JP6003072B2 - Microscope equipment - Google Patents

Description

  The present invention relates to a microscope apparatus that generates fluorescence by irradiating a sample with excitation light, and detects the fluorescence to generate an image of the sample.

  2. Description of the Related Art Conventionally, a microscope apparatus that obtains an image of a sample based on fluorescence emitted from the sample by irradiating the sample with a fluorescent dye, fluorescent protein, or the like with excitation with laser light has been used. The sample image is required to have a high resolution, and therefore a STED microscope using a plurality of laser beams is used. An example of this STED microscope is disclosed in Patent Document 1.

  The STED microscope shown in this document uses two laser beams of excitation light and suppression light (STED light). The excitation light is laser light having a wavelength for exciting the sample. The suppression light is laser light having a wavelength that suppresses the fluorescence generation of the sample. By bringing the suppression light close to the periphery of the excitation light at the focal point of the sample, the region where the excitation light fluoresces is substantially narrowed. By narrowing the fluorescent region, the resolution of the sample image is increased.

US Pat. No. 5,731,588

  The STED microscope of Patent Document 1 needs to use two laser beams of excitation light and suppression light in order to obtain a high-resolution sample image. Therefore, the microscope must be equipped with two laser light sources each emitting different wavelengths. Thereby, the whole microscope apparatus becomes large.

  Further, the combination of the laser beams of the two laser light sources is limited. The fluorescent dyes of the sample have various colors, and it becomes difficult to correspond to each fluorescent dye by combining the wavelengths of two laser beams. Furthermore, the suppression light must be in the vicinity of the excitation light at the focal point of the sample. For this reason, it is necessary to strictly adjust the optical axes of the excitation light and the suppression light, and this optical axis adjustment work becomes complicated.

  Accordingly, an object of the present invention is to acquire a high-resolution sample image using a single laser beam.

In order to solve the above problems, the microscope apparatus of the present invention is
A light source that oscillates excitation light that excites the sample;
A light source controller that controls the light source to irradiate the excitation light to the same focal position in the sample at least twice;
A detection unit for detecting fluorescence generated by irradiating the sample with the excitation light as return light;
An arithmetic unit that performs arithmetic operations on the return light at least twice;
Based on the calculation result of the calculation unit, an image generation unit for generating an image of the sample;
Equipped with a,
The light source control unit controls the light source to oscillate the excitation light having the same intensity of excitation light that causes fading in a part of the sample at least twice;
The calculation unit calculates a calculation result by calculating a difference between a return light when the excitation light is irradiated for the first time and a return light amount when the excitation light is irradiated for the second time,
The image generation unit generates an image of the sample based on the calculation result .

According to this microscope apparatus, the same focal position in the sample is irradiated with excitation light at least twice. By irradiating the sample with the excitation light, the return light can be detected at least twice, and an operation is performed on the return light. Thereby, the information in the focus of a sample can be made into high resolution.
And since the return light which a part of sample discolored by the 1st irradiation of excitation light can be acquired, and the return light after a color fading can be acquired by the 2nd irradiation of excitation light, the sample is calculated by calculating the difference. The information at the focal point can be of high resolution.

  The light source control unit controls the light source to oscillate the excitation light having different excitation light intensities at least twice, and the calculation unit controls fluorescence of the sample based on at least two return lights. The calculation result is calculated by calculating the saturation information of the molecule, and the image generation unit generates an image of the sample based on the calculation result.

  When the sample is irradiated with excitation light having different excitation light intensities from the light source, if the excitation light intensity becomes saturated, a part of the sample is saturated. By calculating the saturation information at this time, the information at the focal point of the sample can have high resolution. The saturation information includes saturation point information in addition to saturation amount information.

  Further, the light source oscillates the excitation light having a wavelength corresponding to the fluorescent dye of the sample.

  Thereby, excitation light having a wavelength corresponding to the fluorescent dye of the sample can be irradiated, and the fluorescent dye can be excited to generate an image regardless of the type of the sample.

  A scanning unit that scans the sample with the excitation light; and a calculation result storage unit that stores the calculation result in association with a focal position when the scanning unit scans the image generation unit. The imaging of the sample is performed based on the calculation result associated with the focal position.

  Thereby, it is possible to generate an image of a predetermined region (scanned region) of the sample, and it is possible to generate an image of the generated sample with high resolution.

  Further, a pinhole is disposed in the optical path of the return light.

  By arranging the pinhole in the optical path of the return light, only the light within the focal range passes, so that a confocal image with high resolution in the optical axis direction can be generated.

  Also, a microlens disk in which a plurality of microlenses are spirally arranged in multiple stripes, and a pinhole disk that is provided at a position spaced from the microlens disk and in which a plurality of pinholes are arranged in the same pattern as the microlens, A scanning unit including a rotating unit that integrally rotates the microlens disk and the pinhole disk, and the image generation unit is configured to scan a plurality of images captured by scanning with the scanning unit. In this case, the sample is imaged by obtaining positional information of fading and saturated fluorescent molecules.

  By rotating the microlens disk and the pinhole disk integrally, the microlens disk and the pinhole disk can be used as a Nipkow disk type confocal microscope, and a high-resolution image can be generated at high speed.

  The present invention irradiates at least two excitation lights to the same focal position in a sample, and performs an operation on at least two return lights that are fluorescent by the excitation light, thereby generating a high-resolution sample image. It can be carried out.

It is a schematic block diagram of a microscope apparatus. It is a block diagram which shows the structure of a controller. It is a figure which shows the light quantity distribution of the XY direction in embodiment. It is a figure which shows the light quantity distribution of the Z direction in embodiment. It is a figure explaining the state to which the focus in the modification 1 is shifted. It is a figure explaining the state which shifts the focus in the modification 2. FIG. FIG. 10 is a configuration diagram showing a configuration of a microscope apparatus in Modification 3. It is a conceptual diagram of the microscope apparatus in the modification 3. It is a figure which shows the light quantity distribution of the XY direction in the modification 3. It is a figure which shows the focus and fading area | region in the modification 3. It is a graph which shows the relationship between the excitation light intensity | strength and fluorescence intensity in the modification 4.

  Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows a microscope apparatus 1. The microscope apparatus 1 includes a laser light source 2, a shutter 3, a dichroic mirror 4, an optical scanning unit 5, a pupil relay lens 6, a mirror 7, an imaging lens 8, an objective lens 9, a stage 10, a fluorescent filter 11, a focusing lens 12, and a pinhole. 13, a detector 14, a controller 15, and a display 16. A sample S into which a fluorescent indicator (fluorescent dye) has been introduced is placed on the stage 10.

  The laser light source 2 is a light source that oscillates and outputs a laser beam L as excitation light. As the wavelength of the laser light L, a wavelength for exciting the fluorescent dye introduced into the sample S is used. The laser light L oscillated and output from the laser light source 2 is converted into parallel light by a collimator lens (not shown) and guided to the shutter 3. The shutter 3 allows the laser light L to pass through the opening control at a predetermined timing. The laser light L that has passed through the shutter 3 enters the dichroic mirror 4.

  The dichroic mirror 4 is an optical element that reflects the laser light L from the laser light source 2 and transmits the return light (fluorescence) R from the sample S. Therefore, the laser light L oscillated and output from the laser light source 2 is reflected by the dichroic mirror 4 and guided to the optical scanning unit 5. The optical scanning unit 5 is a scanning unit having a first variable mirror 5a and a second variable mirror 5b.

  The first variable mirror 5a and the second variable mirror 5b are configured to be rotatable around one axis. Thereby, the reflection angle of the laser beam L is controlled, and the focal point of the laser beam L is scanned in the XY direction (horizontal plane direction) of the sample S placed on the stage 10.

  The laser light L output from the optical scanning unit 5 enters the pupil relay lens 6 and is then reflected by the mirror 7. The laser light L is focused on the sample S placed on the stage 10 by the objective lens 9. Since the pupil of the objective lens 9 is relayed to the position of the optical scanning unit 5 by the pupil relay lens 6, the laser light scanned by the optical scanning unit 5 is scanned at the pupil position of the objective lens 9. The focus by 9 scans the sample S.

  A fluorescent dye is introduced into the sample S, and the fluorescent dye of the sample S is excited by irradiating the laser beam L. Thereby, return light (fluorescence) R is generated. The generated return light R enters the objective lens 9 and travels in the opposite direction to the laser light L. Then, the light enters the dichroic mirror 4 through the objective lens 9, the imaging lens 8, the mirror 7, the pupil relay lens 6, and the optical scanning unit 5.

  The dichroic mirror 4 has a characteristic of transmitting the fluorescence wavelength of the sample S. Therefore, the return light R passes through the dichroic mirror 4 and enters the fluorescent filter 11. The fluorescent filter 11 is a filter that selectively transmits light having a specific wavelength component, that is, a fluorescent wavelength.

  As a result, the return light R is selectively transmitted by the fluorescent filter 11 with the fluorescent wavelength component and is focused on the detector 14 by the focusing lens 12. A pinhole 13 is disposed in the optical path of the return light R, particularly in the preceding stage of the detector 14. When the return light R passes through the pinhole 13, only the light in the focal range is allowed to pass, and other light is removed. The image generated by the pinhole 13 becomes a confocal image with high resolution in the optical axis direction.

  The return light R that has passed through the pinhole 13 is input to the detector 14. The detector 14 is a detector that detects the return light R. That is, the light amount of the received return light R is photoelectrically converted into an electric signal. The converted electric signal becomes digital data (light amount data) of the light amount of the return light R. The light amount data converted by the detector 14 is input to the controller 15. FIG. 2 shows the configuration of the controller 15. The controller 15 includes a system control unit 21, a light source control unit 22, a shutter control unit 23, an optical scanning unit control unit 24, an input unit 25, a calculation unit 26, a calculation result storage unit 27, and an image generation unit 28. Yes.

  The system control unit 21 performs overall control of the controller 15. The light source controller 22 controls the laser light source 2. The laser light source 2 oscillates the laser light L under the control of the light source control unit 22. The shutter control unit 23 performs opening / closing control of the shutter 3. The optical scanning unit controller 24 controls the optical scanning unit 5, and the focal point of the laser light L is scanned with the sample S by performing this control.

  The input unit 25 inputs light amount data from the detector 14. The calculation unit 26 performs a predetermined calculation based on the input light quantity data. Here, the difference (difference value) in the light amount data between the return light R fluorescent by the first laser light L irradiation and the return light R fluorescent by the second laser light L irradiation is calculated as a calculation result.

  The calculation result calculated by the calculation unit 26 is stored in the calculation result storage unit 27. The calculation result storage unit 27 stores the calculation result in association with the focal position when the laser beam L is irradiated. The focal position of the laser beam L is controlled by the optical scanning unit 5, and the system control unit 21 grasps the focal position. Therefore, under the control of the system control unit 21, the calculation result storage unit 27 stores the calculation result in association with the focal position of the laser light L.

  The image generation unit 28 generates an image of the sample S based on the calculation result (difference value) stored in the calculation result storage unit 27 in association with the focal position. The generated image data may be stored in an internal memory (not shown). The display 16 is image display means connected to the controller 15. The display 16 displays the image generated by the image generation unit 28. As a result, the image of the sample S is displayed on the display 16.

  Next, the operation of this embodiment will be described. In the present embodiment, the same focal position in the sample S is irradiated twice with the laser light L having the same excitation light intensity. For this reason, the laser beam L is irradiated twice with the focus of the sample S fixed. That is, the first irradiation and the second irradiation are performed on the same portion of the sample S. First, the first irradiation will be described.

  The time for irradiating the sample S with the laser light L is a predetermined time (unit irradiation time) set in advance. Therefore, the fluorescent dye is excited by the laser light L focused on the sample S during the unit irradiation time. This excitation generates fluorescence. The generated fluorescence is guided to the detector 14 as return light R.

  At this time, the light source control unit 22 adjusts the oscillation intensity (excitation light intensity) when the laser light source 2 oscillates the laser light L. 3 shows the intensity distribution (curve 31) of the laser beam L in the XY direction (horizontal plane direction) in the sample S, and FIG. 4 shows the intensity distribution (curve 41) of the laser beam L in the Z direction (vertical direction). ing. As shown in FIGS. 3 and 4, the intensity distribution of the laser light L is a Gaussian distribution in which the intensity at the center of the optical axis is high and the intensity at the periphery is low.

  Here, when the intensity of the laser light L applied to the sample S becomes higher than a predetermined value, the fluorescent dye of the sample S is faded. The threshold value at this time is the threshold value 32 in FIG. 3 and the threshold value 42 in FIG. That is, in the XY direction, when the intensity is higher than the threshold value 32, the color fading occurs and the color fading area 33 is generated. When the intensity is higher than the threshold value 42 in the Z direction, fading occurs and a fading area 43 is generated.

  Therefore, the intensity of the laser light L oscillated by the laser light source 2 is adjusted so as to be higher than the intensity that causes a part of the sample S to fade, that is, the intensity higher than the threshold values 32 and 42. This intensity adjustment is performed by the light source controller 22. When the sample S is irradiated with the first laser beam L, fading occurs in a part of the sample S.

  Next, the laser beam L is irradiated for the second time only during the unit irradiation time with the focus of the laser beam L being fixed. The intensity of the second laser beam L is the same as the intensity of the first laser beam L. In the first irradiation, all light quantities indicated by the intensity distributions in FIGS. 3 and 4 are detected. However, at this time, fading occurs in a partial region of the sample S. On the other hand, in the second irradiation, the light quantity in the areas excluding the fading areas 33 and 43 among the light quantities shown in the intensity distributions of FIGS. 3 and 4 is detected.

  That is, the light quantity indicated by the light quantity data obtained by the second irradiation is reduced by the amount of fading than the light quantity indicated by the light quantity data obtained by the first irradiation. The calculation unit 26 calculates the difference between the light amount data for the first irradiation and the light amount data for the second irradiation. This calculation result is used as a difference value. Further, since the fading amount at this time is proportional to the amount of the original fluorescent molecule, the calculated difference value indicates the amount of fluorescent molecule in the fading region of the sample S, that is, the fluorescence intensity.

  As shown in FIGS. 3 and 4, when the sample S is irradiated with the laser beam L only once, the light amount of all the regions of the intensity distribution is detected. That is, the amount of light in the region indicated by the width 34 is detected in the XY direction shown in FIG. 3, and the amount of light in the region indicated by the width 44 is detected in the Z direction shown in FIG.

  On the other hand, when the focal point is fixed and the sample S is irradiated with the laser beam L twice and the difference is detected, the difference value is detected as the amount of light in the faded area. That is, the amount of light in the region indicated by the width 35 is detected in the XY direction shown in FIG. 3, and the amount of light in the region indicated by the width 45 is detected in the Z direction shown in FIG.

  The width 35 in the XY direction is a limited region that is narrower than the width 34. Further, the width 45 in the Z direction is a limited region narrower than the width 44. That is, the amount of light in a limited region that is narrower than the region detected by the single laser beam L irradiation is detected. Since the region for detecting the light amount can be a narrow region, the light amount data of the sample S at the focal position of the laser light L can have high resolution. In other words, with the focus of the laser light L fixed, the laser light L having an excitation light intensity that causes a part of the sample S to fade is irradiated twice to detect the difference in the light amount data of the return light R. Thus, information with high resolution can be obtained for the focal position. Note that the laser beam L may be irradiated three times or more.

  Then, the optical scanning unit 5 is controlled by the control of the optical scanning unit controller 24 to change the focal position of the laser light L. Thereby, the focus of the laser beam L on the sample S placed on the stage 10 is scanned in the XY directions. As described above, the calculation result storage unit 27 stores the difference value in association with the focal position of the laser beam L.

  Therefore, the image generation unit 28 generates an image of the sample S based on the difference value corresponding to the focal position. Since the difference value at this time has high resolution, the generated image of the sample S also has high resolution. For this reason, a detailed image of the sample S with high resolution can be obtained.

  Moreover, since only the laser beam L is irradiated twice with the focal point fixed, the required number of laser light sources 2 is one. Therefore, it is not necessary to use a plurality of laser light sources 2, and the entire configuration of the microscope apparatus 1 can be made compact. In addition, since two lights such as excitation light and suppression light are not used, it is not necessary to perform complicated processing such as adjustment of the optical axis. Further, since one laser beam L is used, there is no difficulty in combining the laser beams L of two wavelengths with the sample S.

  In the above, the laser light source 2 may be configured to oscillate and output laser light L having a plurality of wavelengths. The wavelength of the laser light L to be excited differs depending on the fluorescent dye (color) of the sample S. Therefore, the laser light source 2 can oscillate laser beams L having a plurality of types of wavelengths so as to oscillate the laser beam L that excites the fluorescent pigment in accordance with the fluorescent pigment of the sample S.

  Alternatively, the stage 10 may be moved in the Z direction to image another surface of the sample S in the Z direction. In the example of FIG. 1, the pinhole 13 is arranged in front of the detector 14. As a result, light from other than the focal plane of the return light R is removed. For this reason, a high resolution confocal image can be obtained. However, since the faded area is limited to a narrow area as described above, the pinhole 13 may not be disposed. Alternatively, the opening diameter of the pinhole 13 may be increased.

  Further, if the laser light L having the excitation light intensity that causes fading to a part of the sample is oscillated from the laser light source 2, the excitation light intensity may be arbitrarily set. Since the fading regions 33 and 43 change depending on the excitation light intensity of the laser light L, the excitation light intensity of the laser light L is changed to control the width 35 in FIG. 3 and the width 45 in FIG. May be.

  Further, a near-infrared pulse laser may be used as the laser light L oscillated from the laser light source 2. When near infrared light is used as the laser light L, an optical system such as the dichroic mirror 4 is also used. Thereby, the microscope apparatus 1 can be used as a two-photon excitation microscope. Since a two-photon excitation microscope is used, two-photon excitation occurs only near the focal point. For this reason, the influence of the fluorescence generated in the middle of the optical path and the fading caused by it in deep observation or the like can be extremely reduced, and an image with higher resolution can be obtained.

  Next, Modification 1 will be described. In Modification 1, scanning of the laser light L in the XY directions will be described. FIG. 5 shows a region of the focal point F (Fa, Fb,...) Of the laser light L in the sample S. The focal point F is a region in the XY direction having a width 34 shown in FIG. The optical scanning unit 5 scans the focal point F shown below.

  First, the first irradiation with the laser beam L is performed at the focal point Fa. As a result, the central region (fading region Ga) narrower than the focal point Fa is faded. In the above-described embodiment, the focus of the laser beam L is fixed and the second irradiation is performed. However, in the present modification, the first irradiation does not overlap with the focus Fa in one of the XY directions (X direction). The focal point Fd is scanned so as to be close to and close.

  As a result, the fading region Gd at the center of the focal point Fd fades. Then, the focus F is sequentially scanned so as not to overlap with the previous focus F in the X direction. This scanning is performed by the optical scanning unit 5. After the scanning of one row in the X direction is completed, the laser beam L is returned to the focal point Fa again. Then, the scanning is sequentially performed again from the focal point Fa so that the focal point F does not overlap and is close to the focal point.

  That is, the laser beam L is irradiated twice at the same focal point F. Thereby, a difference value between the first irradiation and the second irradiation at each focal point F is generated. Next, the optical scanning unit 5 sets the focal point of the laser light L to the position Fb. The focal point Fb is set at a position where the fading region Gb that fades when the laser beam L is irradiated to the focal point Fb and the fading region Ga at the focal point Fa that has already been irradiated do not overlap and are close to each other.

  Then, the focal points F are sequentially scanned so that the focal points F do not overlap in the X direction and are close to each other. After the scanning of one row in the X direction is completed, the second laser light L is irradiated again from the focal point Fb, and the difference value is detected. Similarly, scanning is performed in the X direction starting from the focal point Fc in FIG. 5, and a difference value between the first irradiation with the laser beam L and the second irradiation with the laser beam L is detected.

  Thereby, discrete scanning is performed, and a fading region G (Ga, Gb,...) Fading as shown in FIG. 5 is formed. Each fading area G is narrower than the area of the focus F, and the fading areas G are arranged so as not to overlap each other. This narrow fading area G is detected as a difference value and used for imaging. Therefore, the resolution in the X direction can be increased.

  After the scanning in the X direction is completed, the column to be scanned in the Y direction is shifted and the same scanning is performed again. Thereby, the fading areas G can be arranged close to each other in the Y direction, and the resolution in the Y direction can be increased. That is, the fading area G narrower than the focal point F is arranged at a narrow interval, and the light amount of the fading area G is detected as a difference value. The difference value indicating the fading amount is the amount of the original fluorescent molecule. Since it is proportional, the amount of fluorescent molecules in the XY directions, that is, the fluorescence intensity is shown. Thereby, high resolution can be obtained in the XY directions.

  Of course, in the Z direction, regardless of the scanning in the XY direction, the fading area is detected as a difference value, so that high resolution can be obtained. Therefore, it is possible to obtain an image of the sample S having a high resolution in three dimensions in the XY direction and the Z direction.

  Next, Modification 2 will be described with reference to FIG. In the first modification, the part to be scanned next to the focal point Fa is the focal point Fd so as not to overlap with the focal point Fa. However, in this modified example, the laser beam L is applied to the focal point Fb next to the focal point Fa. Let it scan. That is, the laser beam L is scanned with the focal point F so that the fading region Ga of the focal point Fa and the fading region Gb of the focal point Fb do not overlap with each other.

  The scanning of the laser beam L in the X direction is performed so that the fading areas G do not overlap and are close to each other, and are sequentially shifted in the X direction. After the scanning of one column in the X direction is completed, the scanning is performed in the X direction again by shifting one column in the Y direction. Thereby, the first irradiation of the laser beam L is performed in the XY directions.

  Next, the laser beam L is set again at the focal point Fa, and the second irradiation of the laser beam L is performed. Then, scanning is performed in one column in the X direction, and a column scanned in the Y direction is sequentially shifted, so that the second scanning is performed in the XY direction. Therefore, the difference value between the first irradiation and the second irradiation can be detected, and high-resolution images can be obtained in the XY direction and the Z direction as in the first modification.

  In the first modification, since the focal points F are scanned so as not to overlap and close to each other, it is necessary to repeatedly reciprocate the position of the focal point F in the X direction. In this regard, in the second modification, it is only necessary to sequentially shift in the X direction, and there is no need to reciprocate. Therefore, since the number of reciprocations of the laser beam L can be reduced, the scanning time can be increased in this modification. Thereby, the speed of image generation is also increased. However, the accuracy of the generated image is higher in the first modification.

  As shown in FIG. 6, in the second irradiation, the region of the focal point Fa includes the fading regions Gb, Gg, and Gh (parts) of the other focal points Fb, Fg, and Fh. Therefore, when the difference between the light amount data at the first irradiation of the focal point Fa and the light amount data at the second irradiation is detected, the light amount is reduced by the above-mentioned fading areas Gb, Gg, and Gh.

  Therefore, the exact amount of light in the fading area Ga at the focal point Fa is not detected. This slightly reduces the accuracy of the image. In the first modification, since a certain focal point F does not include the fading region G of another focal point F, the accuracy of the image is lower than that in the first modification.

  However, as also shown in FIGS. 3 and 4, the intensity distribution of the laser light L is a Gaussian distribution, and the amount of light in the region other than the center of the optical axis is small. Therefore, even if another fading area G is included in this area, the influence is very low. In this respect, although there is a slight influence on the accuracy of the image, the second modification that places importance on the high speed of image generation may be employed.

  Further, correction may be performed so as to eliminate the influence of the other fading area G at the focal point F. Since the other fading area G included in the focal point F can be recognized in advance, the light quantity of the recognized other fading area G is multiplied by a predetermined coefficient (a coefficient corresponding to the light quantity of the Gaussian distribution) and reduced. Thus, it is possible to perform correction to eliminate the influence of the other fading area G. As a result, the image generation speed can be increased while improving the accuracy of the image.

  Next, Modification 3 will be described. 7 and 8 show a microscope apparatus 1 according to this modification. This microscope apparatus 1 is a Niipou disk type confocal microscope apparatus, and includes a laser light source 2, a scanning unit 50, a microscope 60, and a camera 70.

  The laser light source 2 is the laser light source already described, and oscillates and outputs the laser light L. The laser light L oscillated by the laser light source 2 is transmitted to the scanning unit 50 through the optical fiber 2F. The scanning unit 50 includes a microlens disk 51, a pinhole disk 52, a rotating drum 53, a motor 54, a collimator lens 55, a dichroic mirror 56, and a relay lens 57.

  As shown in FIG. 8, the microlens disk 51 is a rotating disk in which a plurality of microlenses 51M having a spiral pattern are arranged. In addition, the spiral pattern may not be four strips, but may be a multiple strip spiral pattern. The pinhole disk 52 is a rotating disk in which a plurality of pinholes 52P are arranged in the same pattern as the pattern of the microlens 51M of the microlens disk 51.

  A predetermined gap is provided between the microlens disk 51 and the pinhole disk 52. The rotating drum 53 rotates the microlens disk 51 and the pinhole disk 52 integrally. The motor 54 is a rotating unit that applies a rotational force to the rotating drum 53. The rotating drum 53 and the motor 54 constitute a rotating part. The collimating lens 55 converts the laser light L guided by the optical fiber 2F into parallel light. The laser light L is focused at a plurality of pinholes 52P by a plurality of microlenses 51M.

  A dichroic mirror 56 is disposed between the microlens 51M and the pinhole 52P. The dichroic mirror 56 is an optical element having a characteristic of transmitting light having the wavelength of the laser light L oscillated by the laser light source 2 and reflecting light having the wavelength of the fluorescence of the sample S (return light R). Therefore, the laser light L is transmitted from the microlens 51M to the pinhole 52P.

  Laser light L enters from each microlens 51M toward the pinhole 52P. The laser beam L that has passed through the pinhole 52P enters the microscope 60. The microscope 60 includes an imaging lens 61 and an objective lens 62. The laser light L that has passed through the pinhole 52P is converted into parallel light having a predetermined inclination by the imaging lens 61. This parallel light is incident on the objective lens 62.

  The laser beam L incident on the objective lens 62 is focused on the sample S of the stage 10. In the sample S, the fluorescent dye is excited by the laser light L, and fluorescence is generated. This fluorescence becomes return light R, passes through the microscope 60 and the pinhole 52P, and is reflected by the dichroic mirror 56. The reflected return light R is imaged by the image sensor of the camera 70 by the relay lens 57. A fluorescence filter (not shown) is provided in front of the camera 70, and only the fluorescence of the sample S is selectively imaged on the camera 70.

  In FIG. 7, two optical paths of the laser light L are indicated by a solid line and a broken line. Of these, the optical path indicated by the solid line and the optical path indicated by the broken line are light passing through different microlenses 51M. Yes. That is, the laser beam L at the center of the optical axis of the laser beam L and the laser beam L shifted from the center of the optical axis are shown. In FIG. 8, the microscope 60 is schematically shown as a single lens.

  The operation of this modification will be described. In this operation, the image acquisition operation is performed twice. First, the first image acquisition operation will be described. When the motor 54 rotates the rotary drum 53, the microlens disk 51 and the pinhole disk 52 rotate integrally.

  By oscillating the laser light L from the laser light source 2, the laser light L passes through the pinhole 52P from the microlens 51M. The microlens disk 51 and the pinhole disk 52 rotate integrally. Thereby, the laser beam L is scanned at high speed on the sample S of the stage 10. When the sample S is focused on the laser light L, the fluorescent dye of the sample S emits light.

  This fluorescence becomes return light R and forms an image on the camera 70. Of the return light R, light other than the focal plane cannot pass through the pinhole 52P and therefore does not reach the camera 70. As a result, the camera 70 captures a confocal image of only the focal range. Since the laser light L is scanned by the sample S, the return light R is scanned on the light receiving surface of the camera 70.

  Each pixel is formed in the camera 70 in an XY (vertical and horizontal) direction, and the return light R is scanned by each pixel. Thereby, the light quantity data of each pixel is obtained, and one image of the sample S is acquired. In this modification, not the detector 14 but the image data (image data) acquired by the camera 70 is input to the input unit 25 shown in FIG. 2, and the calculation unit 26 holds this image data.

  FIG. 9 shows an intensity distribution (curve 71) in the XY direction in the vicinity of the focal point when the sample S is irradiated with the laser light L using the Niipou disc type confocal microscope apparatus. As shown in FIG. 9, the spread is shown in the range of the width 72.

  FIG. 10 schematically shows the pixels P arranged in the XY directions constituting the camera 70. The return light R is scanned over each pixel P of the camera 70. When there is a minute fluorescent molecule at the vertex 73 of the focal point, the return light has a predetermined spread, and is thus projected on the camera in a range of Ha. Here, the range Ha is projected so that the intensity increases toward the center according to the point spread function. In these ranges, a plurality of unillustrated focal points are scanned in the X direction as the pinhole disk is scanned.

  As described in the above-described embodiment, the first laser beam L is scanned from the laser light source 2. By scanning the sample S with the laser light L, the sample S fluoresces and the return light R is generated. The excitation light intensity of the first laser beam L at this time is set to an intensity at which a part of the sample S fades. Thereby, image data of the first sample S is acquired.

  Next, the second oscillation of the laser light L from the laser light source 2 is performed. Then, the image data of the second sample S is acquired by scanning the sample 2. The fluorescent molecules present at the vertex 73 of the focal spot are faded by the first laser beam and do not emit fluorescence. Thereby, the fluorescence from this fluorescent molecule is not recorded in the second image data. The calculation unit 26 calculates the difference between the first and second image data and stores the difference in the calculation result storage unit 27. Thereby, the intensity distribution of the faded area Ha in FIG. 10 is stored.

  Further, the calculation unit 26 calculates the center position Ga by fitting the intensity distribution of the region Ha with a two-dimensional Gaussian function, and sets the center position as the position of the fluorescent molecule in the calculation result storage unit 27. Remember me.

  In addition, since the fading of these fluorescent dyes is generated steadily, there are very few dyes that fade in one scan. Therefore, the above image acquisition is repeated, and the calculation of the difference between the images is repeated to calculate the position of each fluorescent molecule, and based on this, the image generation unit 28 constructs a high-resolution fluorescent image, and the display 16 is displayed. Alternatively, a high-resolution fluorescent image may be constructed by repeating the above image acquisition while gradually increasing the excitation light intensity.

  In addition, since the present configuration is a Nipkow disk type confocal microscope, it is excellent in Z resolution and can generate an image at a very high speed.

  Since the amount of fading caused by the irradiation with the laser light L is proportional to the amount of the original fluorescent molecules, the difference value calculated by the calculating unit 26 indicates the amount of fluorescent molecules in the fading area of the sample S. As a result, the image generated by the image generation unit 28 becomes a high-resolution image. In addition, since it is applied to a Niipou disc type confocal microscope, image generation can be performed at a very high speed.

  Next, Modification 4 will be described. In the fourth modification, the microscope apparatus having the configuration shown in FIG. 1 is used. The laser light L is oscillated and output from the laser light source 2, but the laser light L is oscillated at least twice with different intensities in a state where the focus of the laser light L is fixed as in the embodiment. FIG. 11 shows an example.

  Here, the laser beam L is oscillated four times with the excitation light intensity of A, B, C, and D with respect to the same focal position of the sample S. The excitation light intensities A, B, C, and D have a relationship of A <B <C <D, and first oscillate the laser light L having the excitation light intensity A. The laser light L is excited by being irradiated on the sample S, and generates return light R having a fluorescence intensity a. This return light R is detected by the detector 14. This is a point 90 shown in FIG.

  Then, the laser beam L having the second excitation light intensity B is oscillated with the focal point fixed. Thereby, the return light R with the fluorescence intensity b is detected. This is a point 91 shown in FIG. Similarly, the laser light L having the third excitation light intensity C is oscillated. Thereby, the return light R with the fluorescence intensity c is detected. This is a point 92 shown in FIG. Finally, the laser light L having the fourth excitation light intensity D is oscillated. Thereby, the return light R with the fluorescence intensity d is detected. This is a point 93 shown in FIG.

  The light source control unit 22 gradually increases the excitation light intensity of the laser light L oscillated from the laser light source 2 from A to D at the same focal position. As shown in FIG. 11, the excitation light intensity and the fluorescence intensity have a proportional relationship (that is, a linear relationship) up to the excitation light intensity E. However, the excitation light intensity and the fluorescence intensity are separated from each other with the excitation light intensity E as a boundary. The relationship changes to a non-linear relationship. This excitation light intensity E becomes the excitation light intensity (saturation intensity E) that saturates the fluorescent molecules of the sample S.

  When the fluorescent molecules of the sample S are saturated, saturation excitation occurs at the center of the laser light L irradiated to the sample S. This is due to the fact that the number of fluorescent molecules in focus is finite and the amount of fluorescence is suppressed due to a delay called fluorescence lifetime before the molecules are excited to generate fluorescence.

  Therefore, by irradiating the sample S with the laser beam L with the saturation intensity E at which the fluorescent molecules of the sample S are saturated, a very narrow region at the center of the laser beam L irradiated to the sample S is saturated. This saturated region corresponds to, for example, the region of the width 35 in the XY direction shown in FIG. 3 and the region of the width 45 shown in FIG. These regions of width 35 and width 45 are narrow regions within the focal range and reflect the amount of saturated fluorescent molecules.

  The computing unit 26 of the controller 15 has the intensities (fluorescence intensities) a to d of the return light R when the laser beams L with the excitation light intensities A to D are irradiated. Based on the excitation light intensities A to D and the fluorescence intensities a to d, the calculation unit 26 calculates the excitation light intensity E (saturation intensity E) at which the fluorescent molecule is saturated. That is, a calculation is performed to estimate a point 94 where the excitation light intensity and the fluorescence intensity are in a non-linear relationship from a linear relationship.

  Based on the excitation light intensities A to D and the fluorescence intensities a to d, the calculator 26 calculates a straight line L1 having a linear relationship (proportional relationship) and a curve C1 having a nonlinear relationship. Then, the calculation unit 26 calculates a fluorescence intensity f corresponding to an intensity higher than the saturation intensity E (excitation light intensity F) based on the curve C1. In FIG. 11, the excitation light intensity F and the fluorescence intensity f become a point 95.

  As shown in FIG. 11, since saturation occurs in the excitation light intensity F, a difference is generated between the straight line L1 and the curve C1. This difference becomes the nonlinear component NL. This nonlinear component NL indicates the amount of saturated fluorescent molecules (saturation amount) and corresponds to the width 35 in the XY direction shown in FIG. 3 and the width 45 shown in FIG.

  Therefore, the calculation result storage unit 27 stores the result calculated by the calculation unit 26 (nonlinear component NL: saturation amount). The saturation amount at this time is a value for one focal position, and the optical scanning unit controller 24 controls the optical scanning unit 5 to scan the sample S in the X and Y directions. At this time, the optical scanning unit controller 24 performs scanning so that saturated regions (width 35 in FIG. 3) do not overlap.

  Therefore, the image generation unit 28 generates an image of the sample S by associating the scanned focus position and the saturation amount (nonlinear component NL) with each other and storing them in the calculation result storage unit 27. At this time, image generation is performed based on the saturation amount (width 35 in FIG. 3 and width 45 in FIG. 4). Since the saturation amount is a narrow region at the center of the focal region of the sample S, a high-resolution image of the sample S can be generated.

  Here, the fluorescence intensity f when the sample S is irradiated with the excitation light intensity F higher than the saturation intensity E is obtained. As a result, a saturation amount (nonlinear component NL) in a predetermined region is generated. By performing image generation based on this saturation amount, high-resolution image generation can be performed.

  Here, at the saturation intensity E, it means that the fluorescence of the focal point and the fluorescent molecule has just reached saturation. This is equivalent to the case where the width is reduced to the limit of the width 35 and the width 45 in FIG. 3 and means that the vertexes of the curves in FIGS. 3 and 4 have reached saturation. That is, the information on the saturation point 94 means that the fluorescence of the fluorescent molecule at the center of the focal point is just saturated. Accordingly, since the information such as the saturation intensity E at the saturation point 94 and the fluorescence intensity e at the saturation intensity E is correlated with the amount of fluorescent molecules at the center of the focal point, even if the information on these saturation points is used, high resolution is achieved. Image generation can be performed. Thus, imaging may be performed using not only the saturation amount but also the saturation point information.

  As described above, in the present modification, the excitation light intensity is changed in four stages (A to D), but may be other than four stages. Further, although the excitation light intensity of the laser light L oscillated from the laser light source 2 is increased stepwise, it may be decreased stepwise. Further, a neutral density filter may be inserted in the optical path of the return light R in order to control the saturation amount generated when the sample S is irradiated with the laser light L. In this case, the calculation unit 26 may calculate the saturation amount by performing calculation in consideration of the effect.

  In the example of FIG. 11, when the sample S is irradiated with the laser light L a plurality of times, the excitation light intensity is increased uniformly. However, the first excitation light intensity A is the same, but the second and subsequent times. The excitation light intensity of may be changed. That is, the second and subsequent excitation light intensities B to D may be changed according to the point 90 of the excitation light intensity A. For example, when the fluorescence intensity a at the point 90 in the excitation light intensity A is large, the slope of the straight line L1 becomes large. When the slope of the straight line L1 is large, the density of the fluorescent molecules is high and the fluorescence intensity E at which the fluorescent molecules are saturated is presumed to be high. You may make it make it. In addition, when the fluorescence intensity a at the point 90 in the first excitation light intensity A is smaller than a predetermined value, it is assumed that there is no fluorescent molecule in this region, so that the calculated value may be stored as 0.

  In this modification, the focus is fixed and the laser light L is irradiated four times with the excitation light intensities A to D, and the scanning is performed after the return light R is detected. After scanning the sample S, the sample S may be scanned with the excitation light intensity B, then the sample S may be scanned with the excitation light intensity C, and finally the sample S may be scanned with the excitation light intensity D.

  Therefore, by oscillating the excitation light intensity of the laser light L with a plurality of different intensities with respect to the same focus of the sample S, fluorescence saturation is caused at the center of the focus. When performing image generation, since image generation is performed based on the saturation amount at this time, high-resolution image generation can be performed.

  Next, Modification 5 will be described. In the modification 4, high-resolution image generation is performed by causing the fluorescent molecules of the sample S to be saturated with the microscope apparatus having the configuration shown in FIG. 1, but in the Niipou disc type confocal microscope as shown in the modification 3 as well. Then, saturation is generated in the fluorescent molecules of the sample S, and high-resolution image generation is performed.

  In this modification, the laser light source 2 oscillates the laser light L having an excitation light intensity A and rotates the microlens disk 51 and the pinhole disk 52, thereby scanning the sample S at high speed. Thereby, the first image data based on the return light R of each focus at the excitation light intensity A is generated.

  Next, the excitation light intensity of the laser light L is oscillated with F, and the microlens disk 51 and the pinhole disk 52 are rotated, thereby scanning the sample S at high speed and generating the second image data. .

  At this time, the calculation unit 26 calculates the saturation amount of fluorescence (nonlinear component NL) in each pixel of the two pieces of image data. Here, when one fluorescent molecule existing at the focal point is saturated, the distribution of the saturation amount of the fluorescent molecule is reflected in the region Ha in FIG. Here, the range Ha is projected so that the saturation amount becomes stronger toward the center according to the point spread function.

Further, the calculation unit 26 calculates the center position Ga by fitting the intensity distribution of the region Ha with a two-dimensional Gaussian function, and sets the center position as the position of the fluorescent molecule in the calculation result storage unit 27. Remember.
In addition, since the saturation of these fluorescent dyes is caused by the laser light intensity, the above-mentioned image acquisition is repeated while gradually changing the laser light intensity, and the calculation of the saturation amount between the images is repeated, so that The position is calculated, and based on this, the image generation unit 28 constructs a high-resolution fluorescent image and displays it on the display 16. Thereby, the image of the sample S with high resolution in the XY directions can be generated.

  In addition, since the Niipou disk type confocal microscope shown in FIGS. 7 and 8 is used, the scanning speed can be increased and image generation can be performed at high speed.

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Laser light source 3 Shutter 4 Dichroic mirror 5 Optical scanning unit 6 Pupil relay lens 7 Mirror 8 Imaging lens 9 Objective lens 10 Stage 11 Fluorescence filter 12 Focusing lens 13 Pinhole 14 Detector 15 Controller 16 Display 21 System control part 22 light source control unit 23 shutter control unit 24 optical scanning unit control unit 25 input unit 26 calculation unit 27 calculation result storage unit 28 image generation unit 50 scanning unit 51 micro lens disk 52 pinhole disk 53 rotating drum 54 motor 56 dichroic mirror 60 microscope 61 Imaging lens 62 Objective lens 70 Camera

Claims (6)

A light source that oscillates excitation light that excites the sample;
A light source controller that controls the light source to irradiate the excitation light to the same focal position in the sample at least twice;
A detection unit for detecting fluorescence generated by irradiating the sample with the excitation light as return light;
An arithmetic unit that performs arithmetic operations on the return light at least twice;
Based on the calculation result of the calculation unit, an image generation unit for generating an image of the sample;
With
The light source control unit controls the light source to oscillate the excitation light having the same intensity of excitation light that causes fading in a part of the sample at least twice;
The calculation unit calculates a calculation result by calculating a difference between a return light when the excitation light is irradiated for the first time and a return light amount when the excitation light is irradiated for the second time,
The microscope device characterized in that the image generation unit generates an image of the sample based on the calculation result. The light source control unit controls the light source to oscillate the excitation light having different excitation light intensities at least twice,
The calculation unit calculates saturation information of fluorescent molecules of the sample based on at least two of the return lights, and calculates a calculation result,
The microscope apparatus according to claim 1, wherein the image generation unit generates an image of the sample based on the calculation result. The microscope apparatus according to claim 1, wherein the light source oscillates the excitation light having a wavelength corresponding to a fluorescent dye of the sample. A scanning unit for scanning the sample with the excitation light;
A calculation result storage unit that stores the calculation result in association with the focal position when the scanning unit scans,
The image generating unit imaging the sample based on the calculation result associated with the focal position;
The microscope apparatus according to claim 3. The microscope apparatus according to claim 4, wherein a pinhole is disposed in an optical path of the return light. A microlens disk in which a plurality of microlenses are spirally arranged in multiple stripes;
A pinhole disk that is provided at a position separated from the microlens disk, and in which a plurality of pinholes are arranged in the same pattern as the microlens,
A scanning unit comprising a rotating unit that integrally rotates the microlens disk and the pinhole disk;
Wherein the image generation unit, a plurality of images captured by being scanned by the scanning unit, and obtain the position information of the fading and saturated fluorescent molecules, have rows imaging of the sample,
The microscope apparatus according to claim 2, wherein the light source oscillates the excitation light having a wavelength corresponding to the fluorescent dye of the sample .

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