of the excitation light, and the pulse excitation light scans each predetermined area according to the scanning speed calculated by the scanning speed calculation means. And scanning control means for controlling, characterized by comprising a.

  According to a fourth aspect of the present invention, there is provided a fluorescence lifetime measuring apparatus according to the above invention, wherein a time correlation unit for calculating the fluorescence lifetime based on a time from when the pulsed excitation light is irradiated until the fluorescence photons are emitted. A calculation means for calculating the fluorescence lifetime using one counting method is provided.

  According to a fifth aspect of the present invention, there is provided the fluorescence lifetime measuring apparatus according to the above invention, wherein the fluorescence lifetime is calculated based on the number of fluorescence photons counted in a plurality of time zones after the pulse excitation light is irradiated. An arithmetic means for calculating the fluorescence lifetime using a gate method is provided.

  The fluorescence lifetime measuring apparatus according to the present invention has an effect that the irradiation intensity of the excitation light is changed in accordance with the emission probability of the fluorescence photons, and a uniform error fluorescence lifetime distribution image can be acquired in a short time.

  Exemplary embodiments of a fluorescence lifetime measuring apparatus according to the present invention will be described below in detail with reference to the accompanying drawings.

(Embodiment 1)
FIG. 1 is a block diagram showing a schematic configuration of a fluorescence lifetime measuring apparatus 100 according to Embodiment 1 of the present invention. In FIG. 1, this fluorescence lifetime measuring apparatus 100 includes a pulse laser light source 1, lens systems 2, 5, and 7, a beam splitter 3, a scanner 4, a detector 8, a control unit 9, and a display unit 10. have. Further, the control unit 9 includes a signal processing unit 91, a counter 92, a scanner control unit 93, and a calculation unit 94.

  First, under the control of the control unit 9, pulse excitation light is emitted from the pulse laser light source 1 at a predetermined light intensity and at a predetermined time interval. The pulse excitation light is condensed on the sample 6 through the lens system 2, the beam splitter 3, the scanner 4, and the lens system 5 in this order. The condensed pulsed excitation light excites the sample 6, and fluorescent photons are emitted from the sample 6. The fluorescent photons emitted from the sample 6 are reflected by the beam splitter 3 through the lens system 5 and the scanner 4 in order, and enter the detector 8 through the lens system 7. The detector 8 outputs an electrical signal S1 corresponding to the light intensity of the incident fluorescent photon to the control unit 9.

  The signal processing unit 91 in the control unit 9 receives the electric signal S <b> 1, binarizes it with a predetermined threshold value, and outputs a binarized signal to the counter 92. The counter 92 inputs a binarized signal, counts the fluorescence photons, and when the counted number of fluorescent photons reaches a preset number of photons, the counted number of fluorescent photons reaches the preset number of fluorescent photons. The effect is output to the scanner control unit 93 and the calculation unit 94. When the scanner controller 93 inputs that the counted number of fluorescent photons has reached the preset number of photons, the scanner control unit 93 variably controls the scanner 4 to scan the irradiation position of the pulse excitation light to the next irradiation position. When the calculation unit 94 inputs that the accumulated number of fluorescent photons has reached a preset number of photons, the calculation unit 94 calculates the fluorescence lifetime based on the measured number of fluorescent photons, and outputs it to the display unit 10. The controller 9 outputs and displays a fluorescence lifetime distribution image via the display unit 10 when the fluorescence lifetime for one screen is calculated. The calculation unit 94 is a single correlation time counting method that calculates the fluorescence lifetime based on the time required for emitting the number of fluorescent photons after irradiation with pulsed excitation light, or fluorescence emitted by irradiation with pulsed excitation light. A photon is measured in a plurality of time zones, and the fluorescence lifetime is calculated using any one of the time gate methods for calculating the fluorescence lifetime from the time width of the plurality of time zones and the time difference between the measurement times.

  Next, the operation of the control unit 9 and each part in the control unit 9 will be described with reference to the flowchart shown in FIG. First, the scanner control unit 93 sets the scanning position of the pulse excitation light to the initial scanning position (step S101), and the control unit 9 resets the counter 92 (step S102) to oscillate the pulse excitation light (step S102). S103). The counter 92 starts counting fluorescent photons (step S104), and determines whether or not the counted number of fluorescent photons has reached a preset number of fluorescent photons (step S105). When the counted number of fluorescent photons has reached the preset number of fluorescent photons (step S105, Yes), the counter 92 outputs to the calculation unit 94 that the counted number of fluorescent photons has reached the preset number of fluorescent photons. . When the counted number of fluorescent photons has not reached the preset number of fluorescent photons (step S105, No), the control unit 9 oscillates the pulse excitation light again (step S103). When the calculation unit 94 inputs that the counted number of fluorescent photons has reached a preset number of fluorescent photons, the calculating unit 94 calculates the fluorescence lifetime based on the measured number of fluorescent photons (step S106). Next, the control unit 9 determines whether or not the fluorescence lifetime calculated by the calculation unit 94 has been completed for one screen (step S107), and when the display has been completed for one screen (step S107, Yes), the display unit 10 is changed. The distribution image of the fluorescence lifetime is output and displayed (step S108), and if one screen is not completed (step S107, No), the irradiation position of the pulse excitation light is set to the next irradiation position via the scanner controller 93. (Step S109), and the counter 92 is reset (step S102).

  FIG. 3 is a schematic diagram illustrating how the scanner 4 is variably controlled by the scanner control unit 93 and the irradiation position of the pulse excitation light is scanned with time. As shown in FIG. 3, the irradiation position of the pulse excitation light is scanned in units of one pixel, but the time that remains at the irradiation position corresponding to one pixel is not constant. For example, when the emission probability of the fluorescent photons of the pixels corresponding to the irradiation positions P0 to P1 is large and the emission probability of the fluorescent photons of the pixels corresponding to the irradiation positions P1 to P2 is small, the time Δt1 is made longer than the time Δt2. . In this way, for pixels corresponding to the irradiation positions P0 to P1 having a low emission probability of fluorescent photons, the light intensity is increased by increasing the time during which the excitation light stays, and the irradiation positions having a high emission probability of fluorescent photons. For the pixels corresponding to P1 and P2, the time during which the excitation light stays is shortened. Thus, if the time during which excitation light stays is varied in accordance with the emission probability of fluorescent photons, the number of fluorescent photons emitted from the pixels corresponding to all irradiation positions becomes the same.

  In general, the error in fluorescence lifetime is small when the number of counted fluorescence photons is large, and is large when the number of counted fluorescence photons is small. Therefore, if the number of fluorescent photons counted per pixel is set in advance and the number of fluorescent photons counted in each pixel matches the preset number of fluorescent photons, the distribution image of the fluorescence lifetime with uniform error can be obtained. You can get it.

  In the first embodiment, the number of fluorescent photons counted from the excitation light irradiation position corresponding to one pixel is set in advance, and the next pixel is reached when the counted number of fluorescent photons reaches the set number of fluorescent photons. The irradiation position is scanned at a position corresponding to. Therefore, a fluorescence lifetime distribution screen with uniform error can be obtained.

  In the first embodiment, the irradiation position of the pulse excitation light is fixed at one place so as to correspond to one pixel. However, the irradiation position of the pulse excitation light is within an area corresponding to one pixel. May be moved.

  In the first embodiment, the irradiation position of the pulse excitation light is scanned after the calculation of the fluorescence lifetime of the region corresponding to one pixel. However, the pulse excitation light is scanned first. After measuring the number of fluorescent photons for the screen, the fluorescence lifetime may be calculated collectively.

(Embodiment 2)
Next, the second embodiment will be described. In the first embodiment, the number of fluorescent photons counted from the irradiation position corresponding to one pixel is set in advance, and the error in the fluorescence lifetime of each pixel is made uniform. Second, scan the pulsed excitation light at a predetermined speed to obtain a fluorescence intensity distribution image and calculate the scanning speed at which the number of fluorescent photons counted based on the fluorescent intensity is set, The second time, the pulse excitation light is scanned according to the calculated scanning speed, and a distribution image of the fluorescence lifetime with uniform error is acquired.

  FIG. 4 is a block diagram showing a schematic configuration of a fluorescence lifetime measuring apparatus 200 according to Embodiment 2 of the present invention. In FIG. 4, the fluorescence lifetime measuring apparatus 200 includes a control unit 9A instead of the control unit 9, and the control unit 9A includes a signal processing unit 91, a counter 92, a scanner control unit 93a, and a calculation unit 94. The scanning speed calculation unit 95 and the storage unit 96 are provided. In addition, the same code | symbol is attached | subjected to the component same as FIG.

  First, under the control of the control unit 9A, pulse excitation light is emitted from the pulse laser light source 1, and the pulse excitation light is scanned under the control of the scanner control unit 93a. The scanner control unit 93a scans the pulse excitation light at a predetermined scanning speed. The detector 8 outputs an electric signal S2 corresponding to the fluorescence intensity of the fluorescence emitted from the sample 6 to the control unit 9A by scanning with the pulse excitation light. The control unit 9A outputs the input electric signal S2 to the scanning speed calculation unit 95 and the display unit 10. The display unit 10 converts the input electrical signal S2 into a display signal, and outputs and displays a fluorescence intensity distribution image.

  The scanning speed calculation unit 95 calculates the scanning speed of the pulse excitation light so that the number of fluorescent photons counted based on the input electrical signal S2 becomes a preset number of fluorescent photons, and outputs it to the storage unit 96. . The storage unit 96 stores the input scanning speed.

  Next, pulsed excitation light is emitted from the pulsed laser light source 1 under the control of the control unit 9A, and the pulsed excitation light is scanned under the control of the scanner control unit 93a. The scanner control unit 93 a scans the pulse excitation light according to the scanning speed stored in the storage unit 96. By the scanning of the pulse excitation light, the number of fluorescent photons is sequentially counted through the signal processing unit 91 and the counter 92, and the counted number of fluorescent photons is output to the calculation unit 94. The calculation unit 94 calculates the fluorescence lifetime based on the number of input fluorescence photons, and outputs the calculation result to the display unit 10. The display unit 10 accumulates the fluorescence lifetime input from the calculation unit 94, and outputs and displays a fluorescence lifetime distribution image when the fluorescence lifetime for one screen is accumulated.

  With reference to the flowchart shown in FIG. 5, the operation of the control unit 9A and each part in the control unit 9A will be described. First, the control unit 9A oscillates pulse excitation light (step S201), the scanner control unit 93a scans the pulse excitation light at a predetermined scanning speed (step S202), and the control unit 9A passes through the detector 8. Then, the electric signal S2 corresponding to the fluorescence intensity is input (step S203), and the electric signal S2 is output to the scanning speed calculation unit 95 and the display unit 10. The scanning speed calculation unit 95 calculates a scanning speed for scanning for each pixel based on the electrical signal S2 corresponding to the fluorescence intensity (step S204), and outputs the calculation result to the storage unit 96. The display unit 10 outputs and displays a fluorescence intensity distribution image based on the electrical signal S2 corresponding to the fluorescence intensity (step S205). The controller 9A proceeds to the measurement of the fluorescence lifetime when the scanning for one screen is completed.

  Next, measurement of fluorescence lifetime will be described. First, the scanner controller 95 sets the scanning position of the pulse excitation light to the initial scanning position (step S206), and the controller 9A resets the counter 92 (step S207) to oscillate the pulse excitation light (step S207). S208). The counter 92 starts counting fluorescent photons via the signal processing unit 91 (step S209), and outputs the counted number of fluorescent photons to the arithmetic unit 94. The scanner control unit 95 scans the pulse excitation light according to the scanning speed stored in the storage unit 96 (step S210), determines whether one pixel is scanned (step S211), and scans one pixel. Is completed (step S211, Yes), the calculation unit 94 calculates the fluorescence lifetime based on the input number of fluorescent photons (step S212). When scanning for one pixel is not completed (step S211, No), the control unit 9A oscillates pulse excitation light (step S208). Next, the control unit 9A determines whether or not the calculation of the fluorescence lifetime has been completed for one screen (step S213), and when the calculation has been completed for one screen (step S213, Yes), the fluorescence lifetime is displayed via the display unit 10. When the calculation of the fluorescence lifetime has not been completed for one screen (step S213, No), the pulse excitation light is sent to the irradiation position of the next pixel via the scanner control unit 95. Scanning is performed (step S215), and the counter 92 is reset (step S207).

  6A to 6D are schematic diagrams illustrating the relationship among excitation light intensity, fluorescence intensity, and scanning speed of excitation light. As illustrated in FIG. 6A, the scanner control unit 93a scans the excitation light at a constant scanning speed for the first time. That is, since the scanning speed is constant, the excitation light intensity L1 is constant. As shown in FIG. 6B, the fluorescence intensity La is acquired through the detector 8 by this scanning. On the other hand, the predetermined fluorescence intensity Lt required for measuring the fluorescence lifetime is constant. As shown in FIG. 6-3, the excitation light intensity L2 necessary for measuring the fluorescence lifetime in each pixel is proportional to the value obtained by dividing the fluorescence intensity Lt by the fluorescence intensity La (L2∝Lt / La). The scanning time T necessary for measuring the fluorescence lifetime in each pixel is proportional to L2, and the scanning speed SP of the excitation light is inversely proportional to T. The scanning speed calculation unit 95 calculates the scanning time T or the scanning speed SP from the fluorescence intensity La (T∝La / Lt, SP∝Lt / La). As shown in FIG. 6-4, the scanning speed SP is fast where the fluorescent light intensity La is large, and the scanning speed SP is slow where the fluorescent light intensity La is small. In addition, the scanning speed calculation unit 95 arbitrarily changes the measured fluorescence lifetime error by changing the proportional coefficient C of the relational expression (SP = C (Lt / La)) between the scanning speed SP and the fluorescence intensity La. Can be set.

  FIG. 7 is a schematic diagram showing that the pulse excitation light is scanned while changing the scanning speed for each pixel. In FIG. 7, the scanning speed of the pulse excitation light is represented by the slope of a straight line, and this scanning speed follows the speed calculated by the scanning speed calculation unit 95. Therefore, the scanning speed of the pulse excitation light is high in the pixel area where the emission probability of the fluorescent photon is high, and the scanning speed of the pulse excitation light is low in the pixel area where the emission probability of the fluorescence photon is small.

  In the second embodiment, the first time is scanned with pulse excitation light at a constant scanning speed to display the fluorescence intensity and the scanning speed for scanning each pixel is calculated. The second time is the calculated scanning speed. Therefore, the fluorescence lifetime is calculated by scanning the pulse excitation light. In this way, a fluorescence intensity distribution image is acquired by the first scan, and a uniform error fluorescence lifetime distribution image is acquired by the second scan.

  In the second embodiment, the fluorescence intensity distribution image obtained by the first scan of the pulse excitation light is displayed. However, the display may be selectable. Further, the time during which the pulse excitation light stays at the irradiation position may be determined by changing the number of irradiation times of the pulse excitation light, or may be determined by changing the irradiation time of the pulse excitation light. This is because the irradiation intensity is changed in any case.

  In the second embodiment, the pulse excitation light is used both when acquiring the fluorescence intensity distribution image and when acquiring the fluorescence lifetime distribution image. However, the fluorescence intensity distribution image is acquired. In this case, a continuous oscillation light source may be used instead of a pulse oscillation light source.

It is a functional block diagram which shows the fluorescence lifetime measuring apparatus concerning Embodiment 1 of this invention. It is a flowchart which shows operation | movement of the control part concerning Embodiment 1 of this invention. It is a schematic diagram which shows the relationship between the irradiation position of the pulse excitation light concerning Embodiment 1 of this invention, and time. It is a functional block diagram which shows the fluorescence lifetime measuring apparatus concerning Embodiment 2 of this invention. It is a flowchart which shows operation | movement of the control part concerning Embodiment 2 of this invention. It is a schematic diagram which shows the relationship between the light intensity of the excitation light concerning Embodiment 2 of this invention, and a scanning position. It is a schematic diagram which shows the relationship between the fluorescence intensity concerning Embodiment 2 of this invention, and predetermined | prescribed fluorescence intensity. It is a schematic diagram which shows the relationship between the light intensity of the excitation light concerning Embodiment 2 of this invention, and a scanning position. It is a schematic diagram which shows the relationship between the scanning speed of the excitation light concerning Embodiment 2 of this invention, and a scanning position. It is a schematic diagram which shows the relationship between the irradiation position of the pulse excitation light concerning Embodiment 2 of this invention, and time.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Pulse laser light source 2, 5, 7 Lens system 3 Beam splitter 4 Scanner 6 Sample 8 Detector 9, 9A Control part 91 Signal processing part 92 Counter 93, 93a Scanner control part 94 Calculation part 95 Scan speed calculation part 96 Storage part 100 , 200 Fluorescence lifetime measuring device

Claims (5)

Fluorescence lifetime measurement that scans a sample while irradiating it with pulse excitation light, counts the fluorescence photons emitted from the sample excited by the pulse excitation light, and measures the fluorescence lifetime based on the counted number of fluorescence photons In the device
A fluorescence lifetime measuring apparatus comprising scanning control means for scanning the irradiation position of the pulse excitation light to the next irradiation position every time the counted number of fluorescent photons reaches a predetermined number.   The fluorescence lifetime measuring apparatus according to claim 1, wherein the irradiation position corresponds to a predetermined region in one pixel. Fluorescence lifetime measurement that scans a sample while irradiating it with pulse excitation light, counts the fluorescence photons emitted from the sample excited by the pulse excitation light, and measures the fluorescence lifetime based on the counted number of fluorescence photons In the device
Fluorescence intensity measurement means for scanning the fluorescence lifetime measurement region of the sample in advance with excitation light of a predetermined light intensity and measuring the fluorescence intensity distribution of the fluorescence lifetime measurement region;
Scanning that calculates the scanning speed of the excitation light corresponding to the intensity of the excitation light so that the number of fluorescent photons emitted from each predetermined area in the fluorescence lifetime measurement area exceeds a predetermined number based on the fluorescence intensity distribution Speed calculation means;
Scanning control means for controlling the pulse excitation light to scan each predetermined area according to the scanning speed calculated by the scanning speed calculation means;
A fluorescence lifetime measuring apparatus comprising:   Computation means for computing the fluorescence lifetime using a time-correlated single counting method for measuring the fluorescence lifetime based on the time from the irradiation of the pulsed excitation light to the emission of the fluorescence photons. The fluorescence lifetime measuring apparatus according to any one of claims 1 to 3.   Comprising computing means for computing the fluorescence lifetime using a time gate method for measuring the fluorescence lifetime based on the number of fluorescence photons counted in a plurality of time zones after irradiation with the pulse excitation light. The fluorescence lifetime measuring apparatus according to any one of claims 1 to 3.

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