WO2018138794A1

WO2018138794A1 - Optical-scanning-type microscope device and method for measuring distance between spots in optical-scanning-type microscope device

Info

Publication number: WO2018138794A1
Application number: PCT/JP2017/002482
Authority: WO
Inventors: 厚志土井
Original assignee: オリンパス株式会社
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2018-08-02

Abstract

This optical-scanning-type microscope device (100) is provided with a light source unit (1) for outputting two illumination lights (L1, L2) having a relative angle to each other, a scanning unit (2) for scanning the two illumination lights (L1, L2), an objective optical system (3) for radiating the scanned two illumination lights (L1, L2) to a sample (A) and condensing the illumination lights (L1, L2) at mutually different condensing positions, a detection optical system (6) for detecting a signal light generated in the condensing position, and an angle detection unit (7) for detecting the relative angle between the two illumination lights (L1, L2) in an approximate pupil position of the objective optical system (3).

Description

Optical scanning microscope apparatus and spot distance measuring method in optical scanning microscope apparatus

The present invention relates to an optical scanning microscope apparatus that scans two illumination lights condensed at different positions, and a spot-to-spot distance measuring method in the optical scanning microscope apparatus.

Conventionally, there has been proposed an optical scanning microscope in which two spots are formed by condensing two illumination lights at different positions of a sample and scanning the two spots (see, for example, Patent Document 1).

International Publication No. 2015/161631

In the optical scanning microscope of Patent Document 1, accurate control of the distance between two spots is important, but it is difficult to grasp the distance between the two spots. In particular, in the absence of a sample, it is more difficult to grasp the distance between two spots.

The present invention has been made in view of the above-described circumstances, and is an optical scanning microscope apparatus that scans two illumination lights condensed at different positions, and two illuminations even in the absence of a sample. An object of the present invention is to provide an optical scanning microscope apparatus that can measure the distance between spots of light and a method for measuring the distance between spots in the optical scanning microscope apparatus.

In order to achieve the above object, the present invention provides the following means.
According to an aspect of the present invention, a light source unit that outputs two illumination lights having a relative angle to each other, a scanning unit that scans the two illumination lights output from the light source unit, and the scan that is scanned by the scanning unit An objective optical system that irradiates the sample with two illumination lights and collects them at different condensing positions; a detection optical system that detects signal light generated at the condensing position; and the pupil optical system at the substantially pupil position of the objective optical system. An optical scanning microscope apparatus including an angle detection unit that detects a relative angle between two illumination lights.

According to this aspect, when two illumination lights emitted from the light source unit are irradiated onto the sample from the objective optical system while being scanned by the scanning unit, signal light is generated at each irradiation position of the illumination light, and the signal Light is detected by a detection optical system. Therefore, an image of the sample can be generated by associating the detected intensity of the signal light with the irradiation position of the illumination light.

In this case, two illumination lights having a relative angle are incident on the pupil of the objective optical system at different angles, and are condensed at two condensing positions separated by a distance corresponding to the relative angle to form a spot. . Therefore, based on the relative angle between the two illumination lights detected by the angle detector at the approximate pupil position of the objective optical system, the distance between the spots can be measured even in the absence of the sample.

In the above aspect, an angle adjustment unit that adjusts a relative angle between the two illumination lights at a substantially pupil position of the objective optical system may be provided. The angle adjustment unit may automatically adjust the relative angle between the two illumination lights to a predetermined angle based on the relative angle detected by the angle detection unit.
By doing in this way, based on the relative angle between the two illumination lights detected by the angle detector, the relative angle can be adjusted manually or automatically.

In the above aspect, the angle detection unit is disposed at a position optically conjugate with a substantially pupil position of the objective optical system, and captures an interference fringe image by photographing the interference fringes of the two illumination lights. You may have.
A pattern of interference fringes corresponding to the relative angle between the two illumination lights is formed at the position of the camera conjugate with the pupil position of the objective optical system. Therefore, the relative angle between the two illumination lights can be detected based on the interference fringe pattern in the interference fringe image. When the two illumination lights are pulse lights, interference fringes are formed when the pulse timings of the two illumination lights are simultaneous, and no interference fringes are formed when the pulse timings of the two illumination lights are non-simultaneous. Therefore, the simultaneous and non-simultaneous detection of the pulse timings of the two illumination lights can be detected based on the presence or absence of interference fringes.

In the above aspect, the angle detection unit may calculate the relative angle based on the interference fringe image.
In this way, the relative angle between the two illumination lights can be quantitatively detected from the interference fringe image.

In the above aspect, the angle detection unit may include a polarization changing element that changes a polarization state of the two illumination lights incident on the camera.
Two illumination lights whose polarization directions are orthogonal to each other do not form interference fringes. Even when the polarization directions of the two illumination lights from the light source unit are orthogonal to each other, the polarization changing element is adjusted so that the polarization directions of the two illumination lights are not orthogonal to each other, thereby forming interference fringes between the spots. Distance measurements can be made.

In the above aspect, the angle detection unit may include an angle changing element that changes a relative angle between the two illumination lights incident on the camera.
By doing in this way, this interference fringe can be adjusted so that the interference fringe suitable for the detection of a relative angle is formed.

In the said aspect, you may provide the optical path length difference calculation part which calculates the optical path length difference of said two illumination light based on the contrast of the said interference fringe image.
When the two illumination lights are pulse lights, the contrast of light and darkness of the interference fringes changes according to the relative pulse timing of the two illumination lights, that is, the optical path length difference between the two illumination lights. Therefore, the optical path length difference between the two illumination lights can be calculated based on the contrast of the interference fringe image.

In the above aspect, an optical path length difference adjustment unit that adjusts an optical path length difference between the two illumination lights based on the optical path length difference calculated by the optical path length difference calculation unit may be provided.
By doing in this way, the relative pulse timing of the two illumination lights in a sample can be adjusted based on the optical path length difference calculated by the optical path length difference calculation part.

Another aspect of the present invention is an optical scanning microscope apparatus that scans the two illumination lights that irradiate the sample with two illumination lights having relative angles to each other from the objective optical system and collect the lights at different collection positions. A spot-to-spot distance measuring method for measuring a distance between the two illumination light spots formed at the condensing position, wherein a relative angle between the two illumination lights at a substantially pupil position of the objective optical system is detected. It is the distance measurement method between spots in the optical scanning microscope apparatus including the process to do.

According to the present invention, in an optical scanning microscope apparatus that scans two illumination lights condensed at different positions, the distance between two illumination light spots can be measured even in the absence of a sample. There is an effect.

1 is an overall configuration diagram of an optical scanning microscope apparatus according to a first embodiment of the present invention. It is a figure explaining two laser beams which inject into an objective lens and a camera. It is a block diagram of the excitation light production | generation part in the optical scanning microscope apparatus of FIG. It is a block diagram of the modification of the excitation light production | generation part in the optical scanning microscope apparatus of FIG. It is a flowchart which shows the distance measurement method between spots by the optical scanning microscope apparatus of FIG. It is a block diagram of the modification of the interference fringe measurement part in the optical scanning microscope apparatus of FIG. It is a block diagram of the other modification of the interference fringe measurement part in the optical scanning microscope apparatus of FIG. It is a block diagram of the other modification of the interference fringe measurement part in the optical scanning microscope apparatus of FIG. It is a figure explaining the change of the pattern of an interference fringe accompanying the change of the relative angle between two laser beams. It is a figure which shows an example of the interference fringe image acquired with the camera. It is a figure which shows the FFT result of the interference fringe image of FIG. 8A. It is a whole block diagram of the optical scanning microscope apparatus which concerns on the 2nd Embodiment of this invention. It is a block diagram of the excitation light production | generation part in the optical scanning microscope apparatus of FIG. It is a figure which shows an example of the interference fringe formed in the position of a camera, when the pulse timing of the 1st and 2nd laser beam is simultaneous. It is a figure which shows an example of the image of the 1st and 2nd laser beam formed in the position of a camera when the pulse timing of a 1st and 2nd laser beam is non-simultaneously.

(First embodiment)
An optical scanning microscope apparatus 100 according to a first embodiment of the present invention will be described with reference to FIGS. 1 to 8B.
The optical scanning microscope apparatus 100 according to this embodiment irradiates a sample A with laser beams (illumination beams) L1 and L2, and excites a fluorescent substance having an excitation wavelength substantially equal to the wavelength of the laser beams L1 and L2. It is a laser scanning confocal microscope apparatus for excitation fluorescence observation.

As shown in FIG. 1, the optical scanning microscope apparatus 100 outputs two laser beams L1 and L2 and adjusts the relative angle between the two laser beams L1 and L2 (light source unit). ) 1, the scanning unit 2 that scans the laser beams L 1 and L 2, and the fluorescence generated by irradiating the sample A with the laser beams L 1 and L 2 scanned by the scanning unit 2 and at the focusing positions of the laser beams L 1 and L 2 An objective lens (objective optical system) 3 that collects (signal light) and pupil projection that is arranged between the scanning unit 2 and the objective lens 3 and relays the laser beams L1 and L2 from the scanning unit 2 to the pupil position of the objective lens 3 A lens 4 and an imaging lens 5; a detection optical system 6 that detects fluorescence collected by the objective lens 3 and converts it into a detection signal; and laser light L1, at a position optically conjugate with the pupil position of the objective lens 3. Measure L2 interference fringes Interference fringe measurement unit (angle detection unit) 7 and a processing device (angle detection unit) that creates a fluorescence image of the sample A based on the detection signal and calculates the relative angle between the laser beams L1 and L2 based on the interference fringe ) 8.

The configuration of the excitation light generator 1 will be described in detail later.
The scanning unit 2 is, for example, a proximity galvano scanner having two galvanometer mirrors arranged close to each other so as to be swingable about a non-parallel axis, and the laser beams L1 and L2 are used as the optical axes of the laser beams L1 and L2. Scan in two intersecting directions.
The pupil projection lens 4 and the imaging lens 5 are configured such that the scanning unit 2 is disposed at a position optically conjugate with the pupil position of the objective lens 3, and the laser beams L 1 and L 2 scanned by the scanning unit 2 are provided. Relay to the pupil position of the objective lens 3.

As shown in FIG. 2, the first and second laser beams L1 and L2 having a relative angle θ are incident on the objective lens 3 at different angles and are condensed at different condensing positions P1 and P2, respectively. A spot is formed. The distance between the two spots (between the condensing positions P1 and P2) is determined according to the relative angle θ between the laser beams L1 and L2. Fluorescence generated in the two spots is collected by the objective lens 3 and travels in the opposite direction along the same optical path as the laser beams L1 and L2.

The detection optical system 6 is disposed between the excitation light generation unit 1 and the scanning unit 2 and transmits the laser beams L1 and L2, and reflects the fluorescence to split the fluorescence from the optical path of the laser beams L1 and L2. A mirror 61, a condensing lens 62 for condensing the fluorescence branched by the dichroic mirror 61, a pinhole 63 disposed at a position optically conjugate with the condensing position P1 of the first laser light L1, And a photodetector 64 that detects the fluorescence that has passed through the pinhole 63 and transmits a detection signal corresponding to the intensity of the fluorescence to the processing device 8.

The interference fringe measuring unit 7 includes a non-polarizing beam splitter 71 disposed between the imaging lens 5 and the objective lens 3, and a digital camera 72 disposed at a position optically conjugate with the pupil position of the objective lens 3. It has.
The non-polarizing beam splitter 71 branches a part (for example, 1%) of the laser beams L 1 and L 2 emitted from the imaging lens 5 toward the objective lens 3 toward the camera 72.

Since the pupil of the objective lens 3 and the camera 72 are optically conjugate to each other, the relative angle between the first and second laser beams L1 and L2 at the pupil position of the objective lens 3 and the position of the camera 72 The relative angles between the first and second laser beams L1 and L2 are equal to each other. Therefore, interference fringes corresponding to the relative angle θ between the first and second laser beams L1 and L2 at the pupil position are formed at the position of the camera 72. The camera 72 captures the interference fringe to acquire the interference fringe image, and transmits the interference fringe image to the processing device 8.

The camera 72 may be arranged at another position optically conjugate with the pupil position of the objective lens 3. For example, the laser beam L1, L2 may be relayed from the camera 72 arranged at an arbitrary position to the scanning unit 2.

The processing device 8 generates a fluorescent image by associating the detection signal received from the photodetector 64 with the scanning position of the laser light L1 by the scanning unit 2.
Further, the processing device 8 analyzes the interference fringe image received from the interference fringe measurement unit 7 and calculates the relative angle θ between the first and second laser beams L1 and L2 at the pupil position of the objective lens 3. Since there is a certain relationship between the light-dark cycle of the interference fringes and the relative angle θ between the laser beams L1 and L2, the relative angle θ is calculated based on the light-dark cycle of the interference fringes in the interference fringe image. be able to. Next, the processing device 8 converts the relative angle θ into a distance between two spots, and outputs a control signal for adjusting the relative angle θ so that the obtained distance matches a predetermined distance. 1 to send. The predetermined distance is, for example, a distance set by the user via an input unit (not shown).
The processing device 8 that executes such image generation, calculation, and control processing is realized by, for example, a computer.

Next, the excitation light generation unit 1 will be described.
As shown in FIG. 3, the excitation light generation unit 1 uses a single laser light source 11 that emits continuous-wave laser light, and the beam diameter of the laser light emitted from the laser light source 11 as the pupil diameter of the objective lens 3. A beam diameter adjusting optical system 12 that adjusts the size in combination, a first polarization beam splitter (PBS) 13 that divides the laser light from the laser light source 11 into two according to the polarization, the laser light source 11 and the first PBS 13. And a half-wave plate 14 that adjusts the polarization direction of the laser light incident on the first PBS 13 and the two laser lights L1 and L2 divided by the first PBS 13 are coaxially multiplexed. A second polarization beam splitter (PBS) 15 and a relay lens 16 that relays the combined laser beams L1 and L2 from the second PBS 15 to the scanning unit 2 are provided. Between the second PBS 15 and the relay lens 16, a quarter wavelength plate 17 for converting the laser beams L1 and L2 into circularly polarized light may be provided.

The light quantity ratio between the two laser beams L1 and L2 divided by the first PBS 13 is determined according to the polarization direction of the laser beam incident on the first PBS 13. For example, the half-wave plate 14 adjusts the polarization direction of the laser light to 45 ° with respect to the two polarization axes of the first PBS 13 so that the light amounts of the two laser lights L1 and L2 are equal.

Of the two laser beams L1 and L2 divided by the first PBS 13, the first laser beam L1 passes through the second PBS 15, and the second laser beam L2 is reflected by the second PBS 15. The first and second laser beams L1 and L2 are combined with each other. Reference numeral 20 denotes a mirror that forms an optical path of the first and second laser beams L1 and L2.

The second PBS (angle adjustment unit) 15 is provided so as to be swingable around a swing axis in a direction perpendicular to the optical axes of the laser beams L1 and L2 (a direction perpendicular to the paper surface of FIG. 3). When the tilt angle around the oscillation axis of the second PBS 15 changes, the emission angle of the first laser light L1 from the second PBS 15 is negligibly small, but the second laser from the second PBS 15 The emission angle of the light L2 changes. Therefore, the relative angle between the first and second laser beams L1 and L2 can be adjusted by the tilt angle of the second PBS 15.

The second PBS 15 is provided with a motor 18 for changing the tilt angle of the second PBS 15 by swinging the second PBS 15 about the swing axis. The motor 18 is driven in accordance with a control signal from the processing device 8 to change the tilt angle of the second PBS 15, so that the relative angle θ between the first and second laser beams L1 and L2 is automatically adjusted. It has become.

The second PBS 15 is disposed at a position optically conjugate with the scanning unit 2 by the relay lens 16. That is, since the second PBS 15 is disposed at a position optically conjugate with the pupil position of the objective lens 3 via the scanning unit 2, the second PBS 15 is positioned at the pupil position of the objective lens 3 depending on the tilt angle of the second PBS 15. The distance between the spots can be controlled by adjusting the relative angle between the first and second laser beams L1 and L2.

Note that the optical path configuration of the excitation light generation unit 1 shown in FIG. 3 is an example, and an arbitrary optical path configuration is adopted as long as two laser beams L1 and L2 having a relative angle with each other can be output. Can do. For example, FIG. 3 shows a Mach-Zehnder type as an example of a split optical path of laser light, but other types of optical paths such as a Michelson type may be adopted. Further, a non-polarizing beam splitter may be used instead of the

PBSs

13 and 15 for dividing and multiplexing the laser light. Further, instead of a configuration in which a single laser beam is divided to generate two laser beams L1 and L2, as shown in FIG. 4, a first laser beam L1 and a second laser beam L2 are emitted, respectively. A configuration including two

laser light sources

11A and 11B may be employed.

Next, the operation of the optical scanning microscope apparatus 100 configured as described above will be described.
In order to perform fluorescence observation of the sample A using the optical scanning microscope apparatus 100 according to the present embodiment, the user sets a distance between two spots as shown in FIG. 5 (step S1). The processing device 8 sets the relative angle between the first and second laser beams L1 and L2 to an angle corresponding to the distance set by the user, and the set relative angle is set to the two laser beams L1 and L2. The tilt angle of the second PBS 15 is set so as to be given (step S2).

Next, output of laser light from the laser light source 11 is started (step S3). In the excitation light generation unit 1, the polarization direction of the laser light output from the laser light source 11 is adjusted by the half-wave plate 14, divided into two by the first PBS 13, and then multiplexed by the second PBS 15. Is done. At the time of multiplexing, the relative angles set in step S2 are given to the two laser beams L1 and L2.

The two laser beams L1 and L2 given relative angles in the second PBS 15 are relayed to the scanning unit 2 by the relay lens 16, scanned by the scanning unit 2, and from the scanning unit 2 to the pupil position of the objective lens 3. Relayed by the pupil projection lens 4 and the imaging lens 5. The two laser beams L1 and L2 are incident on the objective lens 3 in a state having a relative angle, and are respectively connected to two condensing positions P1 and P2 separated by a distance set in step S1 to form a spot.

The fluorescence generated at the two condensing positions P1 and P2 of the sample A is collected by the objective lens 3, and passes through the non-polarizing beam splitter 71, the imaging lens 5, the pupil projection lens 4, and the scanning unit 2, and then the dichroic mirror 61. , And is separated from the optical paths of the laser beams L1 and L2 by the dichroic mirror 61.

For example, when applied to Patent Document 1, the fluorescence generated at the condensing position P1 of the first laser beam L1 optically conjugate with the pinhole 63 is condensed by the condensing lens 62 and passes through the pinhole 63. Passed and detected by the photodetector 64, a detection signal is transmitted to the processing device 8. In the processing device 8, a fluorescence image of the sample A is generated based on the detection signal. On the other hand, the fluorescence generated at the condensing position P2 of the second laser beam L2 that is optically unconjugated with the pinhole 63 cannot pass through the pinhole 63 and is blocked at the pinhole 63.

Here, the relative angle θ between the laser beams L1 and L2 is detected by the interference fringe measuring unit 7 and the processing device 8 using a part of the laser beams L1 and L2 before entering the objective lens 3. That is, a part of the laser beams L 1 and L 2 is branched toward the camera 72 by the non-polarizing beam splitter 71 before the objective lens 3. At the position of the camera 72 that is optically conjugate with the pupil position of the objective lens 3, the laser light L1 and the laser light L2 interfere with each other to form an interference fringe pattern according to the relative angle θ. Is acquired by the camera 72 (step S4). Then, in the processing device 8, the relative angle θ is calculated from the acquired interference fringe image (step S5), and when the relative angle θ is deviated from the angle set in step S2, it matches the set angle. The tilt angle of the second PBS 15 is automatically adjusted by the motor 18 (step S6). Thereby, the distance between the spots is automatically controlled so as to be maintained at the distance set in step S1.

Thus, according to the present embodiment, the relative angle θ between the laser beams L1 and L2 is determined by measuring the interference fringes of the laser beams L1 and L2 at a position optically conjugate with the pupil position of the objective lens 3. There is an advantage that the distance between two spots can be grasped from the measured relative angle θ. Further, by feedback controlling the tilt angle of the second PBS 15 so that the actually measured relative angle θ matches the set angle, the distance between the spots can be accurately controlled to the distance set by the user. There is an advantage. Further, since the interference fringes of the laser beams L1 and L2 before being irradiated onto the sample A are used, there is an advantage that the distance between the two spots can be easily grasped regardless of the presence or absence of the sample A.

In the pupil position of the objective lens 3, interference fringes appear when the two laser beams L1 and L2 are relatively tilted or shifted. However, since the PBS 15 is optically conjugated to the pupil position of the objective lens 3, even when the tilt angle of the PBS 15 is changed, the relative shift of the laser beams L1 and L2 at the pupil position of the objective lens 3 does not occur. The relative angle between the lights L1 and L2 has a dominant influence on the interference fringes. Therefore, there is an advantage that the distance between the spots can be accurately measured based on the relative angle between the laser beams L1 and L2 obtained from the measurement of the interference fringes.

Here, the measurement accuracy of the distance between spots using the interference fringes of the laser beams L1 and L2 will be described.
Assume that the objective lens 3 having a numerical aperture of 1.05, a magnification of 30 times, and a pupil diameter of 12.6 mm and laser beams L1 and L2 having a wavelength λ = 488 nm are used. In the analysis of the interference fringe image acquired by the camera 72, the contrast of light and darkness of the interference fringe having the λ / 10 period can be detected. The relative angle θ corresponding to the interference fringes of λ / 10 period is a geometric relationship between the optical path length difference between the first and second laser beams L1 and L2 incident on the pupil having a diameter of 12.6 mm and the relative angle θ. Is calculated as follows.
θ = arctan ((488 [nm] / 10) /12.6 [mm])
= 2.2 × 10 ⁻⁴ [deg]
When the relative angle θ = 2.2 × 10 ⁻⁴ is converted into the distance between the two spots, it is about 23 nm. That is, it can be seen that a change in the distance between the spots sufficiently smaller than the wavelength λ of the laser beams L1 and L2 can be detected using the interference fringe image, and the distance between the spots can be measured with high accuracy.

Note that the second laser light L2 condensed at the condensing position P2 that is optically non-conjugated with the pinhole 63 is used, for example, for noise removal of the fluorescent image based on the fluorescence generated at the condensing position P1.
Specifically, the first laser light L1 generates fluorescence not only in the condensing position P1, but also in the path to the condensing position P1 in the sample A. Therefore, the fluorescence detected through the pinhole 63 and detected by the photodetector 64 includes a part of the fluorescence generated at a position other than the condensing position P1 as noise light.

On the other hand, the second laser beam L2 also generates fluorescence not only in the condensing position P2 but also in the path to the condensing position P2 in the sample A. The fluorescence generated at the condensing position P2 that is optically non-conjugated with the pinhole 63 is blocked without passing through the pinhole 63, but a part of the fluorescence generated at a position other than the condensing position P2 is: The light passes through the pinhole 63 and is detected by the photodetector 64. Most of the fluorescence generation range detected at this time coincides with the noise light generation range of the first laser beam L1.

Therefore, a noise image including only noise light information can be obtained based on the fluorescence detected by the photodetector 64 while irradiating the sample A with only the second laser light L2. And the noise contained in a fluorescence image can be removed using a noise image. In order to irradiate the sample A with only the second laser light L2, for example, the half-wave plate 14 so that the light quantity ratio between the first laser light L1 and the second laser light L2 is 0: 100. What is necessary is just to adjust the rotation angle.

In the present embodiment, the interference fringe measuring unit 7 includes a variable magnification optical system 74, a polarization changing element 75, and an angle changing element between the non-polarizing beam splitter 71 and the camera 72, as shown in FIGS. 6A to 6C. 76 may be provided. The variable magnification optical system 74, the polarization changing element 75, and the angle changing element 76 may be used in appropriate combination.
The interference fringe measuring unit 7 in FIG. 6A includes a variable magnification optical system 74 for optically enlarging or reducing the interference fringes formed at the position of the camera 72.

The interference fringe measuring unit 7 in FIG. 6B includes a polarization changing element 75 that changes the polarization state of the first and second laser beams L1 and L2. When the polarization direction of the first laser light L1 and the polarization direction of the second laser light L2 are orthogonal to each other, no interference fringes are generated between the first laser light L1 and the second laser light L2. In such a case, interference fringes are observed by adjusting the polarization direction of the first laser light L1 and the polarization direction of the second laser light L2 to be non-orthogonal by the polarization changing element 75. be able to.

The interference fringe measuring unit 7 in FIG. 6C includes an angle changing element 76 that changes the relative angle between the first and second laser beams L1 and L2 between the non-polarizing beam splitter 71 and the polarization changing element 75, for example, Lotion. A polarizing prism is provided. The Rochon polarizing prism causes one of two laser beams incident on the Rochon polarizing prism and whose polarization directions are orthogonal to each other to go straight and emits the other obliquely. Therefore, an offset angle is added to the relative angle θ between the first and second laser beams L1 and L2 that have passed through the Rochon polarizing prism. The angle changing element 76 may be configured to be able to select whether to use or to change the offset angle.

The light-dark cycle and contrast of the interference fringes change according to the optical path length difference between the laser beams L1 and L2. Therefore, by adjusting the optical path length difference by giving an offset to the relative angle between the two laser beams L1 and L2 by the Lotion polarization prism so that an interference fringe suitable for detection of the relative angle θ is formed. The detection accuracy of θ can be improved. Further, even when the laser beams L1 and L2 are overlapped (the relative angle given by the second PBS 15 is zero), it is possible to detect the spot position by generating interference fringes. In this modification, the relative angle θ is calculated with the offset angle taken into account.

FIG. 7 shows an example of a change in interference fringes accompanying a change in the relative angle θ between the laser beams L1 and L2 in the modification including the angle changing element 76 in FIG. 6C.
Even when the relative angle θ applied to the laser beams L1 and L2 by the second PBS 15 is zero, an offset angle is given to the laser beams L1 and L2 incident on the camera 72 by the angle changing element 76. As a result, interference fringes (see “no tilt” in FIG. 7) occur.

When the relative angle θ is changed by the second PBS 15, the light / dark cycle of the interference fringes (see “X direction tilt (+)” and “X direction tilt (−)” in FIG. 7) becomes smaller or larger. The number of fringes in the interference fringe image increases or decreases. Due to misalignment of the optical elements on the optical paths of the laser beams L1 and L2, the laser beams L1 and L2 are in a direction (Y direction) perpendicular to the tilt direction of the second laser beam L2 by the second PBS 15. When tilted, interference fringes rotated clockwise or counterclockwise (see “Y direction tilt (+)” and “Y direction tilt (−)” in FIG. 7) are obtained. Therefore, the relative angle θ between the first and second laser beams L1 and L2 and the tilt angle in the Y direction can be specified based on the bright / dark cycle and rotation angle of the interference fringes.

In the present embodiment, fast Fourier transform (FFT) may be used for the analysis of the interference fringe image by the processing device 8. FIG. 8B shows the FFT result of the interference fringes shown in FIG. 8A. As shown in FIG. 8B, spots Q1 and Q2 appear in the Fourier space at positions corresponding to the spatial frequency of the interference fringes in addition to the DC component. The distance d between the spots Q1 and Q2 corresponds to the relative angle θ, and the inclination angle δ of the line segment connecting the spots Q1 and Q2 corresponds to the tilt direction of the second laser light L2. Therefore, the relative angle θ and the tilt direction can be easily detected by FFT analysis of the interference fringe image.

In the present embodiment, the processing device 8 automatically adjusts the tilt angle of the second PBS 15 based on the relative angle θ calculated from the interference fringe image, but instead of or in addition to this, The user may be configured to adjust the tilt angle of the second PBS 15. For example, the user may adjust the tilt angle of the second PBS 15 while observing the interference fringe image displayed on the display.

(Second Embodiment)
Next, an optical scanning microscope apparatus 200 according to a second embodiment of the present invention will be described with reference to FIGS. 9 to 11B. In the present embodiment, a configuration different from that of the first embodiment will be described, and the same reference numerals are given to configurations common to the first embodiment, and description thereof will be omitted.

The optical scanning microscope apparatus 200 according to the present embodiment is a laser scanning microscope apparatus for two-photon excitation fluorescence observation that excites a fluorescent material having an excitation wavelength that is approximately ½ times the wavelength of the laser beams L1 and L2. . In the two-photon excitation, since the pulse laser beams L1 and L2 are used as the excitation light, it is necessary to control the timing between the two pulse laser beams L1 and L2, and therefore, adjustment of the optical path length difference between the laser beams L1 and L2 is required. Is required. The optical scanning microscope apparatus 200 is mainly different from the first embodiment in that it further includes a mechanism for adjusting the optical path length difference between the laser beams L1 and L2.

As shown in FIG. 9, the optical scanning microscope apparatus 200 includes an excitation light generation unit 10, a scanning unit 2, an objective lens 3, a pupil projection lens 4, an imaging lens 5, and a detection optical system 60. The interference fringe measuring unit 7 and the processing device 8 are provided.
The detection optical system 60 includes a dichroic mirror 61, a condenser lens 62, and a photodetector 64, and does not include a pinhole.

As shown in FIG. 10, the excitation light generation unit 10 includes a laser light source 111 that emits pulsed laser light, a beam diameter adjusting optical system 12, a first PBS 13, a half-

wave plate

14, 2 PBS 15, relay lens 16, and optical path length adjusting optical system (optical path length difference adjusting unit) 19 provided in the optical path of the second laser beam L 2 between the first PBS 13 and the second PBS 15. I have. A quarter wavelength plate 17 may be provided between the second PBS 15 and the relay lens 16.

The optical path length adjusting optical system 19 changes the optical path length of the second laser light L2 by moving the pair of

mirrors

19a and 19b in the direction of arrow B by a motor (not shown). Thereby, the first and second laser beams L1 and L2 are combined so that the timings of the pulses of the first and second laser beams L1 and L2 after being combined by the second PBS 15 are the same or non-simultaneous. The optical path length difference is adjusted.

When the optical path length difference between the laser beams L1 and L2 is zero and the pulse timings of the two laser beams L1 and L2 at the pupil position of the objective lens 3 are the same, as shown in FIG. Since the interference fringes are formed, the contrast of light and dark in the interference fringe image becomes high. On the other hand, when there is an optical path length difference between the laser beams L1 and L2 and the pulse timings of the two laser beams L1 and L2 at the pupil position of the objective lens 3 are not simultaneous, as shown in FIG. Since no interference fringe is formed at the position of, the contrast of light and dark in the interference fringe image is lowered.

The processing device 8 drives the motor of the optical path length adjusting optical system 19 so that the pulse timings of the laser beams L1 and L2 at the pupil position are the same or non-simultaneous based on the contrast intensity of the dark and light in the interference fringe image. The optical path length difference between the laser beams L1 and L2 is adjusted.

Further, when the optical path length of the second laser light L2 is gradually changed by the optical path length adjusting optical system 19, the intensity of the contrast of the light and darkness of the interference fringes is gradually increased in a period corresponding to the pulse width of the laser lights L1 and L2. When the first laser beam L1 and the second laser beam L2 completely coincide with each other in time, the contrast becomes maximum, and the first laser beam L1 and the second laser beam L2 vary with time. The contrast is minimized when completely separated. That is, the contrast intensity of the interference fringe image correlates with the optical path length difference between the laser beams L1 and L2. Based on the correlation between the contrast of the interference fringe image and the optical path length difference, the processing device (optical path length calculation unit) 8 calculates the optical path length difference between the laser beams L1 and L2 from the interference fringe image. Also good.

The profile of the contrast intensity of the interference fringe image acquired while the optical path length is gradually changed by the optical path length adjusting optical system 19 includes the pulse width of the laser beams L1 and L2 and the time difference between the pulses of the laser beams L1 and L2. Information is included. The processing device 8 may measure the pulse width of the laser beams L1 and L2 using the profile, or may measure the overlap width of the pulses between the laser beams L1 and L2.

According to the present embodiment, by measuring the interference fringes of the laser beams L1 and L2 at a position optically conjugate with the pupil position of the objective lens 3, the simultaneous and non-simultaneous timing of the pulse timing of the laser beams L1 and L2 can be grasped. There is an advantage that you can. In addition, since the interference fringes of the laser beams L1 and L2 before irradiating the sample A are used, the simultaneous and non-simultaneous timing of the pulse timings of the two laser beams L1 and L2 is grasped regardless of the presence or absence of the sample A There is an advantage that can be.
Since the operation and other effects of this embodiment are the same as those of the first embodiment, description thereof will be omitted.

In the present embodiment, the processing device 8 causes the optical path length adjustment optical system 19 to automatically adjust the optical path length difference based on the interference fringe image. Instead of this, or in addition to this, the user The optical path length adjustment optical system 19 may be moved so that the optical path length difference can be adjusted. For example, the user may operate the optical path length adjustment optical system 19 while observing the interference fringe image displayed on the display.

In the first and second embodiments, the optical

scanning microscope apparatuses

100 and 200 for fluorescence observation have been described. However, light other than excitation light is used as illumination light, and light other than fluorescence is detected as signal light. Also good.

1,10 Excitation light generation unit (light source unit)
2 Scanning unit 3 Objective lens (objective optical system)
6,60 Detection optical system 7 Interference fringe measurement unit (angle detection unit)
8 Processing device (angle detector, optical path length difference calculator)
15 Second polarization beam splitter (angle adjustment unit)
19 Optical path length adjustment optical system (Optical path length difference adjustment unit)
72 Camera 75 Polarization changing element 76

Angle changing element

100, 200 Optical scanning microscope apparatus

Claims

A light source unit that outputs two illumination lights having relative angles to each other;
A scanning unit that scans the two illumination lights output from the light source unit;
An objective optical system for irradiating the sample with the two illumination lights scanned by the scanning unit and condensing them at different condensing positions;
A detection optical system for detecting the signal light generated at the condensing position;
An optical scanning microscope apparatus comprising: an angle detection unit configured to detect a relative angle between the two illumination lights at a substantially pupil position of the objective optical system.
The optical scanning microscope apparatus according to claim 1, further comprising an angle adjustment unit that adjusts a relative angle between the two illumination lights at a substantially pupil position of the objective optical system.
3. The optical scanning microscope apparatus according to claim 2, wherein the angle adjustment unit automatically adjusts the relative angle between the two illumination lights to a predetermined angle based on the relative angle detected by the angle detection unit.
The angle detector is provided at a position optically conjugate with a substantially pupil position of the objective optical system, and includes a camera that captures an interference fringe image of the two illumination lights and acquires an interference fringe image. The optical scanning microscope apparatus according to claim 3.
The optical scanning microscope apparatus according to claim 4, wherein the angle detection unit calculates the relative angle based on the interference fringe image.
The optical scanning microscope apparatus according to claim 4 or 5, wherein the angle detection unit includes a polarization changing element that changes a polarization state of the two illumination lights incident on the camera.
The optical scanning microscope apparatus according to any one of claims 4 to 6, wherein the angle detection unit includes an angle changing element that changes a relative angle between the two illumination lights incident on the camera.
The optical scanning microscope apparatus according to any one of claims 4 to 7, further comprising an optical path length difference calculation unit that calculates an optical path length difference between the two illumination lights based on a contrast of the interference fringe image.
The optical scanning microscope apparatus according to claim 8, further comprising: an optical path length difference adjusting unit that adjusts an optical path length difference between the two illumination lights based on the optical path length difference calculated by the optical path length difference calculating unit.
In the optical scanning microscope apparatus that scans the two illumination lights that irradiate the sample with two illumination lights having relative angles to each other from the objective optical system and collect the two illumination lights at different collection positions. A method for measuring a distance between spots, which measures a distance between spots of the two illumination lights,
A spot-to-spot distance measuring method in an optical scanning microscope apparatus including a step of detecting a relative angle between the two illumination lights at a substantially pupil position of the objective optical system.