JPH09189612A - Time-resolved emission imaging device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively receive the emission generated by the emission of a pulse exciting light outputted from a pulse laser beam source having a high repeated frequency, and perform the time-resolved measurement of the emission from a sample in a short period. SOLUTION: The pulse light outputted from a pulse laser beam source 1 is partially reflected by a beam splitter 2, and the remainder is transmitted thereby. The transmitted pulse light is incident on a second harmonic generator 3, and converted into 1/2 wavelength to form a pulse exciting light 5, which is then emitted to a sample 8. The emission 10 generated from the sample 8 is incident on an optical car gate 12 through a polarizer 11. On the other hand, the pulse light reflected by the beam splitter 2 is emitted to the optical car gate 12 as a gate pulse light 18. When the gate pulse light 18 is incident, the emission 10 becomes an elliptically polarized light when transmitted by the optical car gate 12, and it is partially transmitted by an analyzer 13, and detected by a two-dimensional optical detector 20. Since the gate pulse light 18 is incident on the optical car gate 12 with a delay according to the position of a movable right-angled mirror 14, the time-resolved measurement of the emission 10 can be performed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a time-resolved luminescence imaging apparatus for irradiating a probe labeled on a sample to be measured with excitation light and time-resolving and measuring an image of luminescence from the probe.

[0002]

2. Description of the Related Art Conventionally, an imaging intensifier with a gate function has been used in time-resolved measurement of an image of luminescence generated from a labeled sample. FIG.
It is a block diagram of the conventional time-resolved luminescence imaging device.

The pulse excitation light 5 output from the pulse laser light source 1 has its beam diameter expanded by the beam expander 4, is reflected by the dichroic mirror 6, passes through the objective lens 7, and is emitted to the luminescence-marked sample 8. Incident. The sample 8 is irradiated with the pulsed excitation light 5 to excite the luminescent label in the sample 8, and the emitted light is passed through the objective lens 7, the dichroic mirror 6, the excitation light absorption filter 9 and the imaging lens 19, and then has a gate function. An image is formed on the photocathode of the attached imaging intensifier 49.

The gate of the imaging intensifier 49 with the gate function is provided with a delay circuit 5 for the output timing of the pulsed pump light 5 in the pulsed laser light source 1.
It is opened by the gate control circuit 51 for a fixed time Δτ after the elapse of a predetermined delay time τ given by 0.
Therefore, the light emission image formed on the photocathode of the imaging intensifier 49 with a gate function during the period from time τ to time τ + Δτ with reference to the time of generation of the pulse excitation light 5 is the imaging intensity with a gate function. The secondary electrons that have been multiplied by the micro channel plate of the fire 49 are made incident on the fluorescent screen and are converted into an optical image again. Then, this optical image passes through the lens 52 and
The optical image detected by the three-dimensional photodetector 20 is processed by the processing device 21 and displayed. The above process is repeated for a large number of pulsed pump lights until a sufficiently good S / N ratio is obtained, and the delay time τ is changed by the delay circuit 50 to perform time-resolved measurement.

[0005]

The above-mentioned conventional example has the following problems. The imaging intensifier with a gate function usually has a maximum gate opening / closing repetition frequency of about 10 kHz, with a limit of several hundreds kHz at most. On the other hand, some pulsed laser light sources have an output repetition frequency of pulsed excitation light of about 70 MHz to 80 MHz. Therefore, the imaging intensifier with a gate function can receive only a small part of the emitted light generated by the irradiation of the pulsed excitation light output from the pulsed laser light source, and a large amount of the pulsed excitation light is wasted. Is bad.

Particularly when observing light emission from a biological sample,
If only a very small amount of light emitted by the irradiation of pulsed excitation light can be received, it will take a long time for observation, damage to the biological sample will be large, and the physiological state of the biological sample will change. Can not be accurately observed. Also, when examining the dynamics of biological samples,
The observation period of the imaging intensifier with gating function may not be sufficient for observing fast changes in biological samples.

The present invention has been made in order to solve the above problems, and effectively emits light emitted by the irradiation of pulsed excitation light output from a pulsed laser light source having a high repetition frequency, so that It is an object of the present invention to provide a time-resolved luminescence imaging apparatus capable of observing the luminescence of light in a short period.

[0008]

A time-resolved luminescence imaging apparatus according to the present invention comprises (1) a pulsed light source for outputting pulsed light, and (2) a first light beam and a second light beam for branching the pulsed light. And (3) an excitation optical system for forming pulse excitation light based on the first light flux and irradiating the sample under measurement with the pulse excitation light, and (4) pulse excitation for the sample under measurement. A light emitting optical system that guides light emitted by light irradiation to a predetermined position, (5) A gate optical system that forms gate pulse light based on the second light flux and guides the light to a predetermined position, (6) It is arranged at a predetermined position and transmits the light emission when the gate pulse light is incident, and does not transmit the light emission otherwise.
Optical gate means of (7), first photodetection means for detecting light emitted from the first optical gate means, and (8)
An optical path length varying means for setting an optical path length difference between the optical path lengths of the excitation optical system and the emission optical system and the optical path length of the gate optical system is provided.

The operation of this device is as follows. First, the pulsed light output from the pulsed light source is split into the first and second light fluxes by the light flux splitting means. The pulsed excitation light formed by the excitation optical system based on the first light flux is applied to the sample to be measured, and the light emission generated by this is applied to the first optical gate means arranged at a predetermined position by the light emission optical system. Be guided. Further, the gate pulse light formed by the gate optical system based on the second light flux is guided to the first optical gate means. The emitted light is transmitted through the first optical gate means when the gate pulse light is incident, and cannot be transmitted otherwise.
The emitted light emitted from the first optical gate means is detected by the first light detection means. Then, the optical path length difference between the optical path lengths of the excitation optical system and the emission optical system and the optical path length of the gate optical system is variably set by the optical path length varying means, whereby the time of light emission generated from the sample to be measured is set. Decomposition measurement is possible.

The optical path length changing means includes a pair of reflecting mirror groups disposed on the optical path of the gate optical system so as to face each other, and at least one of the pair of reflecting mirror groups of the luminous fluxes arriving from the luminous flux splitting means. A moving unit that moves in a direction parallel to the optical axis, and reciprocates the light flux that has arrived from the light flux splitting unit at least once between the pair of reflecting mirror groups, and then emits it toward a predetermined position. It may be one. Further, the optical path length varying means is arranged on the optical path of the excitation optical system so as to face each other.
The pair of reflecting mirror groups and the moving means for moving at least one of the pair of reflecting mirror groups in the direction parallel to the optical axis of the light flux arriving from the light flux splitting means are provided. It may be one that reciprocates at least once between the pair of reflecting mirror groups and then emits the light toward the sample to be measured. Further, the optical path length varying means includes a pair of reflecting mirror groups arranged to face each other on the optical path of the light emitting optical system, and a light beam of a light flux that reaches at least one of the pair of reflecting mirror groups from the sample to be measured. A moving means that moves in a direction parallel to the axis,
The light flux reaching from the sample to be measured may be reciprocated at least once between the pair of reflecting mirror groups and then emitted toward a predetermined position. In either case, the timing between the light emission reaching the optical gate means and the gate pulse light is greatly changed by only slightly changing the distance between the pair of reflecting mirror groups.

Further, the optical path length varying means may increase one of the optical path lengths of the gate optical system and the excitation optical system or the optical path lengths of the gate optical system and the light emitting optical system and decrease the other. In this case, For both the light emission and the gate pulse light that reach the optical gate means, one can be advanced and the other can be delayed, so that the arrival timings of the both greatly change.

(1) A light-emission branching unit which is provided on the optical path of the light-emission optical system, reflects a part of the light emission, transmits the remaining light, and makes the transmitted light incident on the first optical gate unit.
(2) Gate pulse light branching means provided on the optical path of the gate optical system for reflecting a part of the gate pulse light and transmitting the rest, and for making the transmitted gate pulse light incident on the first optical gate means, 3) The light emission reflected by the light emission branching means and the gate pulse light reflected by the gate pulse light branching means are made incident, and the light emission is transmitted when the gate pulse light is incident, and the light emission is not transmitted otherwise. It further comprises a second optical gate means, and (4) a second photodetecting means for detecting the light emitted from the second optical gate means, each of the first and second photodetecting means. , Linearly polarized light components in different directions of light emission may be detected. In this case, the anisotropy of light emission can be obtained.

Each of the first and second optical gate means may have a flat plate shape, and the light emission and the gate pulse light may be made to be incident symmetrically with respect to the flat plate surface. In this case, each of the first and second optical gate means transmits the luminescence generated at the same time at each position on the measured sample.

The first optical gate means includes (1) a polarizer for emitting light emission and emitting a linearly polarized light beam in the first direction, and (2) a gate pulsed light beam and a light beam emitted from the polarizer. Is incident, and the birefringence changes according to the amount of gate pulse light, and the light beam emitted from the polarizer is converted into elliptically polarized light with an ellipticity according to the amount of gate pulse light and then emitted. An optical cargate that utilizes the effect, and (3) an analyzer that enters a light beam emitted from the optical cargate and emits a linearly polarized light beam in a second direction orthogonal to the first direction. May be. Further, a saturable absorber that transmits light emission when the gate pulse light is incident and does not transmit light emission when the gate pulse light is not incident may be provided. The same applies to the second optical gate means. In either case, each of the first and second optical gate means transmits the light emission only when the gate pulse light is incident, and the gate opening / closing operation can be performed at high speed.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

(First Embodiment) First, a first embodiment will be described. FIG. 1 is a configuration diagram of a time-resolved luminescence imaging apparatus according to the first embodiment.

The pulsed light output from the pulsed laser light source 1 is partially reflected by the beam splitter (beam splitting means) 2 and the rest is transmitted and split into two beams. As the pulse laser light source 1, for example, a mode-locked titanium sapphire laser light source is used.

One of the light beams split by the beam splitter 2 is applied to the sample 8 through the excitation optical system. That is, first, this light flux is converted into a wavelength of ½ by the second harmonic generator 3, and is converted into a wavelength suitable for exciting the luminescent marker contained in the sample 8. The pulsed excitation light 5 output from the second harmonic generator 3 is transmitted to the beam expander 4
The beam diameter is expanded by the dichroic mirror 6
Then, the sample 8 labeled with luminescence is irradiated through the objective lens 7 after being reflected. The dichroic mirror 6 used here reflects the pulsed excitation light 5 and transmits the luminescence generated from the luminescence marker in the sample 8.

When the sample is irradiated with the pulsed excitation light 5 to excite the luminescent label, luminescence 10 is generated from the luminescent label. This emitted light 10 reaches an optical Kerr gate 12, which is a main component of the optical gate means, through a light emitting optical system. That is, the light emission 10 passes through the objective lens 7, the dichroic mirror 6, the excitation light absorption filter 9, the polarizer 11, the linearly polarized light, and the optical cargate 12. The excitation light absorption filter 9 transmits the emitted light 10 and blocks the pulse excitation light 5, and prevents the scattered light of the pulse excitation light 5 applied to the sample 8 from reaching the photodetector 20. It is provided in.

Further, when the strong optical polarization light (gate pulse light 18) is applied to the optical Kerr gate 12, different refractive index changes occur between a direction parallel to the linear polarization direction and a direction perpendicular to the direction. The birefringence is composed of an optical Kerr medium having a Kerr effect which is proportional to the light quantity of the gate pulse light 18, and as the optical Kerr medium, for example, carbon disulfide (C
S ₂ ) and nitrobenzene are used. Light car gate 1
2 is arranged with its optical axis in a plane perpendicular to the optical axis of the light emission 10 and at an angle of 45 degrees with the transmission polarization azimuth. Therefore, the gate pulse light 1 is applied to the optical Kerr gate 12.
8 is not illuminated, the optical Kerr medium is isotropic without causing birefringence.
The linearly polarized light emission 10 incident on 2 is emitted with the linearly polarized state thereof being maintained. On the other hand, when the optical car gate 12 is irradiated with the gate pulse light 18,
Birefringence occurs in the optical Kerr medium, and the light emission 10 has a different phase difference with respect to each component of the optical axis of the optical Kerr medium, and is emitted as elliptically polarized light having an ellipticity corresponding to the phase difference. .

Light emission 10 emitted from the optical car gate 12.
Subsequently enters the analyzer 13. This analyzer 13
The linearly polarized light beam orthogonal to the direction of the linearly polarized light beam emitted from the polarizer 11 is transmitted. Therefore, when the emitted light 10 is emitted from the optical Kerr gate 12 and enters the analyzer 13 while maintaining its polarization state, the emitted light 10 cannot pass through the analyzer 13. But,
When the emitted light 10 is elliptically polarized and is emitted from the optical Kerr gate 12 and is incident on the analyzer 13, a light flux having a light amount corresponding to the ellipticity, that is, the light amount of the gate pulse light 18 is emitted from the analyzer 13. In this way, the polarizer 11,
The optical Kerr gate 12 and the analyzer 13 function as an optical gate. Then, the light emission 10 that has passed through the analyzer 13
Is a two-dimensional photodetector (for example, a two-dimensional C
The pulse laser light source 1 is imaged on the light receiving surface of the CD) 20.
The image of each pulse output from the two-dimensional photodetector 2
The light is received at 0, and the image data is sent to the processing device 21 and stored or processed.

The gate pulse light 18 applied to the optical Kerr gate 12 is formed from the other light beam split by the beam splitter 2, passes through the gate optical system, and is sent to the optical Kerr gate 12.
Incident on. That is, the other light beam split by the beam splitter 2 is a movable right-angle mirror (optical path length changing means) 1
4 and then by the plane mirror 15, 1 /
The polarization direction is adjusted by the two-wave plate 16, the beam diameter is expanded by the beam expander 17, and the optical Kerr gate 12 is irradiated with the gate pulse light 18.

Here, the movable right-angle mirror 14 is composed of two plane mirrors arranged at right angles to each other, and a light beam incident perpendicularly to the intersection of the plane mirror surfaces of the two plane mirrors. Are sequentially reflected by and are emitted in the opposite direction on the optical axis parallel to the optical axis of the incident light beam. Further, the movable right-angle mirror 14 can move in a direction parallel to the optical axis of the incident light beam. Even if the movable right-angle mirror 14 moves, the optical axis of the emitted light beam does not change. Therefore,
By moving the movable right-angle mirror 14, the delay of the gate pulse light 18 can be variably set. Assuming that the position change amount of the movable right-angle mirror 14 is Δx and the light speed is c, the change amount Δt of the delay time is represented by Δt = 2Δx / c (1).

As described above, the gate pulse light 18 emits the gate pulse light 18 at the time corresponding to the moving position of the movable right-angle mirror 14.
The light emission 10 which is irradiated on the light beam 2 and reaches the optical cargate 12 at that time passes through the analyzer 13, and the image of the light emission 10 is received by the two-dimensional photodetector 20. Therefore, the time when the gate pulse light 18 reaches the optical cargate 12 can be changed by moving the movable right-angled mirror 14, so that the image of the light emission 10 generated from the sample 8 can be time-resolved and observed. it can.

Further, since the response speed of the optical Kerr gate 12 used in this embodiment is about several picoseconds, not only the measurement with high time resolution can be performed but also the pulse laser light source 1 can be used.
The output repetition cycle of the pulsed light output from
Even if it is z or more, the pulsed light can be effectively used.

(Second Embodiment) Next, a second embodiment will be described. FIG. 2 is a configuration diagram of a time-resolved luminescence imaging apparatus according to the second embodiment. In this embodiment, the optical system that gives a delay to the gate pulse light is different,
It is possible to give a large change in delay time with a slight movement of the movable right-angle mirror.

In the present embodiment, (1) the pulsed pump light 5 generated from the pulsed laser light source 1 and split by the beam splitter 2 into one half wavelength by the second harmonic generator 3 Is an excitation optical system that irradiates the sample 8 through the beam expander 4, the dichroic mirror 6, and the objective lens 7, and (2) the light emission 10 generated by the sample 8.
After passing through the objective lens 7, the dichroic mirror 6, the excitation light absorption filter 9 and the polarizer 11, the optical cargate 1
2, and (3) An optical system in which the light emission 10 emitted from the optical cargate 12 is imaged and detected by the two-dimensional photodetector 20 via the analyzer 13 and the imaging lens 19. , Are the same as those in the first embodiment.

This embodiment is different from the first embodiment in the configuration for delaying the gate pulse light 18. The other light beam split by the beam splitter 2 enters the polarization beam splitter 22, the p-polarized light component (the electric field vector of which is parallel to the incident surface) is transmitted, and the movable right-angle mirror (reflection mirror group) 2
3 and the fixed right-angled mirror (reflecting mirror group) 24 make three reciprocations, pass through the quarter-wave plate 25 perpendicularly and are reflected by the fixed flat mirror 26, and again the quarter-wave plate 25 is reflected. Through,
The movable right-angle mirror 23 and the fixed right-angle mirror 24 make three round trips to reach the polarization beam splitter 22.

The optical axis of the quarter-wave plate 25 is
It is at an angle of 45 degrees with respect to the linear polarization direction of the incident p-polarized light. Therefore, since the light flux passes through the quarter-wave plate 25 twice, the p-polarized light flux output from the polarization beam splitter 22 is rotated by 90 degrees in the direction of linear polarization to the polarization beam splitter 22. When it reaches again, it becomes s-polarized light (the electric field vector is perpendicular to the incident surface) and is reflected by the polarization beam splitter 22. The beam diameter of the light beam reflected by the polarization beam splitter 22 is expanded by the beam expander 17, and the optical car gate 12 is irradiated with the gate pulse light 18.

Each of the movable right-angle mirror 23 and the fixed right-angle mirror 24 is composed of two plane mirrors arranged at right angles to each other, and a light beam incident perpendicularly on the intersection line of the two plane mirror surfaces is The light is sequentially reflected by each of the two plane mirrors and emitted in the opposite direction on the optical axis parallel to the optical axis of the incident light beam. In addition, the movable right-angle mirror 2
The line of intersection of the two plane mirror surfaces that form 3 and the line of intersection of the two plane mirror surfaces that form the fixed right-angle mirror 24 are parallel to each other, and from the polarization beam splitter 22 to the movable right-angle mirror 23. The distances from the optical axis of the traveling light beam are different from each other. Therefore, the light beam can reciprocate between the polarization beam splitter 22 and the fixed plane mirror 26.

Furthermore, the movable right-angle mirror 23 can move in a direction parallel to the optical axis of the incident light beam from the polarization beam splitter 22. Even if the movable right-angle mirror 23 moves, the optical axis of the emitted light beam does not change. Therefore, the delay of the gate pulse light 18 can be variably set by moving the movable right-angle mirror 23. The change amount Δt of the delay time with respect to the position change amount Δx of the movable right-angle mirror 23 is represented by Δt = 12Δx / c (2). Comparing this with the expression (1) in the first embodiment, even if the position change amount of the movable right-angle mirror is the same, the delay time of the gate pulse light 18 can be changed by a factor of 6 in the present embodiment. You can That is, it is possible to measure the time change of light emission over a long period of time with a high time resolution and a short cycle.

Further, by changing the arrangement of the movable right-angle mirror 23, the fixed right-angle mirror 24, the quarter-wave plate 25, and the fixed plane mirror 26, it is possible to increase the number of reciprocations of the light beam during this period, and to increase the gate size. Pulsed light 18
Can be obtained. Further, the optical system for delaying the gate pulse light 18 is not limited to this structure, and various modifications are possible. The point is that a large delay time change is given to the gate pulse light 18 by reciprocating the light beam many times between the first mirror group and the second mirror group and moving at least one of the mirror groups. .

(Third Embodiment) Next, a third embodiment will be described. FIG. 3 is a configuration diagram of a time-resolved luminescence imaging apparatus according to the third embodiment. In the present embodiment, an optical system that not only delays the gate pulse light but also delays the pulsed pump light is provided.
It is possible to increase (decrease) one delay time and decrease (increase) the other delay time.

The light beam output from the pulse laser light source 1 and partially transmitted by the beam splitter 2 is a plane mirror 34, 3
Polarization beam splitter 2 which is sequentially reflected at 5 and 36
2 is transmitted and becomes p-polarized light. This p-polarized light beam is
In the same manner as in the above embodiment, the movable right-angle mirror 23 and the fixed right-angle mirror 24 make three reciprocating movements, vertically enter the fixed plane mirror 26 via the quarter-wave plate 25, are reflected, and again.
After passing through the quarter-wave plate 25, the movable right-angle mirror 23 and the fixed right-angle mirror 24 make three round trips to reach the polarization beam splitter 22. At this time, since the light flux is s-polarized light, it is reflected by the polarization beam splitter 22 and the beam diameter is expanded by the beam expander 17, so that the optical cargate 1
2 is irradiated.

On the other hand, the light beam output from the pulse laser light source 1 and partially reflected by the beam splitter 2 is sequentially reflected by the plane mirrors 27 and 28, transmitted through the polarization beam splitter 29, and becomes p-polarized light. The p-polarized light beam also makes three round trips between the movable right-angle mirror 30 and the fixed right-angle mirror 31, passes through the quarter-wave plate 32, is vertically incident on the fixed plane mirror 33, and is reflected again. After passing through the four-wave plate 32, the movable right-angle mirror 30 and the fixed right-angle mirror 31 make three round trips and reach the polarization beam splitter 29. At this time, since the light flux is s-polarized light, it is reflected by the polarization beam splitter 29, is incident on the second harmonic generator 3 and has a half wavelength, and the beam diameter is expanded by the beam expander 4. , And becomes the pulse excitation light 5. The subsequent steps are the same as those in the first embodiment.

Here, the optical axis of the light beam traveling from the polarization beam splitter 22 to the movable right-angle mirror 23 and the optical axis of the light beam traveling from the polarization beam splitter 29 to the movable right-angle mirror 30 are parallel to each other, and the movable right angle is also. Mirror 23
The movable right-angle mirror 30 is fixedly arranged on the movable stage 37, and they can move together in a direction parallel to the optical axis of the light beam traveling from the polarization beam splitter 22 to the movable right-angle mirror 23.

Therefore, when the movable stage 37 is moved by Δx, the time when the light emission 10 reaches the optical car gate 12 is changed by 12Δx / c, and the gate pulse light 18 is emitted.
Arrives at the optical car gate 12 at -12Δx / c
Only change. That is, with respect to the position change amount Δx of the movable stage 37, the change amount Δ of the time difference when each of the light emission 10 and the gate pulse light 18 reaches the optical car gate 12.
t is represented by Δt = 24Δx / c (3). Comparing this with the expression (2) in the second embodiment, even if the position change amount of the movable right-angle mirror is the same, the present embodiment can change the delay time of the gate pulse light 18 twice as much. You can This makes it possible to observe a luminescent label with a longer life.

(Fourth Embodiment) Next, a fourth embodiment will be described. FIG. 4 is a configuration diagram of a time-resolved luminescence imaging apparatus according to the fourth embodiment. In the present embodiment, the optical Kerr gate 12 has a flat plate shape as compared with the first embodiment, the light emission 10 is incident on one surface of the optical Kerr gate 12, and the gate pulse light 18 is incident on the other surface of the optical Kerr gate 12. They are different in that they are incident on a surface and have the same incident angle.

In the above-described first embodiment, the light emission 1
0 is incident vertically on the surface of the optical cargate 12, while
The gate pulse light 18 is obliquely incident on the surface of the optical Kerr gate 12. Therefore, since the time when the gate pulse light 18 arrives at each position on the surface of the optical car gate 12 is different, the light emission 10 transmitted at each position on the surface of the optical car gate 12 is generated at a different time. Defocus is generated and is received by the two-dimensional photodetector 20. The problem of temporal blurring becomes more significant as the gate operation of the optical gate means becomes faster. The present embodiment is capable of time-resolved measurement of light emission without causing this temporal blur.

In the present embodiment, (1) the pulsed pump light 5 generated from the pulsed laser light source 1 and split by the beam splitter 2 into one half wavelength by the second harmonic generator 3 Is an excitation optical system that irradiates the sample 8 through the beam expander 4, the dichroic mirror 6, and the objective lens 7, and (2) the light emission 10 generated by the sample 8.
After passing through the objective lens 7, the dichroic mirror 6, the excitation light absorption filter 9 and the polarizer 11, the optical cargate 1
2, and (3) An optical system in which the light emission 10 emitted from the optical cargate 12 is imaged and detected by the two-dimensional photodetector 20 via the analyzer 13 and the imaging lens 19. , Are almost the same as those in the first embodiment. However,
The angle at which the light emission 10 is incident on the optical cargate 12 is a right angle in the first embodiment, whereas in the present embodiment, the angle θ (≠ 90 degrees) with respect to the surface of the optical cargate 12.
And the gate pulse light absorption filter 39 is provided on the optical path between the optical Kerr gate 12 and the two-dimensional photodetector 20.

On the other hand, the other light beam output from the pulse laser light source 1 and split by the beam splitter 2 is reflected by the movable right-angle mirror 14, and the plane mirrors 15, 36 and 3 are reflected.
The beam diameter is expanded by the beam expander 17 and is incident on the optical Kerr gate 12 as the gate pulse light 18. The movable right-angle mirror 14 for delaying the gate pulse light 18 is the same as that in the first embodiment. However, unlike the first embodiment, the gate pulse light 18 is incident on the back surface of the optical Kerr gate 12 at an angle θ. The gate pulse light 18 is partially reflected by the back surface of the optical Kerr gate 12 toward the two-dimensional photodetector 20, but is absorbed by the gate pulse light absorption filter 39, so the two-dimensional photodetector 20. Never reach.

Thus, the light emission 10 and the gate pulse light 1
When 8 and 8 are made to enter symmetrically with respect to the surface of the optical Kerr gate 12, the difference in optical path length between the light emission 10 and the gate pulse light 18 becomes the same at each position on the surface of the optical Kerr gate 12. Therefore, the light emission 10 passing through the optical Kerr gate 12 with the incidence of the gate pulse light 18 is generated at the same time in the sample 8, and therefore the two-dimensional photodetector 20
Can obtain an image of the light emission 10 without temporal blurring. Even when an optical gate means capable of high-speed gate operation is used in which temporal blurring is a particularly noticeable problem, a sharp luminescent image can be measured with high time resolution.

(Fifth Embodiment) Next, a fifth embodiment will be described. FIG. 5 is a configuration diagram of a time-resolved luminescence imaging apparatus according to the fifth embodiment. This embodiment includes two sets of a polarizer, an optical Kerr gate, and an analyzer,
The anisotropy of the emitted light can be measured by receiving light in each of two polarization directions of the emitted light which are orthogonal to each other.

In the present embodiment, the pulsed laser light source 1
The pulse excitation light 5 generated from the second harmonic generator 3 by converting one of the light beams output from the beam splitter 2 into a half wavelength and generating the half beam is a beam expander 4, a half wavelength plate 42, and a dichroic. The light emission 10 emitted from the sample 8 irradiated on the sample 8 through the mirror 6 and the objective lens 7 is
After passing through the objective lens 7 and the dichroic mirror 6, the excitation light absorption filter 9 is reached.

The light emission 10 that has passed through the excitation light absorption filter 9 is split into p-polarized light and s-polarized light by a polarization beam splitter (light-emission splitting means) 40. The p-polarized light flux of the light emission 10 that has passed through the polarization beam splitter 40 passes through the dichroic mirror 41 and then enters the optical cargate 12. Of the light emission 10, the s-polarized light flux reflected by the polarization beam splitter 40 passes through the dichroic mirror 43 and then enters the optical cargate 44.

On the other hand, the other light beam output from the pulse laser light source 1 and split by the beam splitter 2 is reflected by the movable right-angle mirror 14, is reflected by the plane mirror 15, and is expanded in beam diameter by the beam expander 17. The optical system until the gate pulse light 18 is formed is the same as that in the first embodiment.

This gate pulse light 18 has an intensity ratio of 1 by a beam splitter (gate pulse light branching means) 46.
The light is reflected / transmitted by the pair 1 and split into two light beams, one light beam is reflected by the dichroic mirror 41 and is incident on the optical Kerr gate 12, and the other light beam is reflected by the dichroic mirror 43 and is incident on the optical Kerr gate 44. To do.

The arrangement angle of the dichroic mirror 41 is such that the p-polarized component of the light emission 10 reaching from the polarization beam splitter 40 and the gate pulse light 18 transmitted through the beam splitter 46 are incident on the optical Kerr gate 12 at the same incident angle. Therefore, the light emission 10 passing through the optical cargate 12 is generated in the sample 8 at the same time. Then, the light emission 10 that has passed through this optical car gate 12
Goes through the analyzer 13 and passes through the gate pulse light absorption filter 3
9 and the two-dimensional photodetector 2 by the imaging lens 19.
An image is formed on the light receiving surface of 0. In this way, the two-dimensional photodetector 20 receives the light image of the p-polarized component of the light emission 10 generated at the sample 8 at the same time.

Similarly, the s-polarized component of the light emission 10 reaching the optical Kerr gate 44 from the polarization beam splitter 40,
By determining the arrangement angle of the dichroic mirror 43 so that the gate pulse light 18 reflected by the beam splitter 43 is incident at the same incident angle, the optical cargate 4
Light emission 10 passing through 4 is generated in the sample 8 at the same time. Then, the light emission 10 that has passed through the optical car gate 44 passes through the analyzer 45, passes through the gate pulse light absorption filter 47, and is imaged on the light receiving surface of the two-dimensional photodetector 49 by the imaging lens 48. In this way, the two-dimensional photodetector 49 receives the light image of the s-polarized light component of the light emission 10 generated in the sample 8 at the same time.

Here, it is preferable that each of the two-dimensional photodetectors 20 and 49 receives the light generated at the same time in the sample 8 for the same time. By doing so, the anisotropy of light emission can be immediately obtained by comparing the optical images received by the two-dimensional photodetectors 20 and 49. Gate pulse light 1 incident on the optical Kerr gates 12 and 44
8 has the same pulse width, because the same light flux was originally split by the beam splitter 46. Therefore, each of the two-dimensional photodetectors 20 and 49 emits light 1
0 is received for the same time. Further, the difference between the arrival times of the light emission 10 reaching the optical car gate 12 and the gate pulse light 18 and the light emission 10 reaching the optical car gate 44.
By arranging each optical component so that the difference between the arrival times of the gate pulse light 18 and the arrival time of the gate pulse light 18 becomes equal,
Each of the two-dimensional photodetectors 20 and 49 can receive the light emission 10 generated at the same time.

Then, the processing device 21 can input the light images received by the two-dimensional photodetectors 20 and 49, and can obtain the anisotropy of the light emission from the sample 8 based on the difference between the two. Further, by moving the movable right-angled mirror 14, the time variation of its anisotropy can be obtained, and the rotation relaxation time of the light emitting molecule can be obtained from the time variation of the anisotropy. Information about the movement of the light-emitting molecule can be obtained in a short cycle.

The present invention is not limited to the above embodiment, but can be variously modified. For example, the following configurations may be adopted.

In the above embodiment, the optical gate means composed of the polarizer, the optical Kerr gate and the analyzer is used. However, the optical gate means can be similarly realized by using the saturable absorber. The saturable absorber absorbs only weak luminescence generated in the sample when it is incident, but when it is irradiated with a gate pulse light with a large amount of light, the difference in the number distribution of molecules in each of the two levels involved in absorption transition is detected. Becomes smaller and saturation of absorption occurs, so that light emitted from the sample is transmitted without being absorbed. In this way, the saturable absorber can transmit the emitted light when illuminated by the gated pulsed light, ie it can be used as an optical gating means. For example, polymethine cyanide dye is used as the saturable absorber, and the response speed is about several tens of picoseconds. Further, if a polarizer is used together with this saturable absorber, it is possible to detect only a linearly polarized light component in a predetermined direction of light emission.

Further, when it is sufficient to measure only the average time change of the light emission generated from the sample, the photodetector for detecting the light flux passing through the optical gate means converts the light flux into an optical image. Instead of detecting, the light amount may be simply detected.

The optical path length varying means is on the optical path of the excitation optical system between the sample and the beam splitter (beam splitting means) for splitting the pulsed light output from the pulsed laser light source,
Alternatively, it may be provided not only in the gate optical system between the beam splitter (beam splitting means) and the optical gate means but also in the optical path of the light emission optical system between the sample and the optical gate means. In addition, any two of these three optical systems
It may be provided in one optical system, or may be provided in all of these three optical systems. Further, even when provided in a plurality of optical systems, each may set the optical path length independently, or may set the optical path length in conjunction with each other as in the third embodiment. Good.

The structure of the movable right-angle mirror and the fixed right-angle mirror is not limited to the structure described in the above embodiment, and for example, a corner cube-shaped retroreflector composed of three reflecting surfaces is used. Alternatively, a larger number of reflecting mirrors may be combined.

[0057]

As described above in detail, according to the present invention, the pulsed light output from the pulsed light source is first split into the first and second light fluxes by the light flux splitting means. The pulse excitation light formed by the excitation optical system based on the first light flux is applied to the sample to be measured, and the light emission generated by this is guided to the optical gate means by the light emission optical system. On the other hand, the gate pulse light formed by the gate optical system based on the second light flux is guided to the optical gate means. The emitted light is transmitted through the optical gate means and detected by the light detection means when the gate pulse light is incident, but cannot be transmitted otherwise. The optical path length difference between the optical path lengths of the excitation optical system and the emission optical system and the optical path length of the gate optical system is variably set by the optical path length varying means. As an optical gate means,
An optical Kerr medium or a saturable absorber is used.

Since the optical gate means is used in this manner, it is possible to perform a gate operation with a high speed and a short period for transmitting / blocking the light emission, and therefore, it is possible to perform a measurement with a high time resolution and a period of several tens MHz. Even when a pulsed light source that outputs pulsed light is used, all the pulsed light can be effectively used. In particular, the dynamics of a rapidly changing biological sample can be measured with the damage to the biological sample minimized.

Further, by using a pair of reflecting mirror groups, at least one of which is movable and reciprocating the light flux a plurality of times between each other, as the optical path length varying means, a large optical path length difference with respect to a slight amount of movement can be obtained. Therefore, in addition to being able to perform measurement with a high time resolution and a short period, it is also possible to perform time-resolved measurement of light emission over a long period of time.

Further, the optical gate means is formed into a flat plate shape, and the light emission and the gate pulse light are made incident on the flat plate surface symmetrically, so that the light emission generated at each position on the sample to be measured at the same time. Can be transmitted through the optical gate means, so that an image of the light emission can be detected without temporal blurring. Even when an optical gate means capable of high-speed gate operation, in which temporal blurring is a particularly noticeable problem, is used, a sharp luminescent image can be measured with high time resolution.

Further, by providing two sets of optical gate means and light detecting means and detecting linearly polarized light components of the emitted light in mutually different directions, the anisotropy of the emitted light can be obtained. The rotational relaxation time of the light emitting molecule can be obtained from the time change of anisotropy, and by this, information on the motion of the light emitting molecule can be obtained with a high time resolution and a short cycle.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a time-resolved luminescence imaging apparatus according to a first embodiment.

FIG. 2 is a configuration diagram of a time-resolved luminescence imaging apparatus according to a second embodiment.

FIG. 3 is a configuration diagram of a time-resolved luminescence imaging apparatus according to a third embodiment.

FIG. 4 is a configuration diagram of a time-resolved luminescence imaging apparatus according to a fourth embodiment.

FIG. 5 is a configuration diagram of a time-resolved luminescence imaging apparatus according to a fifth embodiment.

FIG. 6 is a configuration diagram of a conventional time-resolved luminescence imaging apparatus.

[Explanation of symbols]

1 ... Pulse laser light source, 2 ... Beam splitter, 3 ... Second harmonic generator, 4 ... Beam expander, 5 ... Pulse excitation light, 6 ... Dichroic mirror, 7 ... Objective lens,
8 ... Sample, 9 ... Excitation light absorption filter, 10 ... Emission, 11
... polarizer, 12 ... optical cargate, 13 ... analyzer, 14 ...
Movable right-angle mirror, 15 ... Planar mirror, 16 ... 1/2 wavelength plate, 17 ... Beam expander, 18 ... Gate pulse light, 19 ... Imaging lens, 20 ... Two-dimensional photodetector, 21 ...
Processing device, 22 ... Polarizing beam splitter, 23 ... Movable right-angle mirror, 24 ... Fixed right-angle mirror, 25 ... Quarter wave plate, 26 ... Fixed plane mirror, 27, 28 ... Planar mirror,
29 ... Polarizing beam splitter, 30 ... Movable right-angle mirror,
31 ... Fixed right-angle mirror, 32 ... Quarter wave plate, 33 ... Fixed plane mirror, 34, 35, 36 ... Plane mirror, 37 ...
Movable stage, 38 ... Planar mirror, 39 ... Gate pulse light absorption filter, 40 ... Polarizing beam splitter, 41 ...
Dichroic mirror, 42 ... 1/2 wave plate, 43 ... Dichroic mirror, 44 ... Optical cargate, 45 ... Analyzer, 46 ... Beam splitter, 47 ... Gate pulse light absorption filter, 48 ... Imaging lens, 49 ... Two-dimensional light Detector.

Claims (12)

[Claims] 1. A pulse light source that outputs pulsed light, a light beam splitting unit that splits the pulsed light into a first light beam and a second light beam, and pulse excitation light based on the first light beam. And an excitation optical system for irradiating the sample to be measured with the pulsed excitation light, and an emission optical system for injecting light emission generated by the pulsed excitation light to the sample to be measured to guide it to a predetermined position, A gate optical system that forms gate pulse light based on a second light flux and guides it to the predetermined position; and a gate optical system that is arranged at the predetermined position and transmits the light emission when the gate pulse light is incident, and otherwise. First optical gate means that does not transmit the emitted light; first light detection means that detects the emitted light emitted from the first optical gate means; optical paths of the excitation optical system and the emission optical system Long and above Time-resolved emission imaging apparatus comprising: the optical path length changing means for setting the optical path length difference, the between the optical path length of over preparative optics. 2. The optical path length varying means comprises: a pair of reflecting mirror groups arranged to face each other on an optical path of the gate optical system; and at least one of the pair of reflecting mirror groups, A moving unit that moves in a direction parallel to the optical axis of the light beam that has arrived from the beam splitting unit, wherein the light beam that has arrived from the light beam splitting unit is reciprocated at least once between the pair of reflecting mirror groups. The time-resolved luminescence imaging device according to claim 1, wherein the time-resolved luminescence imaging device emits the light toward the predetermined position. 3. The optical path length varying means is arranged on the optical path of the excitation optical system so as to face each other.
A pair of reflecting mirror groups; and a moving unit that moves at least one of the pair of reflecting mirror groups in a direction parallel to the optical axis of the light beam that has arrived from the light beam splitting unit. The time-resolved luminescence imaging apparatus according to claim 1, wherein the arrived light flux is reciprocated at least once between the pair of reflecting mirror groups and then emitted toward the sample to be measured. 4. The optical path length varying means is arranged on the optical path of the light emitting optical system so as to face each other.
A pair of reflecting mirror groups, and a moving unit that moves at least one of the pair of reflecting mirror groups in a direction parallel to the optical axis of the light flux reaching from the sample to be measured. The time-resolved luminescence imaging apparatus according to claim 1, wherein the arrived light flux is reciprocated at least once between the pair of reflecting mirror groups and then emitted toward the predetermined position. 5. The optical path length varying means increases one of the optical path lengths of the gate optical system and the excitation optical system or the optical path lengths of the gate optical system and the light emitting optical system and decreases the other. The time-resolved luminescence imaging device according to claim 1. 6. A light emission branching unit which is provided on an optical path of the light emission optical system, reflects a part of the light emission, transmits the remaining light, and makes the transmitted light emission enter the first optical gate unit, Gate pulse light branching means provided on the optical path of the gate optical system for reflecting a part of the gate pulse light, transmitting the rest, and making the transmitted gate pulse light incident on the first optical gate means; The light emission reflected by the light emission branching means and the gate pulse light reflected by the gate pulse light branching means are made incident, the light emission is transmitted when the gate pulse light is made incident, and the light is transmitted otherwise. Further comprising: a second optical gate unit that does not transmit light emission; and a second photodetector unit that detects the light emission emitted from the second optical gate unit. Serial second photodetector means, respectively, for detecting respective different directions of the linearly polarized light component of the light emission, time-resolved emission imaging apparatus according to claim 1, wherein a. 7. The first optical gate means has a flat plate shape, and makes the light emission and the gate pulse light incident symmetrically with respect to the flat plate surface. Time-resolved luminescence imaging device. 8. The second optical gate means has a flat plate shape, and causes the light emission and the gate pulse light to enter symmetrically with respect to the flat plate surface. Time-resolved luminescence imaging device. 9. The first optical gate means includes a polarizer that receives the emitted light and emits a linearly polarized light beam in a first direction, and the gate pulsed light and the light beam emitted from the polarizer. Incident light, the birefringence index changes according to the light amount of the gate pulse light, and the light flux emitted from the polarizer is converted into elliptically polarized light having an ellipticity according to the light amount of the gate pulse light and emitted. An optical Kerr gate that utilizes the optical Kerr effect, and an analyzer that receives the light beam emitted from the optical Kerr gate and emits a linearly polarized light beam in a second direction orthogonal to the first direction. The time-resolved luminescence imaging device according to claim 1. 10. The second optical gating means includes a polarizer that receives the emitted light and emits a linearly polarized light beam in a third direction; and a light beam emitted from the gate pulse light and the polarizer. Incident light, the birefringence index changes according to the light amount of the gate pulse light, and the light flux emitted from the polarizer is converted into elliptically polarized light having an ellipticity according to the light amount of the gate pulse light and emitted. An optical Kerr gate that utilizes the optical Kerr effect, and an analyzer that receives the light beam emitted from the optical Kerr gate and emits a linearly polarized light beam in a fourth direction orthogonal to the third direction. The time-resolved luminescence imaging device according to claim 6. 11. The first optical gate means comprises a saturable absorber that transmits the light emission when the gate pulse light is incident and does not transmit the light emission when the gate pulse light is not incident. The time-resolved luminescence imaging apparatus according to claim 1. 12. The second optical gate means comprises a saturable absorber that transmits the emitted light when the gate pulse light is incident and does not transmit the emitted light otherwise. The time-resolved luminescence imaging device according to claim 6.