JP4184228B2 - Fluorescence lifetime measuring device - Google Patents

Description

  The present invention relates to a fluorescence lifetime measuring apparatus that irradiates a sample with excitation light, measures fluorescence emitted while the excited sample transitions from an excited state to a ground state, and calculates a fluorescence lifetime.

  In recent years, fluorescence observation by a scanning fluorescence microscope has become an effective means for examining structural analysis of genomes, DNAs, genes, protein molecules, and the like in the field of biochemistry and analysis of responses to environmental changes. Among them, the measurement of the fluorescence lifetime has many advantages such that it is independent of the concentration of the fluorescent dye and that the difference can be observed even in a sample where the fluorescence wavelength cannot be separated. The measurement of the fluorescence lifetime according to the present invention is to irradiate the observation object with the excitation light, measure the fluorescence photons emitted from the observation object, and calculate the fluorescence lifetime based on the measured fluorescence photons. Conventionally, the entire sample was scanned uniformly with excitation light, the fluorescence lifetime was calculated regardless of the presence or absence of the observation target, and a fluorescence lifetime distribution image was created. Since it takes a long time to calculate the fluorescence lifetime, a method for calculating the fluorescence lifetime with high efficiency has been proposed (see Patent Document 1).

JP 2003-202292 A

  However, irradiating the entire sample uniformly with excitation light and calculating the fluorescence lifetime results in calculation of the fluorescence lifetime of a region outside the observation target. Such excitation light irradiation is a waste of time. In addition to this, there are problems that the life of the excitation light source is shortened and unnecessary damage to the sample is caused.

  In particular, in a method for calculating the fluorescence lifetime by measuring the number of fluorescence photons in a plurality of time zones (hereinafter referred to as a time gate method), several tens of seconds to several seconds are required to create one fluorescence lifetime distribution image. In some cases, it may take a long time, so when the observation object moves in a short time or when the fluorescence life of the observation object itself changes, the creation of a fluorescence life distribution image cannot be handled quickly, and the fluorescence life There was a problem that the change with time cannot be observed.

  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 capable of acquiring a fluorescence lifetime distribution image in a short time by a simple method.

  In order to solve the above-described problems and achieve the object, a fluorescence lifetime measuring apparatus according to claim 1 irradiates a sample with excitation light, and generates fluorescent photons generated while the sample transitions from an excited state to a ground state. In a fluorescence lifetime measuring apparatus that measures in a plurality of time zones and calculates a fluorescence lifetime from fluorescence photons measured in the plurality of time zones, an excitation light control unit that controls scanning of excitation light irradiated on the sample, and the excitation A region specifying unit for specifying a scanning region of light, a fluorescence lifetime calculating unit for calculating the fluorescence lifetime based on fluorescent photons from the scanning region specified by the region specifying unit, and the excitation light control unit The light is irradiated over a wide area of the sample, the region specifying unit specifies a region for calculating the fluorescence lifetime based on the fluorescence emitted by the irradiation, and the excitation light control unit applies the excitation light only to the specified region. And a control means for controlling the fluorescence lifetime to be calculated based on the measured fluorescence photons by the fluorescence lifetime calculation means measuring the fluorescence photons emitted from the irradiated region. To do.

  According to the first aspect of the present invention, the excitation light is scanned over a wide area of the sample, the region emitting fluorescence is detected, the region for calculating the fluorescence lifetime is specified based on the detected region, and the fluorescence is applied only to the specified region. By irradiating excitation light for lifetime calculation, a fluorescence lifetime distribution image can be acquired in a short time.

  According to a second aspect of the present invention, in the fluorescence lifetime measuring apparatus according to the present invention, the region specifying unit specifies a region that emits fluorescence by the excitation light as the scanning region.

  According to a third aspect of the present invention, in the above-described invention, the region specifying unit specifies a region that emits fluorescence of a predetermined intensity by the excitation light as the scanning region.

  According to a fourth aspect of the present invention, in the fluorescence lifetime measuring apparatus according to the above invention, the region specifying unit specifies a region that emits fluorescence having a predetermined intensity and a predetermined area by the excitation light as the scanning region.

  In addition, the fluorescence lifetime measuring apparatus according to claim 5 is characterized in that, in the above invention, the region specifying means specifies a rectangular region including at least a part of a region emitting fluorescence by the excitation light as the scanning region. To do.

  The fluorescence lifetime measuring apparatus according to the present invention scans excitation light over a wide area of a sample, detects a region emitting fluorescence, specifies a region for calculating a fluorescence lifetime based on the detected region, and detects fluorescence only in the specified region. There is an effect that the excitation light for the lifetime calculation is irradiated and a distribution image of the fluorescence lifetime can be acquired in a short time.

(Embodiment)
Embodiments of a fluorescence lifetime measuring apparatus according to the present invention will be described below in detail with reference to the accompanying drawings.

  FIG. 1 is a block diagram showing a schematic configuration of a fluorescence lifetime measuring apparatus 1 according to an embodiment of the present invention. In response to a control signal from the control unit 2, pulsed, sinusoidal, or continuous excitation light is oscillated from the laser light source 3 and is incident on the dichroic mirror 6 through the collimator lens 4 and the ND filter 5 in order. Since the dichroic mirror 6 reflects the light having the wavelength component of the excitation light and transmits the light having the wavelength component of the fluorescence, the excitation light is reflected by the dichroic mirror 6 and enters the galvanometer mirror unit 7. The galvano mirror unit 7 has a function of variably controlling a pair of mirrors according to a control signal from the control unit 2 and scanning a predetermined surface on the sample 14 with incident excitation light.

  The excitation light that has entered the galvanomirror unit 7 is controlled to have a predetermined optical path and is emitted, and is reflected by the reflection mirror 9 through the pupil projection lens 8. The excitation light reflected by the reflection mirror 9 is condensed on the sample 14 through the observation barrel 10, the imaging lens 11, the half mirror 12, and the objective lens 13 in order. The half mirror 12 reflects the illumination light from the observation illumination unit 15 to illuminate the sample 14 so that the sample 14 can be visually observed from the observation barrel 10.

  The sample 14 is excited by the excitation light collected on the sample 14, and the sample 14 emits fluorescence during the transition from the excited state to the ground state. The fluorescence emitted from the sample 14 travels back to the dichroic mirror 6 through the same optical path as the excitation light passes, passes through the dichroic mirror 6, and is detected through the condenser lens 16, the pinhole 17, and the absorption filter 18 in order. The light enters the container 19.

  In the time gate method, the excitation light is generally a low-speed repetitive pulse wave, and the fluorescence incident on the photodetector 19 is converted into an electric signal, and a gate circuit comprising a plurality of time gates set in a plurality of time zones. The electric signal is counted as the number of fluorescent photons by a counter 21 including a plurality of counters corresponding to the plurality of time gates. The counted number of fluorescent photons is output to the control unit 2. The control unit 2 calculates the fluorescence lifetime based on the input fluorescence photon count and a plurality of time zones in which the fluorescence photon count is counted, and outputs the calculated fluorescence lifetime to the display device 22. A distribution image of the fluorescence lifetime is displayed based on the fluorescence lifetime.

  FIG. 2 is a schematic configuration block diagram showing details of the control unit 2. The control unit 2 includes a fluorescence lifetime calculation control unit 2A, an excitation light control unit 2B, a fluorescence image acquisition unit 2C, a region specifying unit 2D, and a fluorescence lifetime calculation unit 2E.

  The fluorescence lifetime calculation control unit 2A controls the excitation light control unit 2B to control the emission timing of the excitation light, controls the fluorescence lifetime calculation unit 2E to control the calculation timing of the fluorescence lifetime, and also controls the fluorescence lifetime calculation unit 2E. Is input to the display device 22. The excitation light control unit 2B controls the laser light source 3 to control the emission of the laser light that is the excitation light, and controls the galvano mirror unit 7 to control the scanning of the excitation light, and also controls the scanning of the excitation light. The control signal is output to the fluorescence image acquisition unit 2C.

  The fluorescence lifetime calculation unit 2E controls the gate circuit 20 to control a plurality of time gates, calculates the fluorescence lifetime by inputting the number of fluorescence photons from the counter 21, and calculates the calculated fluorescence lifetime to the fluorescence lifetime calculation control unit 2A. Output to.

  The fluorescence image acquisition unit 2C receives the electrical signal converted from the fluorescence to the electrical signal by the light detector 19 and the control signal for the excitation light control unit 2B to perform scanning control of the excitation light, and creates a two-dimensional fluorescence image. Then, the created two-dimensional fluorescence image is output to the region specifying unit 2D. The region specifying unit 2D specifies a region where the fluorescence lifetime is to be calculated from the input two-dimensional fluorescence image, and outputs the specified region to the fluorescence lifetime calculation control unit 2A.

  First, when the fluorescence lifetime calculation control unit 2A outputs an instruction to the excitation light control unit 2B, the excitation light control unit 2B outputs a control signal to the laser light source 3 and the galvanometer mirror unit 7, and the laser light source 3 performs excitation. Laser light, which is light, is oscillated, and the galvanometer mirror unit 7 controls the excitation light so that the excitation light scans a wide area of the sample 14. The excitation light during this operation is desirably continuous light, high-speed pulse light, and / or high pulse peak value excitation light.

  When the excitation light scans a wide area of the sample 14, fluorescence is emitted from the region where the fluorescent substance exists, and the fluorescence travels through the dichroic mirror 6 in the same optical path as the path through which the excitation light passes. 19 is reached. The photodetector 19 converts the reached fluorescence into an electrical signal corresponding to the fluorescence intensity, and outputs the electrical signal to the fluorescence image acquisition unit 2C.

  The fluorescence image acquisition unit 2C receives an electric signal from the photodetector 19 and a control signal for performing scanning control of the excitation light from the excitation light control unit 2B, and the excitation light scans a wide area of the sample 14. A two-dimensional fluorescent image is created and output to the region specifying unit 2D. The region specifying unit 2D specifies a region satisfying a predetermined intensity and a predetermined area from the input two-dimensional image, and outputs the specified region to the fluorescence lifetime calculation control unit 2A. The process so far can be executed at an order of magnitude faster than the time for calculating the fluorescence lifetime for all points on the entire screen.

  FIG. 3 is a diagram showing how the excitation light scans the sample wide area 30. At this time, the nuclei 31A, 31B, and 31C that are the calculation targets of the fluorescence lifetime and the calculation of the fluorescence lifetime that includes them are not included. The existence of certain cells 25A, 25B, and 25C and their existing regions are unknown. Conventionally, since the fluorescence lifetime is calculated for the entire sample wide area 30 regardless of the existence area of the nuclei 31A, 31B, and 31C, it takes a lot of time to obtain a fluorescence lifetime distribution image.

  FIG. 4 shows a two-dimensional fluorescence image created by the fluorescence image acquisition unit 2C. This two-dimensional fluorescence image shows the presence of the cells 25A, 25B, and 25C and the contained nuclei 31A, 31B, and 31C and their existing areas. The fluorescence image acquisition unit 2C outputs the created two-dimensional fluorescence image to the region specifying unit 2D. The area specifying unit 2D selects the input two-dimensional fluorescence image based on the fluorescence intensity and the area satisfying the fluorescence intensity, specifies the nuclei 31A, 31B, and 31C as calculation targets of the fluorescence lifetime, and determines the position and the area. It outputs to the fluorescence lifetime calculation control part 2A. Although the cells 25A, 25B, and 25C that emit fluorescence are also shown in the two-dimensional fluorescence image, these are excluded from the calculation of the fluorescence lifetime because they do not satisfy the predetermined fluorescence intensity.

  The fluorescence lifetime calculation control unit 2A irradiates excitation light only to the regions of the nuclei 31A, 31B, and 31C and calculates the fluorescence lifetime. In the calculation of fluorescence lifetime by the time gate method, the excitation light irradiation is different from the irradiation method for acquiring the fluorescence image described above, and the pulse light with a relatively low peak value is equivalent to the excitation light having a long repetition interval. It is necessary to irradiate a certain number of times. The fluorescence lifetime is calculated based on the number of fluorescent photons emitted by this irradiation. Therefore, first, the fluorescence lifetime calculation control unit 2A outputs an instruction to the excitation light control unit 2B to irradiate a predetermined point of the nucleus 31A for calculation of the fluorescence lifetime, and the excitation light control unit 2B A control signal is output to the light source 3 and the galvanometer mirror unit 7, and the excitation light oscillated from the laser light source irradiates a predetermined point of the nucleus 31A.

  The fluorescence emitted from a predetermined point of the nucleus 31A irradiated with the excitation light reaches the photodetector 19, and the fluorescence is converted into an electrical signal by the photodetector 19, and the converted electrical signal is converted into the gate circuit 20. And the counter 21 sequentially, the number of fluorescent photons incident on the set time gate is counted and output to the fluorescence lifetime calculation unit 2E. The irradiation of excitation light and the counting of the number of fluorescent photons with respect to a predetermined point of the nucleus 31A are repeated a predetermined number of times, and the fluorescence lifetime calculation unit 2E calculates the fluorescence lifetime based on the number of fluorescence photons incident on the set time gate. .

  When the fluorescence lifetime of a predetermined point of the nucleus 31A is calculated, the fluorescence lifetime calculation unit 2E outputs the calculated fluorescence lifetime to the fluorescence lifetime calculation control unit 2A, and the fluorescence lifetime calculation control unit 2A outputs the predetermined fluorescence lifetime of the nucleus 31A. The calculated fluorescence lifetime of the point and information on the point are output to the display device 22, and an instruction output similar to that described above is output to the excitation light control unit 2B in order to calculate the fluorescence lifetime of the next predetermined point of the nucleus 31A. Against. When the calculation of the fluorescence lifetime of the entire nucleus 31A is thus completed, the fluorescence lifetime calculation control unit 2A outputs an instruction to the excitation light control unit 2B so as to calculate the fluorescence lifetime of the entire nucleus 31B. When the calculation of the fluorescence lifetime of the entire 31B is completed, an instruction is output to the excitation light control unit 2B so as to calculate the fluorescence lifetime of the entire nucleus 31C.

  FIG. 5 shows that the excitation light is irradiated to the nucleus 31A, which is the calculation target of the fluorescence lifetime, and the excitation light irradiation and the fluorescence lifetime calculation are sequentially performed on the nuclei 31B and 31C as soon as the calculation of the fluorescence lifetime of the nucleus 31A is completed. In addition, as soon as the calculation of the fluorescence lifetime of the nucleus 31C is completed, the calculation of the fluorescence lifetime of the nucleus 31A is performed again.

  In this way, the fluorescence lifetime calculation control unit 2A inputs the fluorescence lifetimes of the nuclei 31A, 31B, and 31C as a whole, and outputs the input fluorescence lifetime and position information to the display device 22. The display device 22 creates and displays a fluorescence lifetime distribution image based on the input fluorescence lifetime and its position information. FIG. 6 shows a distribution image of the fluorescence lifetime displayed on the display device 22. It can be seen that components having different fluorescence lifetimes exist in the nuclei 31A, 31B, and 31C. Further, as described above, the fluorescence lifetime of the nuclei 31A, 31B, and 31C in a very limited range and the position information thereof are input to the display device 22 with respect to the entire screen, and the nuclei 31A, 31B, and 31C are input. Since the fluorescence lifetime distribution image is formed at a very high speed, it is possible to display a relatively fast change in the fluorescence lifetime with time by repeated measurement. When the regions of the nuclei 31A, 31B, and 31C move, when the calculation of the fluorescence lifetime of the nuclei 31C is completed, the excitation light of the sample wide area 30 is scanned to specify the region again, and the nuclei 31A and 31B , 31C, a fluorescence lifetime distribution image may be displayed following the movement of the region 31C.

  Excitation light is emitted by skipping areas that are not subject to calculation of fluorescence lifetime, so unnecessary irradiation can be avoided and not only can a fluorescence lifetime distribution be created in a short time, but also irradiation of unnecessary excitation light. The life of the laser light source can be extended. In the above-described embodiment, the order of calculation of the fluorescence lifetime is set to the nuclei 31A, 31B, and 31C. However, the order may be changed in accordance with the importance of observation or in accordance with the technical difficulty. Good.

  FIG. 7 is a flowchart for explaining the operation until a fluorescence lifetime distribution image is acquired. First, the excitation light control unit 2B controls the excitation light to scan the sample wide area 30 (step S101). Next, the fluorescence image acquisition unit 2C acquires a two-dimensional fluorescence image by scanning with the excitation light (step S102). Next, the region specifying unit 2D determines whether there is a region having a predetermined fluorescence intensity in the input two-dimensional fluorescence image (step S103). If there is no portion satisfying the predetermined intensity (No at Step S103), it is determined that there is no fluorescence lifetime calculation target, and the sample wide area 30 is scanned again with excitation light (Step S101). In this case, the fluorescence lifetime calculation target can be selected by changing the predetermined intensity. If there is a portion satisfying the predetermined strength (step S103, Yes), it is then determined whether the predetermined area is satisfied (step S104). If there is no portion satisfying the predetermined area (No at Step S104), it is determined that there is no fluorescent lifetime calculation target, and the sample wide area 30 is scanned again with excitation light (Step S101). By using this predetermined area as a reference, it is possible to remove simple bright spots due to noise or stray light. Conversely, by removing this determination (step S104), a new fluorescence lifetime calculation region can be found.

  If there is a portion satisfying the predetermined area (Yes in step S104), it is determined that there is a fluorescence lifetime calculation target, and the region specifying unit 2D specifies a fluorescence lifetime calculation region (step S105). Next, in order to calculate the fluorescence lifetime of only the region specified by the region specifying unit 2D, the fluorescence lifetime calculation control unit 2A instructs the excitation light control unit 2B to output the excitation light, and the excitation light control unit 2B Then, the excitation light is irradiated to the specific area (step S106). Based on the fluorescence emitted from the specific region, the fluorescence lifetime calculation unit 2E calculates the fluorescence lifetime and outputs it to the fluorescence lifetime calculation control unit 2A (step S107). The fluorescence lifetime calculation control unit 2A outputs the input fluorescence lifetime and its position to the display device 22 (step S108), and the display device 22 displays a fluorescence lifetime distribution image based on the fluorescence lifetime and position (step S108). S109). Further, after the distribution image of the fluorescence lifetime is displayed (step S109), the process returns to the operation of irradiating the specific region with the excitation light (step S106) in order to observe the temporal change of the fluorescence lifetime calculation target. Further, in order to cope with the movement of the fluorescence lifetime calculation target, if the specific region is irradiated with excitation light (step S106) and scanning of the sample wide area 30 with the excitation light (step S101), the fluorescence lifetime is combined. Both the change with time and the movement of the calculation object can be observed.

  In the above-described embodiment, the region specifying unit 2D specifies a region based on the fluorescence intensity or the like as a predetermined reference, but it can be changed according to an observation situation or a technical viewpoint. . FIG. 8 shows that the area specifying unit 2D defines a rectangular area including the nucleus 31A as the specific area 32A. This is because when the shape of the nucleus 31A is flat, irradiation with excitation light along an irregular shape and scanning do not increase unnecessary technical processes, and the distribution image of the fluorescence lifetime can be more easily obtained. It is for acquiring.

  FIG. 9 shows a case where the region 33A is specified in a rectangular shape including not only the nucleus 31A but the entire cell 25A. This is because the contrast between the cell 25A and the nucleus 31A can also be seen.

  FIG. 10 shows a case where a region 34A including a predetermined margin in the nucleus 31A is specified. This is because it is possible to cope with the case where the region of the nucleus 31A slightly changes with time.

  In the above-described embodiment, the region specifying unit 2D automatically specifies a region using a predetermined reference. However, the two-dimensional fluorescent image created by the fluorescent image acquiring unit 2C is used as an observer. By observing, the observer may specify the region. It is because it can respond to the individual demands of the observer. Further, although this embodiment has been described by the time gate method, it is obvious that the present invention is effective for measuring all fluorescence lifetimes.

It is a block diagram which shows schematic structure of the fluorescence lifetime measuring apparatus which is embodiment of this invention. It is a block diagram which shows schematic structure of the control part of embodiment of this invention. It is explanatory drawing explaining a mode that the excitation light of embodiment of this invention scans a sample wide area | region. It is explanatory drawing which shows the two-dimensional fluorescence image which the fluorescence image acquisition part of embodiment of this invention acquires. It is explanatory drawing explaining a mode that the excitation light of embodiment of this invention irradiates only a specific area | region. It is explanatory drawing which shows the distribution image of the fluorescence lifetime which the display apparatus of embodiment of this invention displays. It is a flowchart which shows operation | movement of the control part of embodiment of this invention. It is explanatory drawing which shows the area | region which the area | region specific part of embodiment of this invention specifies. It is explanatory drawing which shows the area | region which the area | region specific part of embodiment of this invention specifies. It is explanatory drawing which shows the area | region which the area | region specific part of embodiment of this invention specifies.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fluorescence lifetime measuring apparatus 2 Control part 2A Fluorescence lifetime calculation control part 2B Excitation light control part 2C Fluorescence image acquisition part 2D Area | region identification part 2E Fluorescence lifetime calculation part 3 Laser light source 4 Collimator lens 5 ND filter 6 Dichroic mirror 7 Galvanometer mirror unit 8 Pupil projection lens 9 Reflecting mirror 10 Observation barrel 11 Imaging lens 12 Half mirror 13 Objective lens 14 Sample 15 Observation illumination unit 16 Condensing lens 17 Pinhole 18 Absorption filter 19 Photo detector 20 Gate circuit 21 Counter 22 Display device 25A , 25B, 25C cell 30 sample wide area 31A, 31B, 31C nucleus 32A, 33A, 34A specific area

Claims (4)

In a fluorescence lifetime measuring apparatus that irradiates a sample with excitation light, measures a fluorescence photon emitted while the sample transitions from an excited state to a ground state, and calculates a fluorescence lifetime from the measured fluorescence photon.
Excitation light control means for controlling scanning of excitation light applied to the sample;
Area specifying means for specifying a scanning area of the excitation light;
Fluorescence lifetime calculating means for calculating the fluorescence lifetime at each excitation light irradiation position based on fluorescence photons from the excitation light irradiation position which are a plurality of measurement positions within the scanning region specified by the area specifying means; ,
The excitation light control unit irradiates the sample with the excitation light over a wide area, the region specifying unit specifies a region where the fluorescence lifetime is calculated based on the fluorescence emitted by the irradiation, and the excitation light control unit Only the excitation light irradiation position, which is the measurement position in the specified region, is irradiated with excitation light, and the fluorescence lifetime calculating means measures the fluorescence photons emitted from the irradiated position at each excitation light irradiation position. Control means for performing control to calculate the fluorescence lifetime of each excitation light irradiation position based on the measured fluorescence photons;
Display means for displaying the calculated fluorescence lifetime of each excitation light irradiation position as a two-dimensional distribution image;
The region specifying means specifies, as the scanning region, a region that emits fluorescence having a predetermined intensity by the excitation light. The fluorescence lifetime measuring apparatus according to claim 1, wherein the region specifying unit specifies a region that emits fluorescence having a predetermined intensity and a predetermined area by the excitation light as the scanning region. The fluorescence lifetime measuring apparatus according to claim 1, wherein the region specifying unit specifies a rectangular region including at least a part of a region that emits fluorescence by the excitation light as the scanning region.   The fluorescence lifetime measuring apparatus according to any one of claims 1 to 3, wherein the fluorescence lifetime is calculated by a time gate method in which the number of fluorescence photons is measured in a plurality of time zones.

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