JP2018081265A - Light generator, light irradiator, and light-sheet microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extend the effective length of line light or sheet light and suppress the effect of a side lobe.SOLUTION: A light irradiator R1 comprises a light generator 10, an optical scanner 20 and a light irradiation optical system 30. The light generator 10, designed to generate a line light 4 (or a sheet light), includes a light generation unit having a light source 11 and outputting a light of center wavelength λ and wavelength width w, and a stairstep device 100 for causing an input light L2 of refractive index n to be selectively interfered and converted into an interference light and outputting the resulting light. The stairstep device 100 has a plurality of interference surfaces the mutual surfaces of which assume a stepped shape, the height H between each interference surface satisfying the equation (1) below.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a light generation device, a light irradiation device including the light generation device, and a light sheet microscope including the light irradiation device.

  Non-Patent Document 1 discloses that a sample is irradiated with a laser beam that is linear light (a line of light), the line light is scanned by a scanner, and fluorescence generated thereby is coupled by a detection optical system. A scan-type light sheet microscope that captures an image and picks up an imaged light image is disclosed. In this light sheet microscope, line light is generated using a Gaussian beam, and a line of light (a plane of light) is formed by scanning the line light. Patent Document 1 discloses a light sheet microscope that generates line light using a Bessel beam and forms sheet light inside a sample. In Patent Document 2, a cylindrical lens is used to form planar light (sheet light) inside a sample, and fluorescence or scattered light generated thereby is imaged by a detection optical system. A light sheet microscope for imaging is disclosed.

US Patent Application Publication No. 2014/0198200 JP-A-62-180241

Philipp J. Keller, Annette D. Schmidt, Joachim Wittbrodt and Ernst H.K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy”, SCIENCE, November 14, 2008, VOL 322, p. 1065-1069 Sara Abrahamsson, Satoru Usawa and Mats Gustafsson, “A new approach to extended focus for high-speed, high-resolution biological microscopy”, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, February 14, 2006, Proc. of SPIE Vol. 6090

  In the conventional light sheet microscope, as described above, a Gaussian beam or a Bessel beam is used as light for generating line light. Moreover, in the conventional light sheet microscope, a cylindrical lens is used to generate sheet light. When a Gaussian beam is used in a light sheet microscope, if the light diameter of the laser beam is reduced, a portion with a thin light diameter (effective length of line light) may be shortened. Further, when a Bessel beam is used for a light sheet microscope, the Bessel beam generally has a high side lobe intensity, and therefore, the background in an image obtained by the imaging apparatus may increase. Furthermore, even when sheet light is generated by a cylindrical lens, there is a problem that the effective length of the sheet light is short. As described above, the conventional light sheet microscope has a problem that the effective length of the line light or the sheet light is limited, or the influence of the side lobe becomes large and the background becomes large.

  Therefore, the present invention has been made in view of such problems, and it is possible to lengthen the effective length of line light or sheet light, and to suppress the influence of side lobes, a light generation device, a light irradiation device, And it aims at providing a light sheet microscope.

  In order to solve the above-mentioned problems, the present inventors paid attention to a steer step device (see FIG. 1 to FIG. 4 of Non-Patent Document 2, etc.) in the course of intensive studies. And when this steer step device has a predetermined condition, if line light is generated using the steer step device, the effective length of the line light can be made relatively long and the influence of side lobes can be suppressed. As a result, the present invention has been completed.

を満たす。 That is, the present invention, as one aspect thereof, relates to a light generation device for generating line light or sheet light, and this light generation device has a light source and outputs light having a central wavelength λ and a wavelength width w. And a stair step device that has a refractive index of n and selectively interferes with the light output from the light generation unit and converts the light into interference light. The height H between the first and second interference surfaces is expressed by the following formula (1).

Meet.

  In this light generation device, the steer step device has step-shaped first and second interference surfaces having a height H that satisfies the requirement of the above-described formula (1). In this case, the height H becomes higher (longer) than the coherent length of the input light input to the steer step device, and interference between lights output from different interference surfaces is suppressed, while the light is output from the same interference surface. As light interferes, light output from the light generation unit is selectively interfered to obtain interference light. In other words, in a normal circular aperture or the like, light from different areas of the circle is coherently added, whereas this steer step device uses a point spread function (PSF) by an annular aperture. The light selectively interfered with each interference surface can be superimposed. As a result, when a light irradiation device (or a light sheet microscope) is configured using this light generation device, it becomes possible to increase the effective length of line light or sheet light and reduce the influence of side lobes. . The “wavelength width” used here is the full width at half maximum of the input light.

  In the light generation device described above, the first and second interference surfaces may be either a circular shape or an annular shape. In this case, the effect of the point spread function can be further exerted, and when the light irradiation device (or scan type light sheet microscope) is configured, the effective length of the line light is increased and the influence of the side lobe is further increased. It becomes possible to reduce.

  In the light generation device, the light generation unit may further include an optical filter that converts light output from the light source into light having a center wavelength λ and a wavelength width w. In this case, wavelength selectivity can be improved.

  In the light generation apparatus, the light generation unit may include a light adjustment element that adjusts a size of a cross section of light input to the steer step device. In this case, it is possible to adjust so that the input light is irradiated to a suitable position or region of the steer step device. Note that such a light adjustment element may include at least one of a beam expander and a stop.

  In the light generation device described above, the center wavelength λ of the input light may be any of the range of 300 nm to 850 nm, and the wavelength width w of the input light may be any of the range of 1 nm to 100 nm. The light source may be a light source that outputs low coherent light. In this case, when this light generation device is incorporated into an imaging device such as a light sheet microscope, the inspection object can be observed more suitably.

  In the above light generation device, the steer step device may have a third interference surface, and the height H between the second and third interference surfaces may satisfy the above formula (1). . Thus, by increasing the number of stepped interference surfaces, the effective length of the line light or sheet light can be further increased according to the number of steps. In addition, by providing three or more interference surfaces in this way, the effective length of the line light or sheet light can be increased more remarkably.

  In the light generation device, the area of the first interference surface may be equal to the area of the second interference surface. In this case, the light input to each interference surface becomes uniform, and the interference light generated by the steer step device can be converted into line light or sheet light in a more suitable form.

  Moreover, this invention relates to the light irradiation apparatus for irradiating an observation target object with line light as another aspect, This light irradiation apparatus is the interference light from one of said light generation apparatuses and a light generation apparatus. An optical scanning unit for scanning, a first relay optical system to which interference light scanned by the optical scanning unit is input, and interference light guided by the first relay optical system are collected, and line light is observed. And an objective lens for irradiating the object. In this case, as described above, it is possible to reduce the influence of the side lobe and increase the effective length of the line light.

  In the above light irradiation apparatus, a steer step device may be arranged on the surface of the optical scanning unit. In this case, the steer step device can be easily installed at the position of the pupil of the objective lens.

  The light irradiation device may further include a second relay optical system that further relays the pupil of the objective lens relayed by the first relay optical system, and the steer step device includes the second relay optical system. It may be arranged at the position of the pupil relayed by. In this case, since the pupil position and the phase modulation position can be matched, the influence of diffraction or the like by the element can be suppressed, and the spot shape can be more suitably modified.

を満たし、且つ、ステアステップデバイスの第１及び第２の干渉面が矩形形状であってもよい。この場合、上記同様に、サイドローブの影響を低減して、シート光の有効長を長くすることができる。なお、この光照射装置に用いられる光生成装置については、上述した光生成装置の追加構成の何れかを適宜適用できることが当業者には明らかであり、必要に応じて、適宜追加することが可能である。 Moreover, this invention relates to the light irradiation apparatus for irradiating an observation target object with sheet | seat light as another side surface, This light irradiation apparatus has a light source, and outputs the light of central wavelength (lambda) and wavelength width w Light including a generation unit, a cylindrical lens that receives light output from the light generation unit, and a steer step device that has a refractive index of n and selectively interferes with the light output from the light generation unit and converts the light into interference light A generation device and an objective lens that collects interference light output from the light generation device and irradiates an observation object with sheet light, and the steer step device of the light generation device has a mutual surface. Has at least a first interference surface and a second interference surface having a step shape, and a height H between the first and second interference surfaces is expressed by the following equation (1):

And the first and second interference surfaces of the steer step device may be rectangular. In this case, the effective length of the sheet light can be increased by reducing the influence of the side lobe as described above. In addition, it is obvious to those skilled in the art that any of the above-described additional configurations of the light generation device can be appropriately applied to the light generation device used in this light irradiation device, and can be added as necessary. It is.

  The present invention also relates to a light sheet microscope as another aspect, and this light sheet microscope is an observation object in accordance with any of the light irradiation devices described above and line light or sheet light irradiation by the light irradiation device. A detection optical system that forms an image of observation light generated from the image sensor, and an imaging device that captures an image of the observation light imaged by the detection optical system. The light sheet microscope with such a configuration can observe the object using line light or sheet light with a longer effective length while suppressing the influence of side lobes, and captures clearer images. It becomes possible to do.

  ADVANTAGE OF THE INVENTION According to this invention, the effective length of the line light used for a light sheet microscope or sheet light can be lengthened, and it becomes possible to suppress the influence of a side lobe and to suppress a background.

FIG. 1 is a block diagram showing a schematic configuration of a light sheet microscope according to an embodiment of the present invention. FIG. 2 is a diagram showing a schematic configuration of the light irradiation apparatus according to the first embodiment of the present invention. FIG. 3 is a perspective view showing an appearance of the steer step device shown in FIG. 4A and 4B are views showing the appearance of the steer step device shown in FIG. 2, wherein FIG. 4A shows a side view thereof and FIG. 4B shows a plan view thereof. FIG. 5 is a view showing a modification of the steer step device, in which (a) shows a sectional view thereof and (b) shows a plan view thereof. FIG. 6 is a diagram illustrating a configuration example of the light irradiation apparatus used in the example. FIG. 7 is a diagram showing the degree of elongation of line light, which is an experimental result when line light is created by the light irradiation apparatus shown in FIG. FIG. 8 shows an experimental result when line light is created by the light irradiation device shown in FIG. 6, (a) schematically shows an example of the state of peaks and side lobes, and (b) shows the example and It is a figure which shows the ratio (%) of the side lobe with respect to the peak in a comparative example. FIG. 9 is a diagram showing a schematic configuration of a light irradiation apparatus according to the second embodiment of the present invention. FIG. 10 is a diagram showing a schematic configuration of a light irradiation apparatus according to the third embodiment of the present invention. FIG. 11 is a diagram showing a schematic configuration of a light irradiation apparatus according to the fourth embodiment of the present invention. FIG. 12 is a diagram showing a schematic configuration of a light irradiation apparatus according to the fifth embodiment of the present invention. FIG. 13 is a diagram showing a schematic configuration of a light irradiation apparatus according to a sixth embodiment of the present invention, where (a) shows a view of the light irradiation apparatus and the like from the side surface, and (b) shows the light irradiation apparatus The figure after seeing the structure after the mask of FIG. 3 from the upper surface side is shown, (c) shows the plane of the mask, (d) shows the figure which the cylindrical lens and the steer step device were united. FIG. 14 is a diagram showing another example of the steer step device, where (a) shows a side view thereof and (b) shows a plan view thereof. FIG. 15 is a diagram showing still another example of the steer step device, in which (a) shows a side view thereof and (b) shows a plan view thereof.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description, the same reference numerals are used for the same elements or elements having the same function, and redundant description is omitted.

[First Embodiment]
FIG. 1 is a block diagram schematically showing a configuration of a light sheet microscope according to an embodiment of the present invention. As shown in FIG. 1, the light sheet microscope 1 includes a light generation device 10, an optical scanner 20 (light scanning unit), an irradiation optical system 30, a detection optical system 40, an imaging device 50, and a computer 60. Yes. In the light sheet microscope 1, the light generation device 10, the optical scanner 20, and the imaging device 50 are controlled by the computer 60, and the light from the light generation device 10 is condensed by the irradiation optical system 30 while being scanned by the light scanner 20 by this control. Then, the line light is irradiated to the sample S that is the observation object. Then, the observation optical system 40 having a detection objective lens forms an image of observation light such as fluorescence or scattered light generated on the sample S along with the irradiation of the line light, and an image of the observation light is captured by the imaging device 50. Output image data. The imaging device 50 includes an area image sensor such as a CMOS image sensor or a CCD image sensor. The computer 60 controls the light source 11, the light diameter adjuster, the optical scanner 20, and the imaging device 50 of the light generation device 10, processes image data output from the imaging device 50, and creates an image of the sample S. As the computer 60, for example, a personal computer, a smart device such as a smartphone or a tablet terminal, or a cloud server can be used. In the light sheet microscope 1, each device is generally arranged so that the optical axis of the irradiation optical system and the optical axis of the detection optical system intersect (for example, orthogonally).

  Next, the light irradiation device R1 of the light sheet microscope 1 will be described in more detail with reference to FIG. FIG. 2 is a diagram showing a schematic configuration of the light irradiation apparatus according to the first embodiment of the present invention. The light irradiation device R1 includes a light generation device 10, an optical scanner 20, and an irradiation optical system 30. The light generation apparatus 10 includes a light source 11, a collimator 12, a wavelength selection filter 13, a beam expander 14, a mirror 15, and a steer step device 100. The light source 11, the collimator 12, the wavelength selection filter 13, the beam expander 14, and the mirror 15 constitute a light generation unit that generates and outputs desired light.

  The light source 11 is, for example, a light source that outputs low-coherent (incoherent) light. As the low-coherent light source, for example, a super luminescent diode (SLD), an ASE light source (Amplified Spontaneous Emission), a super continuum light source (SC) or a lamp-type light source can be used. is there. The light source 11 may be a light source such as a laser diode (LD). The collimator 12 collimates the light output from the light source 11 and outputs parallel light L1.

  The wavelength selection filter 13 is an optical filter that selects the wavelength width w of the parallel light L1 output from the light source 11 via the collimator 12 within a predetermined wavelength range. For example, when a low-coherent light source such as a lamp-type light source or a supercontinuum light source is used as the light source 11, the wavelength width of the input light is generally wide, so the wavelength selection filter 13 is used to narrow the wavelength width. be able to. As the input light L2 whose wavelength width is narrowed by the wavelength selection filter 13, for example, the center wavelength λ is in the range of 300 nm (0.30 μm) to 850 nm (0.85 μm), and the input light L2 The wavelength width w is preferably in the range of 1 nm (0.001 μm) to 100 nm (0.1 μm). The “wavelength width” used here means the full width at half maximum of input light. The input light L <b> 2 that is input to the wavelength selection filter 13 and has a predetermined wavelength width is output to the beam expander 14.

  The beam expander 14 adjusts the light diameter of the input light L2 input from the light source 11 via the collimator 12 and the wavelength selection filter 13 so as to correspond to the size of the steer step device 100 described later. An optical element having an adjustment function. Note that a diaphragm (aperture) may be used instead of the beam expander 14 as the light diameter adjusting unit of the input light L2. The input light L <b> 2 expanded to a predetermined light diameter by the beam expander 14 is reflected by the mirror 15 and input to the steer step device 100.

  The steer step device 100 is entirely made of a light transmissive material (for example, a material transparent to input light such as glass, quartz, resin, etc.), and the input light L2 whose light diameter is adjusted by the beam expander 14 is used. , Each optical element that selectively interferes with each flat interference surface, and outputs the interference light L3 obtained by superimposing the light that selectively interferes with the input light L2. The steer step device 100 is arranged so that the optical axis of the input light L2 coincides with the central axis of the steer step device 100. 3 is a perspective view showing the appearance of the steer step device, FIG. 4 is a view showing the appearance of the steer step device, (a) shows a side view thereof, and (b) shows a plan view thereof. . As shown in FIGS. 3 and 4, the steer step device 100 has a stepped shape in which transparent disk-shaped members having different outer diameters are concentrically overlapped, for example, a first interference unit. 101, a second interference unit 102, a third interference unit 103, a fourth interference unit 104, and a fifth interference unit 105. The number of stages N of the steer step device 100 is 5 (N = 5) in the embodiment shown in FIGS. 3 and 4, but is not limited to this, and the number of stages N is 2 or more (integer). I just need it. Note that the effective length of the line light L4 formed by being condensed by the irradiation optical system 30 to be described later can be made longer by increasing the number N of stages.

ここで、上記した式（１）で用いる「λ」は、ステアステップデバイス１００に入力される入力光Ｌ２の中心波長（μｍ）であり、「ｗ」は、ステアステップデバイス１００に入力される入力光Ｌ２の波長幅（μｍ）であり、「ｎ」は、ステアステップデバイス１００の屈折率を意味している。また、第２及び第３の干渉面１０２ａ，１０３ａ間の高さＨ、第３及び第４の干渉面１０３ａ，１０４ａ間の高さＨ、第４及び第５の干渉面１０４ａ，１０５ａ間の高さＨも同様に上記の式（１）を満たすように形成されている。なお、各高さＨは、上記の式（１）を満たせばよく、各高さＨが同じであることが好ましいが、各高さＨが相互に異なっていてもよい。 In the steer step device 100, each of the interference units 101 to 105 has interference surfaces 101a, 102a, 103a, 104a, and 105a on the surface on the input side of the input light L2, and light of the interference surfaces 101a to 105a. The outer shape is formed so that the height H (distance) between the interference surfaces adjacent in the transmission direction (for example, the first and second interference surfaces 101a and 102a) satisfies the following expression (1). That is, the height H (distance between the interference surfaces) is set to be longer than the coherent length of the input light L2. The first to fourth interference surfaces 101a to 104a have an annular shape, and the fifth interference surface 105a has a circular shape.

Here, “λ” used in the above formula (1) is the center wavelength (μm) of the input light L2 input to the steer step device 100, and “w” is an input input to the steer step device 100. The wavelength width (μm) of the light L 2, and “n” means the refractive index of the steer step device 100. Further, the height H between the second and third interference surfaces 102a and 103a, the height H between the third and fourth interference surfaces 103a and 104a, and the height between the fourth and fifth interference surfaces 104a and 105a. Similarly, the height H is formed so as to satisfy the above-described formula (1). Each height H only needs to satisfy the above formula (1), and each height H is preferably the same, but each height H may be different from each other.

  Here, the operation of the steer step device 100 will be described. In the steer step device 100, as described above, the height H between the interference surfaces is set higher (longer) than the coherent length of the input light L2 input to the steer step device 100. Therefore, interference between lights input to the steer step device 100 and output from different interference surfaces is suppressed, while light output from the same interference surface interferes with the input light L2 selectively. Interference light L3 is obtained by interference. That is, in the case of a normal circular aperture or the like, light from different circular areas is coherently added, whereas the steer step device 100 uses a point spread function (PSF) by an annular aperture. The light selectively interfered with each interference surface can be superimposed. As a result, when the light irradiation device R1 (or the light sheet microscope 1) is configured, it is possible to increase the effective length of the line light and reduce the influence of side lobes.

  In this way, the steer step device 100 outputs the interference light L3 selectively interfered with each of the interference surfaces 101a to 105a to the optical scanner 20. Note that if the number of stages N of the steer step device 100 is N (N is a positive integer), the effective length of the line light that is condensed by the irradiation optical system 30 is larger than that when the steer step device 100 is not used. It can be about N times.

  Such a steer step device 100 may be produced by, for example, bonding disc-shaped glasses having different diameters with a predetermined thickness H so that the center thereof is coaxial, or using a cylindrical transparent resin optically. You may cut and produce to the level which can demonstrate performance. Further, the steer step device 100 may be a convex steer step device as shown in FIGS. 3 and 4, or conversely, may be a concave steer step device 110 as shown in FIG. 5. For example, when the number of steps N is 5, the concave steer step device 110 includes the interference portions 111 to 115, and the interference portions 111 to 115 have interference surfaces 111a to 115a, respectively. Can be used. Also in this case, the height H between the interference surfaces adjacent to each other among the interference surfaces 111a to 115a satisfies the above formula (1). Further, the steer step devices 100 and 110 may be of a transmissive type or a reflective type. In the case of the transmissive type, it is preferable to apply antireflection treatment to each interference surface. It is preferable to apply.

  Returning now to FIG. The optical scanner 20 is a member that scans the interference light L <b> 3 generated by the selective interference by the steer step device 100 in at least one direction. As the optical scanner 20, for example, a scanner constituted by a galvanometer mirror, a polygon mirror, or a MEMS mirror can be used. The optical scanner 20 forms sheet light inside the sample S of the observation object by scanning the interference light L3 that becomes the line light L4 irradiated to the sample S of the observation object. More specifically, the interference light L3 is condensed on the sample S via a relay optical system 31 and an objective lens 32, which will be described later, and becomes line light L4. By scanning with the optical scanner 20, sheet light which is a plane of light is formed in a pseudo manner. The interference light L3 scanned by the optical scanner 20 is input to the irradiation optical system 30, more specifically, to the relay optical system 31.

  The relay optical system 31 includes a relay lens. The objective lens 32 of the irradiation optical system 30 and the optical scanner 20 are conjugated to each other, and the pupil position of the objective lens 32 is set to the scanning element surface of the optical scanner 20. Relay to

  The objective lens 32 is an irradiation objective lens, condenses the interference light L3 guided by the relay optical system 31 inside the sample S of the observation object, and is line light extending in the optical axis direction of the objective lens 32. It is a lens that forms L4. Observation light (for example, fluorescence or scattered light) generated from the sample S by the line light L4 collected in the sample S by the objective lens 32 is imaged by the objective lens 41 of the detection optical system 40 or the like. The imaging device 50 captures an image formed by the detection optical system 40. When the imaging device 50 can perform so-called rolling reading (rolling shutter), the computer 60 may synchronize the timing of scanning of the line light by the optical scanner 20 and the rolling reading by the imaging device 50. For example, in the imaging device 50 capable of rolling readout, signal readout by rolling readout is performed for each of a plurality of pixel columns, and a predetermined image is generated. Therefore, by synchronizing the speed of the rolling readout and the scanning speed of the line light L4, it is possible to capture only the optical image accompanying the observation light generated from the sample S by the line light L4. As the detection optical system 40, the imaging device 50, and the like, since a general light sheet microscope can be used as appropriate, a more detailed description is omitted here.

  According to the light irradiation apparatus R1 or the light sheet microscope 1 including the light generation apparatus 10 described above, the height H at which the steer step device 100 (or 110) of the light generation apparatus 10 satisfies the requirement of the above-described formula (1). A plurality of stepped interference surfaces 101a to 105a (or 111a to 115a). For this reason, the height H between the interference surfaces becomes higher (longer) than the coherent length of the input light L2 input to the steer step device 100 (or 110), and output from different interference surfaces 101a to 105a (or 111a to 115a). While the interference between the transmitted light is suppressed, the light output from the same interference surfaces 101a to 105a (or 111a to 115a) interferes with the input light L2 selectively so that the interference light L3 is To get. That is, in the case of a normal circular aperture or the like, light from different regions of the circular shape is coherently added, whereas according to the steer step device 100 (or 110) of the present embodiment, the point spread function based on the annular aperture is used. With (PSF: Point Spread Function), it is possible to superimpose light selectively interfered with each of the interference surfaces 101a to 105a (or 111a to 115a). As a result, when the light irradiation device R1 (or the light sheet microscope 1) is configured using the light generation device 10, the effective length of the line light L4 is increased and the side lobe of the side lobe is increased as shown in the examples described later. The influence can be reduced.

  Here, the effect by this embodiment is demonstrated concretely using the light irradiation apparatus R which simplified the light irradiation apparatus R1 of the structure shown in FIG. However, the present invention is not limited to these.

  First, as an example, a light irradiation apparatus R having the configuration shown in FIG. 6 was prepared. Similar to the light irradiation apparatus R1 of the first embodiment, the light irradiation apparatus R includes the light source 11, the collimator 12, the wavelength selection filter 13, the beam expander 14, the steer step device 100, and the objective lens 32 of the irradiation optical system 30. In addition, a diaphragm 15 a is provided between the beam expander 14 and the steer step device 100. Then, the light L output from the objective lens 32 is imaged by the camera C which is a two-dimensional imaging device, and the expansion and the diameter of the line light are determined based on the expansion of the light (the same Gaussian beam as that in the past) and the light without the steer step device. Evaluation was made in comparison with the diameter. In addition, as a steer step device used for an Example, two types of devices from which a manufacturing method differs were prepared. The steer step device 100 of Example 1 was manufactured by laminating discs having different diameters made of glass, and the steer step device 100 of Example 2 was manufactured by cutting out a cylindrical transparent resin.

  Moreover, as a measuring method, line light was formed on each condition, the camera C fixed to the stage was moved to the optical axis direction of the objective lens 32, and the line light in each position was imaged. The focal length of the objective lens 32 is 100 mm, the pupil diameter (2NAf) of the objective lens is 11 mm, the aperture of the diaphragm is 5.2 mm, the Effective NA is 0.026, and DOF = + / − 466 μm. The beam diameter was 29.6 μm. The wavelength selection filter 13 used was set so that the center wavelength of the transmitted light was 630 nm and the wavelength width was 92 nm.

  First, the extension degree of the sheet beam was determined for Examples 1 and 2 using two types of steer step devices and a comparative example using no steer step device. As a result of obtaining the degree of elongation, as shown in FIG. 7, in the comparative example without a steer step device (without SSD), the FWHM (full width at half maximum) suddenly increased from about 2000 μm. In Example 1 and Example 2 using a step device, it was confirmed that it was suddenly increased from about 7000 μm, and the degree of elongation could be increased to 3 times or more.

  Moreover, about Example 1, 2 using two types of steer step devices, and the comparative example which did not use a steer step device, the ratio of a side lobe (sidelobe) and a peak (side lobe / peak × 100 (%)) ) The reason for determining the ratio between the side lobe and the peak is as follows. That is, generally, ring-shaped side lobes appear around the line light (see FIG. 8A). However, when the intensity of the side lobe is large with respect to the peak intensity of the line light (for example, the ratio is 50% or more), when such a light irradiation apparatus is incorporated in the light sheet microscope, the observation light generated by the side lobe (for example, Fluorescence) becomes noise, making it difficult to obtain a clear image. Therefore, line light with a long portion where the ratio of the side lobe to the peak is small is suitable for the light sheet microscope.

  As a result of obtaining the ratio between the side lobe and the peak, as shown in FIG. 8B, in the comparative example without the steer step device (without SSD), the light from the center of the line light below the threshold (for example, the ratio 50%) The distance was as short as about 2000 μm, and when it exceeded 2000 μm, the strength of the side lobe suddenly increased. On the other hand, in Example 1 using the first steer step device and Example 2 using the second steer step device, the distance from the center of the line light below the threshold (for example, the ratio of 50%) is It was confirmed that the portion with a small ratio of the side lobe to the peak can be increased to 3 times or more as compared with the case where the steer step device is not used, which is a little less than 8000 μm. In other words, the line light generated using the steer step device can increase the effective length of the line light while suppressing the influence of side lobes on the peak, and it can be confirmed that the light irradiation apparatus is more suitable for the light sheet microscope. It was. As described above, according to the light irradiation device R1 including the light generation device 10 illustrated in FIG. 2 and the like, it is possible to increase the effective length of the line light L4 and reduce the influence of side lobes.

  Further, in the light irradiation device R1 including the light generation device 10 or the light sheet microscope 1, each of the interference surfaces 101a to 105a and 111a to 115a has either a circular shape or an annular shape. For this reason, the effect by the point spread function can be further exhibited especially by the annular portion, and when the light irradiation device R1 (or the light sheet microscope 1) is configured, the effective length of the line light L4 is further increased. It is possible to reduce the influence of side lobes while increasing the length.

  In the light irradiation device R1 or the light sheet microscope 1 including the light generation device 10, the light generation device 10 includes a wavelength selection filter 13 that converts the light L1 output from the light source 11 into light having a center wavelength λ and a wavelength width w. In addition. For this reason, wavelength selectivity can be improved.

  In the light irradiation device R1 or the light sheet microscope 1 including the light generation device 10, the light generation device 10 includes a beam expander 14 as a light adjustment element that adjusts the size of the cross section of the input light L2. For this reason, it becomes possible to adjust light so that the input light L2 may be irradiated to the suitable position or area | region of the steer step device 100. FIG. As such a light adjusting element, a diaphragm may be used instead of the beam expander.

  Further, in the light irradiation device R1 including the light generation device 10 or the light sheet microscope 1, the center wavelength λ of the input light L2 is any one in the range of 300 nm to 850 nm, and the wavelength width w of the input light L2 is 1 nm. It can be set to be in the range of ˜100 nm. In this case, when the light generation device 10 is incorporated in an imaging device such as the light sheet microscope 1, the inspection object can be observed more suitably.

  Further, in the light irradiation device R1 including the light generation device 10 or the light sheet microscope 1, the steer step device 100 has five stages of interference surfaces such as the first to fifth interference surfaces 101a to 105a (or 111a to 115a). The height H between the interference surfaces satisfies the expression (1). Thus, by increasing the number of stepped interference surfaces, the effective length of the line light can be further increased according to the number of steps. In addition, by providing three or more interference surfaces in this way, the effective length of the line light can be increased more remarkably. FIG. As shown in Fig. 2, the narrower the annular zone is, the thinner it is placed in the periphery, the narrower and longer the beam is generated. However, the narrow annular zone is placed in the periphery by providing three or more interference surfaces. Thus, the effective length of the sheet beam can be significantly increased.

  Further, in the light irradiation device R1 including the light generation device 10 or the light sheet microscope 1, the areas of the first to fifth interference surfaces 101a to 105a (or 111a to 115a) of the steer step device 100 are equal to each other. Yes. For this reason, the light input to each interference surface becomes uniform, and the interference light L3 generated by the steer step device 100 can be changed to the line light L4 in a more preferable form.

[Second Embodiment]
Next, a light irradiation microscope R2 and a light sheet microscope including the light irradiation device R2 according to the second embodiment of the present invention will be described with reference to FIG. FIG. 9 is a diagram showing a schematic configuration of a light irradiation apparatus according to the second embodiment of the present invention. As illustrated in FIG. 9, the light irradiation device R2 includes a light source 11, a collimator 12, a wavelength selection filter 13, a beam expander 14, a mirror 15, a steer step device 100, as in the light irradiation device R1 of the first embodiment. The optical scanner 20, the relay optical system 31 of the irradiation optical system 30, and an objective lens 32 (not shown in FIG. 9) are provided. Further, a second optical scanner 20 is provided between the optical scanner 20 and the steer step device 100. A relay optical system 16 is provided. The light irradiation device R2 constitutes a light sheet microscope together with the detection optical system 40, the imaging device 50, and the computer 60 shown in FIG. Of the light irradiation device R2, the light generation device 10 includes the light source 11, the collimator 12, the wavelength selection filter 13, the beam expander 14, the mirror 15, the steer step device 100, and the second relay optical system 16. .

  The light irradiation device R2 includes a second relay optical system 16 including a relay lens having a conjugate relationship between the steer step device 100 and the optical scanner 20. For this reason, according to the light irradiation apparatus R2 according to the present embodiment, the pupil of the objective lens 32 is relayed to the position of the steer step device 100 by the second relay optical system 16, and thus the sample to be examined It becomes possible to irradiate S with more ideally condensed line light. Since the configuration other than the above is substantially the same as that of the first embodiment, the description thereof will be omitted. However, when a light sheet microscope is configured using such a light irradiation device R2, line light is formed as in the first embodiment. It is possible to increase the effective length of L4 and reduce the influence of side lobes. The second relay optical system 16 may be a reduction optical system. In this case, the scanning element surface of the optical scanner 20 can be reduced, and the apparatus can be miniaturized. Further, since the pupil position and the phase modulation position can be matched, the influence of diffraction or the like by the element can be suppressed, and the spot shape can be more suitably modified.

[Third Embodiment]
Next, a light irradiation microscope R3 and a light sheet microscope including the light irradiation device R3 according to the third embodiment of the present invention will be described with reference to FIG. FIG. 10 is a diagram showing a schematic configuration of a light irradiation apparatus according to the third embodiment of the present invention. As shown in FIG. 10, the light irradiation device R3 includes a light source 11, a collimator 12, a wavelength selection filter 13, a beam expander 14, a mirror 15, a steer step device 100, as in the light irradiation device R1 of the first embodiment. The optical scanner 20 includes a relay optical system 31 of the irradiation optical system 30 and an objective lens 32 (not shown in FIG. 10). However, in the light irradiation apparatus R3 according to the third embodiment, the steer step device 100 is arranged on the surface of the optical scanner 20. The steer step device 100 may be of a reflective type or of a transmissive type, and a reflective concave steer step device 110 (see FIG. 5) may be disposed on the surface of the optical scanner 20. Such a light irradiation device R3 constitutes a light sheet microscope together with the detection optical system 40, the imaging device 50, and the computer 60 shown in FIG. 1, similarly to the light irradiation device R2 of the second embodiment. In this case, the light generation device may include the light source 11, the collimator 12, the wavelength selection filter 13, the beam expander 14, the mirror 15, the steer step device 100, and the optical scanner 20.

  In the light irradiation apparatus R3, since the steer step device 100 is arranged on the surface of the optical scanner 20, the steer step device 100 can be easily installed at the position of the pupil of the objective lens 32, and the optical system is simplified. can do. For this reason, it is possible to irradiate the sample light S to be inspected more ideally with the line light. Since the configuration other than the above is substantially the same as that of the first embodiment, the description thereof is omitted. However, when a light sheet microscope is configured using such a light irradiation device R3, line light is formed as in the first embodiment. It is possible to increase the effective length of L4 and reduce the influence of side lobes.

[Fourth Embodiment]
Next, a light irradiation microscope R4 and a light sheet microscope including the light irradiation device R4 according to the fourth embodiment of the present invention will be described with reference to FIG. FIG. 11 is a diagram showing a schematic configuration of a light irradiation apparatus according to the fourth embodiment of the present invention. As shown in FIG. 11, the light irradiation device R4 includes a light source 11, a collimator 12, a wavelength selection filter 13, a beam expander 14, a mirror 15, and a steer step device 100 (similar to the light irradiation device R1 of the first embodiment. 100a to 100c), the optical scanner 20, and the relay optical system 31 of the irradiation optical system 30, and further the SSD switching mechanism 17 and the objective lens switching mechanism 33. The light irradiation device R4 is configured such that the objective lenses 32a and 32b having different NAs (high NA and low NA) can be switched by the objective lens switching mechanism 33. In the light irradiation device R4, the objective lens switching mechanism 33 realizes the thin line light L4a in a relatively narrow range by the high NA objective lens 32a, and widens the slightly thick line light L4b by the low NA objective lens 32b. It can be realized in a range. Thereby, it becomes possible to select an objective lens according to the sample S to be observed.

  In the light irradiation apparatus R4, three steer step devices 100a, 100b, and 100c are provided as the steer step devices that generate the interference light L3, and one of the steer step devices is provided by the SSD switching mechanism 17. Configured to be selected. These steer step devices 100a to 100c have different numbers of steps and diameters (rings), and suitable steer steps depending on the objective lenses 32a and 32b, beam conditions, and sample S to be observed. The device can be selected. Since the configuration other than the above is substantially the same as that of the first embodiment, the description thereof will be omitted. However, when a light sheet microscope is configured using such a light irradiation device R4, line light is formed as in the first embodiment. It is possible to increase the effective length of L4 and reduce the influence of side lobes.

[Fifth Embodiment]
Next, a light irradiation microscope R5 and a light sheet microscope including the light irradiation device R5 according to the fifth embodiment of the present invention will be described with reference to FIG. FIG. 12 is a diagram showing a schematic configuration of a light irradiation apparatus according to the fifth embodiment of the present invention. As shown in FIG. 12, the light irradiation device R5 is similar to the light irradiation device R1 of the first embodiment. The light source 11, the collimator 12, the wavelength selection filter 13, the mirror 15, the steer step device 100, and the optical scanner 20 are used. Furthermore, objective lenses 32a and 33b similar to those in the fourth embodiment and an objective lens switching mechanism 33 are further provided. The light irradiation device R5 further includes a zoom beam expander 14a, an electric diaphragm 18, and a zoom pupil relay lens 34. In this light irradiation apparatus R5, it is possible to adjust how many of the steer step devices 100 are used by adjusting the light diameter of the light by the zoom beam expander 14a or the electric diaphragm 18. . When considering the light utilization efficiency, it is preferable to use the zoom beam expander 14a rather than using the motorized diaphragm 18, but it is preferable to use the motorized diaphragm 18 in order to obtain a sharp light diameter. By using the beam expander 14a and the electric diaphragm 18 in combination, the number of stages of the steer step device 100 to be used can be accurately selected. Instead of the electric diaphragm 18, a manual diaphragm mechanism may be used.

  In addition, the light irradiation device R5 includes a zoom pupil relay lens 34 so that the pupil diameters of the objective lenses 32a and 32b can be adjusted. For this reason, in this light irradiation apparatus R4, by using the zoom pupil relay lens 34, an objective lens is used at its maximum NA (that is, used at the maximum pupil diameter of the objective lens), and the objective lens is used. It is possible to realize the thinnest line light L4. If there is no zoom pupil relay lens 34, a steer step device having the same size as the pupil diameter of the objective lens is prepared to generate the thinnest and longest line light. In R5, the zoom pupil relay lens 34 is provided, so that the pupil diameter can be adjusted with respect to the steer step device. That is, it is not necessary to switch the steer step device. However, it is preferable that there is a diaphragm that can change the size of the cross section of the input light in accordance with the changed size of the pupil diameter. Since the configuration other than the above is substantially the same as that of the first embodiment, the description thereof will be omitted. However, when a light sheet microscope is configured using such a light irradiation device R5, line light is formed as in the first embodiment. It is possible to increase the effective length of L4 and reduce the influence of side lobes.

[Sixth Embodiment]
Next, a light irradiation microscope R6 and a light sheet microscope including the light irradiation device R6 according to the sixth embodiment of the present invention will be described with reference to FIG. 13A and 13B are diagrams showing a schematic configuration of a light irradiation apparatus according to the sixth embodiment of the present invention, where FIG. 13A is a view seen from the side, and FIG. 13B is a view seen from the top (after the mask). (C) is a plan view of the mask, and (d) is a perspective view of a member in which the cylindrical lens and the steer step device are integrated. As shown in FIGS. 13A and 13B, the light irradiation device R6 includes a light source 11, a collimator 12, a wavelength selection filter 13, a beam expander 14, a steer, similarly to the light irradiation device R1 of the first embodiment. A step device 120, an objective lens 32, and a lens 35 are provided, and a mask 19a and a cylindrical lens 19b are further provided. In this light irradiation device R6, as shown in FIG. 13C, a mask 19a having a substantially rectangular opening 19c is arranged behind the beam expander 14. Moreover, as shown in FIG.13 (d) and FIG.14 (a) (b), each interference surface 121a-125a of each interference part 121-125 of the steer step device 120 is a rectangular shape. In addition, the height H (distance) between the interference surfaces of the interference surfaces 121a to 125a of the steer step device 120 satisfies the formula (1) as in the first embodiment described above, and the input The light is selectively interfered.

  In such a light irradiation apparatus R6, the input light L2 is adjusted by the mask 19a so as to have a substantially rectangular cross section, and the adjusted input light L2a is input to the cylindrical lens 19b. Then, when the light is condensed by the cylindrical lens 19b, the input light is selectively interfered by the steer step device 120, and the sheet light L4c which is planar light is generated by the objective lens 32. Thus, in the light irradiation apparatus R6 according to the present embodiment, since the rectangular steer step device 120 and the cylindrical lens 19b are used, the sheet light L4c having a relatively long effective length is used as the optical axis direction of the objective lens 32. Can be generated along with The sheet light L4c is irradiated onto the sample S, the observation light is imaged by the objective lens 41 of the detection optical system 40, and the like, and is imaged by the imaging device 50. Also in this light irradiation device R6, the beam of the sheet light L4c can be extended in the direction of the optical axis of the objective lens 32, and the effective field of view of the light sheet microscope can be expanded, as in the first embodiment. The steer step device used here may be a concave rectangular steer step device 130 shown in FIG. 15, and the height between the interference surfaces 131 a to 135 a of the steer step device 130 is high. The length H (distance) satisfies the formula (1) as in the first embodiment described above.

  The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the above-described embodiments, and various modifications are possible.

  DESCRIPTION OF SYMBOLS 1 ... Light sheet microscope, 10 ... Light generation apparatus, 11 ... Light source, 13 ... Wavelength selection filter (optical filter), 14, 14a ... Beam expander (light adjustment element), 16 ... Relay optical system, 17 ... SSD switching mechanism , 18 ... Aperture, 20 ... Optical scanner (optical scanning unit), 30 ... Irradiation optical system, 31 ... Relay optical system, 32, 32a, 32b ... Objective lens, 33 ... Objective lens switching mechanism, 34 ... Zoom pupil relay lens, DESCRIPTION OF SYMBOLS 40 ... Detection optical system, 50 ... Imaging apparatus, 60 ... Computer, 100, 110, 120, 130 ... Steer step device, 101a-105a, 111a-115a, 121a-125a, 131a-135a ... Interference surface, H ... Interference surface L2 ... input light, L3 ... interference light, L4, L4a, L4b ... line light, L4c ... sheet light, R, R1-R6 ... light Morphism device, S ... sample (observation object).

Claims (15)

A light generating device for generating line light or sheet light,
A light generation unit having a light source and outputting light having a center wavelength λ and a wavelength width w;
A steer step device having a refractive index of n and selectively interfering light output from the light generation unit into interference light, and
The steer step device has at least first and second interference surfaces whose surfaces are stepped, and a height H between the first and second interference surfaces is expressed by the following formula (1):

A light generator that satisfies the requirements.   The light generation device according to claim 1, wherein the light generation unit further includes an optical filter that converts light output from the light source into the light having a center wavelength λ and a wavelength width w.   The light generation device according to claim 1, wherein the light generation unit further includes a light adjustment element that adjusts a size of a cross section of light input to the steer step device.   The light generation device according to claim 3, wherein the light adjustment element includes at least one of a beam expander and a diaphragm.   The center wavelength λ of the light input to the steer step device is any one in the range of 300 nm to 850 nm, and the wavelength width w of the light input to the steer step device is in the range of 1 nm to 100 nm. The light generation device according to claim 1, wherein the light generation device is any one of the above.   The light generation apparatus according to claim 1, wherein the light source is a light source that outputs low-coherent light.   The steer step device further includes a third interference surface, and a height H between the second and third interference surfaces satisfies the formula (1). Light generator.   The light generation apparatus according to claim 1, wherein an area of the first interference surface is equal to an area of the second interference surface.   The light generation apparatus according to any one of claims 1 to 8, wherein the first and second interference surfaces have either a circular shape or an annular shape. A cylindrical lens that receives the light output from the light generation unit;
The light generation apparatus according to claim 1, wherein the first and second interference surfaces of the steer step device have a rectangular shape. A light irradiation device for irradiating an observation object with line light,
The light generation device according to any one of claims 1 to 9,
An optical scanning unit that scans the interference light output from the light generation device;
A first relay optical system to which the interference light scanned by the optical scanning unit is input;
An objective lens that collects the interference light guided by the first relay optical system and irradiates the observation object with line light;
A light irradiation apparatus comprising:   The light irradiation apparatus according to claim 11, wherein the steer step device is disposed on a scanning element surface of the optical scanning unit. A second relay optical system that further relays the pupil of the objective lens relayed by the first relay optical system;
The light irradiation apparatus according to claim 11 or 12, wherein the steer step device is disposed at a position of a pupil relayed by the second relay optical system. A light irradiation device for irradiating an observation object with sheet light,
The light generation device according to claim 10;
An objective lens that collects the interference light output from the light generation device and irradiates an observation object with sheet light; and
A light irradiation apparatus comprising: The light irradiation device according to any one of claims 11 to 14,
A detection optical system that forms an image of observation light generated from the observation object in accordance with irradiation of line light or sheet light by the light irradiation device,
An imaging device that captures an image of the observation light imaged by the detection optical system;
Light sheet microscope equipped with.

Images