Illumination setting method, light sheet microscope apparatus, and recording medium

An illumination setting method includes acquiring an image of a sample onto which a light sheet has been radiated; determining, on the basis of the acquired image of the sample, a subordinate ray angle with respect to a width direction of the light sheet; and performing a setting of the illumination optical system according to the determined subordinate ray angle.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority from prior Japanese Patent Application No. 2016-094159, filed May 9, 2016, the entire contents of which are incorporated herein by this reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to an illumination setting method, a light sheet microscope apparatus, and a recording medium.

Description of the Related Art

In the field of fluorescence microscopy, a technology is known that radiates a sample with a laser beam from a direction perpendicular to an optical axis of a detection optical system, so as to form, in the sample, a light sheet perpendicular to the optical axis of the detection optical system. This technology has been attracting attention in recent years because it provides the advantages of, for example, suppressing damage caused to a sample and realizing a high longitudinal resolution.

When the above-described technology is applied, a sample is illuminated from a direction different from a direction of the optical axis of the detection optical system. Thus, if the sample has a portion through which light cannot be easily transmitted due to absorption or a portion in which light is scattered (hereinafter collectively referred to as a light-blocking portion), light will not enter behind the light-blocking portion, and then a striped shadow will be created in the field of view.

A related technology is disclosed in, for example, Japanese Laid-open Patent Publication No. 2008-250303. Japanese Laid-open Patent Publication No. 2008-250303 discloses a technology that radiates a sample material with a radiation component of a sheet light at different angles according to the time by use of an oscillatory movement of a wobble plate or a swing mirror.

SUMMARY OF THE INVENTION

An illumination setting method according to an aspect of the present invention includes acquiring an image of a sample onto which a light sheet emitted from an illumination optical system has been radiated; determining, on the basis of the acquired image of the sample, a subordinate ray angle with respect to a width direction of the light sheet emitted from the illumination optical system; and performing a setting of the illumination optical system according to the determined subordinate ray angle.

An illumination setting method according to another aspect of the present invention includes acquiring an image of a sample onto which a light sheet has been radiated by an illumination optical system; determining, on the basis of the acquired image of the sample, an incident angle at which a principal ray of the light sheet emitted from the illumination optical system enters the sample; and performing at least one of a setting of the illumination optical system and a setting of a direction of the sample according to the determined incident angle.

An illumination setting method according to yet another aspect of the present invention includes acquiring, by a computer and from an imaging device, an image of a sample onto which a light sheet has been radiated by an illumination optical system; determining, by the computer and on the basis of the image of the sample that has been acquired from the imaging device, a subordinate ray angle with respect to a width direction of the light sheet emitted from the illumination optical system; and outputting, by the computer, a control signal that gives an instruction to perform a setting of the illumination optical system that corresponds to the determined subordinate ray angle.

A light sheet microscope apparatus according to yet another aspect of the present invention includes an illumination optical system that radiates a light sheet onto a sample; an imaging device that acquires an image of the sample onto which the light sheet has been radiated by the illumination optical system; a controller that determines, on the basis of the image of the sample that has been acquired by the imaging device, a subordinate ray angle with respect to a width direction of the light sheet emitted from the illumination optical system; and a setting device that performs a setting of the illumination optical system according to the subordinate ray angle determined by the controller.

A non-transitory recording medium according to yet another aspect of the present invention has stored therein a program that causes a computer to execute a process including acquiring, from an imaging device, an image of a sample onto which a light sheet has been radiated by an illumination optical system; determining, on the basis of the image of the sample that has been acquired from the imaging device, a subordinate ray angle with respect to a width direction of the light sheet emitted from the illumination optical system; and outputting a control signal that gives an instruction to perform a setting of the illumination optical system that corresponds to the determined subordinate ray angle.

DESCRIPTION OF THE EMBODIMENTS

An illumination setting that can suppress a stripe effectively differs according to the size of a material causing the stripe (that is, the size of a light-blocking portion). With respect to an effect that suppresses a stripe (hereinafter referred to as a stripe eliminating effect), it is preferable that the illumination setting be performed such that a sample is illuminated at a larger angle if there exists a larger causative material. On the other hand, the illumination setting also inevitably affects a basic illumination performance. For example, there tends to be a greater decrease in, for example, illumination efficiency or uniformity of illumination if a sample is illuminated at a larger angle. Thus, it is preferable that an appropriate illumination setting be performed according to an observation target while balancing an illumination performance and a stripe eliminating effect.

In light of the description above, embodiments of the present invention will now be described.

First Embodiment

FIG. 1illustrates a schematic configuration of a light sheet microscope apparatus1according to a first embodiment. The light sheet microscope apparatus1is, for example, a fluorescence microscope that detects a fluorescence from a sample S such as a biological sample. The light sheet microscope apparatus1is configured to illuminate the sample S with a light sheet.

The light sheet microscope apparatus1includes an illumination optical system10that radiates a light sheet onto the sample S, a detection optical system20that guides, to an imaging device30, detected light (such as a fluorescence) from the sample S, and the imaging device30that acquires an image of the sample S. The light sheet microscope apparatus1further includes a controller40that controls the light sheet microscope apparatus1, and a setting device60that performs a setting of the illumination optical system10. The sample S is arranged around a position at which an optical axis of the illumination optical system10and an optical axis of the detection optical system20intersect.

The illumination optical system10is configured to form a light sheet having a sheet shape substantially perpendicular to the optical axis of the detection optical system20and to radiate the light sheet onto the sample S from a direction substantially perpendicular to the optical axis of the detection optical system20. The illumination optical system10will be described in detail later.

Here, the light sheet is illumination light that forms an illuminated area having a sheet shape. The sheet shape is a shape in which a cross-section of illumination light (hereinafter referred to as a beam cross-section) that is perpendicular to a traveling direction of the illumination light (an optical-axis direction on the exit side of the illumination optical system10) has a two-dimensional shape that has two directions perpendicular to each other, wherein one of the two directions is long and the other is short. In the following description, the long direction in the beam cross-section is referred to as a width direction of a light sheet, and the short direction is referred to as a thickness direction of the light sheet. Further, the sheet shape substantially perpendicular to the optical axis of the detection optical system20is a sheet shape in which a light sheet surface that is defined by the traveling direction and the width direction is substantially perpendicular to the optical axis of the detection optical system20. Being substantially perpendicular includes a perpendicular state from which a person skilled in the art can recognize a setting error or a manufacturing error. In the present embodiment, the traveling direction is defined as an x-axis direction, the width direction is defined as a y-axis direction, and the thickness direction is defined as a z-axis direction. The same applies to the other embodiments with respect to this point.

The detection optical system20is an optical system that collects light (such as a fluorescence and hereinafter referred to as detected light) from the sample S and forms an optical image of the sample S on a light-receiving surface of the imaging device30. The imaging device30is a digital camera that includes a two-dimensional image sensor such as a CCD (charge coupled device) or a CMOS (complementary metal oxide semiconductor). The imaging device30acquires an image of the sample S onto which a light sheet has been radiated by the illumination optical system10and outputs image data of the sample S to the controller40.

The controller40is a microscope controller that controls the light sheet microscope apparatus1. The controller40is configured to output a control signal to various electrical mechanisms provided in a microscope body of the light sheet microscope apparatus1. The setting device60is one of the electrical mechanisms, in the microscope body, which operate according to the control signal from the controller40, and is a device that performs a setting of the illumination optical system10.

FIG. 2is a flowchart that illustrates a procedure of illumination processing according to the first embodiment. The illumination processing performed by the light sheet microscope apparatus1is generally described with reference toFIG. 2.

First, the light sheet microscope apparatus1acquires an image of the sample S onto which a light sheet emitted from the illumination optical system10has been radiated (Step S1). Here, the illumination optical system10radiates a light sheet onto the sample S, and the imaging device30captures an image of the sample S and generates image data of the sample S. The generated image data of the sample S is output to the controller40.

Next, the light sheet microscope apparatus1determines a subordinate ray angle with respect to the width direction of the light sheet on the basis of the acquired image (Step S2). The subordinate ray angle is a maximum angle formed by the optical axis on the exit side of the illumination optical system10and the subordinate ray of a light sheet emitted from the illumination optical system10. Further, the subordinate ray angle with respect to the width direction is a subordinate ray angle in a cross-section that includes the width direction and the traveling direction of the light sheet.

In a light sheet illumination, if the sample S has a light-blocking portion in an illuminated area, a striped shadow will occur behind that portion. However, if the subordinate ray angle of a light sheet is not less than zero degrees, light can enter an area behind the light-blocking portion, which results in being able to suppress the striped shadow. Further, if the light sheet has a larger subordinate ray angle, the light can enter an area closer to the light-blocking portion, which results in being able to suppress the striped shadow more effectively.

On the other hand, as illustrated inFIGS. 3 and 4, in order to realize a uniform illumination on an observation range R, a scanning width will be wider when a light sheet L2having a large subordinate ray angle is radiated, compared to when a light sheet L1having a small subordinate ray angle is radiated. This results in a decrease in illumination efficiency and it takes a long time to acquire an image.

Thus, it is preferable that the subordinate ray angle be determined taking into consideration the balance between a stripe eliminating effect and an illumination performance. Further, even if importance is placed on the stripe eliminating effect, it is preferable that the subordinate ray angle be set to be small as long as a striped shadow is suppressed to the extent acceptable to an observer.

However, the size of a striped shadow differs according to the size of a light-blocking portion, and the size of a light-blocking portion differs according to a sample (in particular, an observed portion in the sample). Thus, the subordinate ray angle that can meet the requirements of the observer also differs according to the sample. Therefore, in Step S2, the controller40determines the subordinate ray angle with respect to the width direction of a light sheet emitted from the illumination optical system10on the basis of an image of the sample S onto which the light sheet has been radiated, the image of the sample S being acquired by the imaging device30. Further, the controller40outputs, to the setting device60, a control signal that gives an instruction to perform a setting of the illumination optical system10that corresponds to the subordinate ray angle determined by the controller40. A method for determining a subordinate ray angle will be described in detail later.

When the subordinate ray angle has been determined, the light sheet microscope apparatus1performs a setting of the illumination optical system10according to the determined subordinate ray angle (Step S3), and radiates a light sheet onto the sample (Step S4). Here, the setting device60performs a setting of the illumination optical system10according to a control signal output from the controller40. In other words, the setting device60performs a setting of the illumination optical system10according to the subordinate ray angle determined by the controller40.

According to the light sheet microscope apparatus1, it is possible to perform a setting for obtaining a sufficient stripe eliminating effect while suppressing a reduction in illumination performance, by determining a subordinate ray angle on the basis of an image. Further, the controller40determines the subordinate ray angle on the basis of the image and the setting device60performs a setting according to the determined subordinate ray angle, so a user can easily perform an appropriate setting even if he/she is not used to manipulating a microscope.

Referring toFIGS. 5A to 9, the present embodiment is further described in detail below.FIGS. 5A and 5Billustrate a configuration of the light sheet microscope apparatus1.FIGS. 5A and 5Beach illustrate the light sheet microscope apparatus1and an illumination beam, as viewed from the thickness direction (z-axis direction) and the width direction (y-axis direction) of a light sheet, respectively.

In addition to the illumination optical system10, the detection optical system20, the imaging device30, the controller40, and the setting device60described above, the light sheet microscope apparatus1further includes a display device51and input devices (a keyboard52and a mouse53) that are connected to the controller40.

The illumination optical system10includes a laser11. The laser11is a light source that emits a laser beam (illumination light) that will be converted into a light sheet. The illumination optical system10further includes, in order from the side of the laser11, a first optical system12, a scanner16, and a scanning optical system17.

The first optical system12is an optical system that is arranged between the laser11and the scanner16and that radiates a laser beam onto the scanner16. The first optical system12includes a lens13, a lens14, and a cylindrical lens15. The cylindrical lens15is a movable lens arranged to be movable in the optical-axis direction. The cylindrical lens15is arranged to have a refractive power in an xy plane and to not have a refractive power in an xz plane.

The scanner16is a scanning unit that scans the sample S with a light sheet in the width direction of the light sheet, and is, for example, a rotatable mirror having a deflection surface that deflects light, such as a galvanometer mirror or a resonant mirror. Further, the scanner16may be, for example, an AOD (acousto-optic deflector) or an EOD (electro-optic deflector). In order to simplify the figures, inFIGS. 5A and 5B, optical elements situated in optical paths of light before and after the light is deflected by the scanner16are described in alignment with one another.

The scanning optical system17includes a cylindrical lens18and a cylindrical lens19, and radiates light deflected by the scanner16onto a sample. The cylindrical lens18is arranged to have a refractive power in the xy plane and to not have a refractive power in the xz plane. The cylindrical lens19is arranged to have a refractive power in the xz plane and to not have a refractive power in the xy plane. In other words, the cylindrical lens18and the cylindrical lens19are arranged such that a plane in which the cylindrical lens18has a refractive power and a plane in which the cylindrical lens19has a refractive power are perpendicular to each other. Further, it is preferable that the cylindrical lens19be arranged such that a rear focal position of the cylindrical lens19is situated in a range of the field of view of the detection optical system20, and it is more preferable that the cylindrical lens19be arranged such that the rear focal position of the cylindrical lens19is situated on the optical axis of the detection optical system20.

The scanning optical system17is further arranged such that the scanner16is situated at a front focal position of the scanning optical system17in a light sheet plane (in the xy plane). In other words, the scanning optical system17is arranged such that the scanner16is situated at a front focal position of the cylindrical lens18arranged closest to an object among the cylindrical lenses of the scanning optical system17. The front focal position of the cylindrical lens18is a position at which light is collected into a line when a collimated beam enters the cylindrical lens18from the side close to a sample.

The detection optical system20includes, in order from the side of the sample S, an objective21, a wavelength selective element22, and a tube lens23. The wavelength selective element22is, for example, a barrier filter for preventing a laser beam from entering the imaging device30.

The setting device60is a device that performs a setting of the illumination optical system10, and specifically, a device that changes the position of the cylindrical lens15in its optical-axis direction. As a structure that moves the cylindrical lens15in the optical-axis direction of the cylindrical lens15, the setting device60includes a ball screw61, a nut62screwed with the ball screw61, a holding unit63that holds the cylindrical lens15, and a motor64that rotates the ball screw61. When the setting device60moves the cylindrical lens15in the optical-axis direction of the cylindrical lens15, the focal length of the first optical system12is changed, which results in changing the subordinate ray angle with respect to the width direction of a light sheet emitted from the illumination optical system10, as illustrated inFIG. 5A.

In the light sheet microscope apparatus1having the configuration described above, a laser beam emitted from the laser11enters the scanner16through the cylindrical lens15after its beam diameter is adjusted in the lens13and the lens14. After that, the laser beam deflected in the scanner16is radiated onto the sample S through the cylindrical lens18and the cylindrical lens19.

The cylindrical lens15and the cylindrical lens18do not substantially act on a laser beam in the xz plane because they do not have a refractive power in the xz plane. Further, the scanner16that deflects light in the width direction also does not substantially act on a laser beam in the xz plane. Thus, as illustrated inFIG. 5B, a laser beam is collected into a certain position by the cylindrical lens19independent of a position of the cylindrical lens15or a deflection angle of the scanner16, as viewed from the width direction (y-axis direction).

Further, the cylindrical lens15and the cylindrical lens18have a refractive power in the xy plane. Thus, as illustrated inFIG. 5A, a laser beam is emitted from the cylindrical lens18in a state in which it has a different subordinate ray angle with respect to the width direction according to the position of the cylindrical lens15, and is radiated onto the sample S through the cylindrical lens19, as viewed from the thickness direction (z-axis direction). However, the scanner16is arranged at the front focal position of the cylindrical lens18, so the direction of the principal ray of the laser beam is constant independent of the angle of the scanner16.

Thus, according to the light sheet microscope apparatus1, it is possible to change a subordinate ray angle with respect to the direction of the width of a light sheet according to the position of the cylindrical lens15. Further, it is possible to illuminate an illumination range uniformly because a sample can be scanned while maintaining the direction of the principal ray.

FIG. 6illustrates a hardware configuration of the controller40. The controller40is, for example, a standard computer. The controller40includes a processor41, a memory42, an input/output interface43, a storage44, and a portable recording medium driving device45into which a portable recording medium46is inserted, wherein these components are connected to one another through a bus47.FIG. 6is an example of a hardware configuration of the controller40, and the controller40is not limited to this configuration.

The processor41is, for example, a CPU (central processing unit), an MPU (micro processing unit), or a DSP (digital signal processor), and executes a program so as to perform programmed processing. The memory42is, for example, a RAM (random access memory), and upon the execution of the program, the memory42temporarily stores therein a program or data recorded in the storage44or the portable recording medium46.

The input/output interface43is a circuit that communicates a signal with a device other than the controller40(such as the imaging device30, the display device51, and the setting device60). The storage44is, for example, a hard disk or a flash memory and is mainly used to record various pieces of data and programs. The portable recording medium driving device45is used to accommodate the portable recording medium46such as an optical disk or a CompactFlash®. The portable recording medium46has a role in assisting the storage44.

FIG. 7illustrates a functional configuration of the controller40. The controller40includes an image acquisition unit40a, an image comparison unit40b, an angle determination unit40c, and an output unit40d. At least one of these units may be configured on the memory42by the processor41loading a program recorded in the storage44or the portable recording medium46into the memory42and executing the loaded program. Alternatively, at least one of these units may be configured by hardware such as an integrated circuit such as an FPGA (field-programmable gate array) or an ASIC (application specific integrated circuit).

The image acquisition unit40aacquires, from the imaging device30, an image of a sample that has been acquired by the imaging device30. The image comparison unit40bcompares a plurality of images of the sample that have been acquired by the imaging device30. The angle determination unit40cdetermines a subordinate ray angle with respect to the width direction of a light sheet on the basis of a result of the comparison performed by the image comparison unit40b. The output unit40doutputs, to the setting device60, a control signal that gives an instruction to perform a setting of the illumination optical system10that corresponds to the subordinate ray angle determined by the angle determination unit40c.

FIG. 8is a flowchart that illustrates a procedure of illumination setting processing.FIG. 9is a flowchart that illustrates a procedure of image acquisition processing. Referring toFIGS. 8 and 9, the illumination setting processing performed in the light sheet microscope apparatus1is specifically described below.

First, the light sheet microscope apparatus1performs an initial setting of a subordinate ray angle of a light sheet emitted from the illumination optical system10(Step S100). Here, the controller40outputs a control signal to the setting device60such that a subordinate ray angle with respect to the width direction of the light sheet is a predetermined angle, and the setting device60performs a setting of the illumination optical system10according to the control signal. It is sufficient if the predetermined angle is an angle at which a stripe occurs behind a light-blocking portion, and for example, the predetermined angle is zero degrees, at which the subordinate ray is parallel to the optical axis.

Next, the light sheet microscope apparatus1acquires an image of the sample S in the setting performed in Step S100(Step S110). In this image acquisition processing, as illustrated inFIG. 9, the light sheet microscope apparatus1scans, using the scanner16, the sample S with the light sheet emitted from the illumination optical system10in the width direction of the light sheet (Step S111), and captures, by the imaging device30, the image of the sample S onto which the light sheet has been radiated (Step S112). Accordingly, the imaging device30generates image data of the sample S and outputs the image data to the controller40, and the controller40acquires the image of the sample S. The image acquired here is an image of the sample S illuminated with uniform brightness. The reason is that, during scanning, the light sheet moves in a parallel fashion in the width direction while maintaining the direction of a principal ray of the light sheet, because the scanner16is arranged at the front focal position of the scanning optical system17.

After that, the light sheet microscope apparatus1changes the setting of the subordinate ray angle of the light sheet (Step S120), and acquires an image of the sample S in a setting after the change (Step S130). In Step S120, the controller40outputs a control signal to the setting device60such that the subordinate ray angle is different than a currently set angle (hereinafter referred to as a current angle), and the setting device60performs a setting of the illumination optical system10according to the control signal. It is sufficient if the angle set in Step S120is an angle at which the size of a stripe is the same as or smaller than the size of a stripe at the current angle, and it may be set to be larger than the current angle by a predetermined value. Step S130is similar to Step S110.

The light sheet microscope apparatus1compares a plurality of images of the sample S (Step S140). Here, the controller40compares a plurality of images of the sample S onto which light sheets with different subordinate ray angles have been radiated, and evaluates a change in image. Specifically, the change in image may be evaluated by comparing values each obtained by integrating pixel values in an image in one axis direction (for example, in the x-axis direction or the y-axis direction). Further, the change in image may be evaluated by comparing values each obtained by integrating differences between adjacent pixels in an image in one axis direction (for example, in the y-axis direction). Furthermore, the change in image may be evaluated by comparing spatial frequency distributions each obtained by Fourier transforming an image.

After that, the light sheet microscope apparatus1determines whether the change in image is small (Step S150). Here, on the basis of a result of the comparison in Step S140, the controller40determines whether a value representative of a change in image is smaller than a predetermined value. The value representative of a change in image may be, for example, a value of a difference between values compared between images in Step S140, or it may be a value obtained by standardizing the difference by use of a change amount of subordinate ray angle.

When the change in image has been determined to not be small, the light sheet microscope apparatus1performs the processes of Step S120to Step S150again. The light sheet microscope apparatus1repeats the processes until the change in image is determined to be small in Step S150.

When the change in image has been determined to be small, the light sheet microscope apparatus1determines the subordinate ray angle (Step S160). Here, for example, the controller40determines, to be the subordinate ray angle that is to be set in the illumination optical system10, an angle smallest among a plurality of angles corresponding to a plurality of images in which the change has been determined to be small. In other words, the subordinate ray angle is determined on the basis of a result of comparing a plurality of images.

Finally, the light sheet microscope apparatus1sets the angle determined in Step S160to be the subordinate ray angle (Step S170), and terminates the illumination setting processing. Here, the controller40outputs a control signal to the setting device60such that the subordinate ray angle is an angle determined in Step S160, and the setting device60performs a setting of the illumination optical system10according to the control signal.

After that, the light sheet microscope apparatus1radiates alight sheet onto the sample S in the setting performed in Step S170and acquires an image of the sample S, the observer observes the sample S.

When the light sheet microscope apparatus1performs the illumination setting processing described above, a value of the subordinate ray angle is determined in which there no longer occurs a change in image even if the subordinate ray angle is made larger than the determined value, and the setting of the illumination optical system10is performed such that the subordinate ray angle of a light sheet emitted from the illumination optical system10is the determined value. The state in which there no longer occurs a change in image even if the subordinate ray angle is changed is a state in which a stripe extending behind a light-blocking portion is sufficiently small and less noticeable. According to the illumination setting processing described above, it is possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance, because the subordinate ray angle is not set to be too large. Thus, it is possible to easily perform an appropriate illumination setting for a light sheet illumination.

In the illumination setting processing illustrated inFIG. 8, the example in which the subordinate ray angle is gradually made larger until there no longer occurs a change in image has been described, but the light sheet microscope apparatus1may gradually make the subordinate ray angle smaller until there occurs a change in image. In this case, it is preferable that the initial setting of the subordinate ray angle be a sufficiently large angle such that a stripe does not occur or is less noticeable.

Further, in the illumination setting processing illustrated inFIG. 8, the example in which images acquired by the imaging device30are compared has been described, but a change in image due to a fluorescent material being faded may be excluded and a change in image due to a change in subordinate ray angle may be evaluated. For this purpose, images to be compared may be corrected before the images are compared. For example, a plurality of images may be compared after the images are corrected such that corresponding areas, in the images, in which a stripe does not occur have the same brightness as one another.

Second Embodiment

A light sheet microscope apparatus according to a second embodiment is different from the light sheet microscope apparatus1in that it includes a controller70instead of the controller40. It is similar to the light sheet microscope apparatus1in regard to the other points.

FIG. 10illustrates a functional configuration of the controller70. The controller70includes an image acquisition unit70a, a width calculation unit70b, an angle determination unit70c, and an output unit70d. The hardware configuration of the controller70is similar to that of the controller40. At least one of the units described above may be configured on the memory42by the processor41loading a program into the memory42and executing the loaded program, and it may be configured by hardware such as an integrated circuit such as an FPGA or an ASIC.

The image acquisition unit70aacquires, from the imaging device30, an image of the sample S that has been acquired by the imaging device30. On the basis of the image of the sample S that has been acquired by the imaging device30, the width calculation unit70bcalculates the width of a stripe that appears in the image of the sample S. The angle determination unit70cdetermines a subordinate ray angle with respect to the width direction of a light sheet on the basis of the width of the stripe that has been calculated by the width calculation unit70b. The output unit70doutputs, to the setting device60, a control signal that gives an instruction to perform a setting of the illumination optical system10that corresponds to the subordinate ray angle determined by the angle determination unit70c. The width of a stripe is the length of a stripe with respect to the width direction of a light sheet.

FIG. 11is a flowchart that illustrates a procedure of illumination setting processing. Referring toFIG. 11, the illumination setting processing performed in the light sheet microscope apparatus according to the present embodiment is described below, focusing on the difference from the illumination setting processing illustrated inFIG. 8.

First, the light sheet microscope apparatus performs an initial setting of a subordinate ray angle of a light sheet emitted from the illumination optical system10(Step S200), and acquires an image of the sample S in the initial setting (Step S210). Step S200and Step S210are similar to Step S100and Step S110ofFIG. 8.

When the image has been acquired, the light sheet microscope apparatus calculates the width of a stripe (Step S220). Here, the controller70calculates the width of a stripe that appears in the image on the basis of the image acquired in Step S210. Specifically, pixel values in the image are integrated in the x-axis direction, and a row of pixels in the image in which an integration value is not greater than a predetermined value is identified. Then, the width of a stripe is calculated from the number of rows situated adjacent to one another, the rows situated adjacent to one another being from among the identified rows of pixels. The fact that an integration value of a row in which a stripe has occurred is smaller than an integration value of a row in which a stripe has not occurred is applied to this calculation method. When there exist a plurality of sets of rows situated adjacent to one another, it is preferable that a set of rows that is constituted of a largest number of rows be identified and that the width of a stripe be calculated from the number of rows included in the set.

When the width of the stripe has been calculated, the light sheet microscope apparatus determines the subordinate ray angle (Step S230). Here, the controller70determines the subordinate ray angle on the basis of the width of the stripe that has been calculated in Step S220. Specifically, the subordinate ray angle may be geometrically calculated, for example, on the basis of the width of the stripe that has been calculated in Step S220and a preset acceptable length of the stripe. The acceptable length of a stripe is the length of the stripe with respect to the optical-axis direction of the illumination optical system10.

Finally, the light sheet microscope apparatus sets the angle determined in Step S230to be the subordinate ray angle (Step S240), and terminates the illumination setting processing. Step S240is similar to Step S170ofFIG. 8. After that, the light sheet microscope apparatus radiates a light sheet onto the sample S in the setting performed in Step S240and acquires an image of the sample S, an observer observes the sample S.

When the light sheet microscope apparatus performs the illumination setting processing ofFIG. 11, the width of a stripe is calculated from an image and a subordinate ray angle is determined on the basis of the width of the stripe. As in the first embodiment, this makes it possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance, because the subordinate ray angle is not set to be too large. Thus, it is possible to easily perform an appropriate illumination setting for a light sheet illumination.

Further, in the illumination setting processing illustrated inFIG. 11, only one image is sufficient. Thus, according to the present embodiment, it is possible to perform an illumination setting in a shorter time than according to the first embodiment in which a plurality of images are acquired and a comparison is performed repeatedly. In addition, it is also possible to suppress damage caused to a sample due to an illumination setting.

The width of a stripe corresponds to the width of a light-blocking portion, so it hardly varies near the light-blocking portion. However, when the subordinate ray has an angle with respect to the optical axis, the width of a stripe is smaller if the distance from the light-blocking portion is longer. Thus, when the width of a stripe is calculated using a value obtained by integrating pixel values in the x-axis direction, a reduction in integration value due to a factor other than the stripe and a reduction in integration value due to the stripe may be falsely recognized if the angle of the subordinate ray is large. In order to prevent this, it is preferable that the subordinate ray angle set in Step S200be smaller. In particular, it is preferable that the subordinate ray angle be set to zero degrees, at which the subordinate ray is parallel to the optical axis.

FIG. 12is a flowchart that illustrates another procedure of illumination setting processing.FIG. 13is a flowchart that illustrates another procedure of width calculation processing. The light sheet microscope apparatus according to the present embodiment may perform illumination setting processing ofFIGS. 12 and 13instead of the illumination setting processing ofFIG. 11.

First, the light sheet microscope apparatus performs an initial setting of a subordinate ray angle of a light sheet emitted from the illumination optical system10(Step S300), and acquires an image of the sample S in the initial setting (Step S310). Steps S300and Step S310are similar to Step S200and Step S210ofFIG. 11.

Next, the light sheet microscope apparatus changes the setting of the subordinate ray angle of the light sheet (Step S320), and acquires an image of the sample S in a setting after the change (Step S330). It is sufficient if the angle set in Step S320is an angle at which a stripe is less likely to occur behind a light-blocking portion, and it is preferable that it be set to be relatively large.

After that, the light sheet microscope apparatus calculates the width of a stripe (Step S340). Here, on the basis of two images acquired in Step S310and Step S330, the controller70calculates the width of a stripe that appears in each of the images. In this width calculation processing, the controller70compares two images (Step S341) and calculates the width of a stripe on the basis of a result of the comparison (Step S342). Specifically, a difference between values of the corresponding pixels in the two images may be taken, and the width of the stripe may be calculated from a distribution of pixels between which the difference is not less than a predetermined value. Further, pixel values in an image are integrated in the x-axis direction, and a row of pixels in which a difference in integration value between the two images is not less than a predetermined value is identified. Then, the width of the stripe may be calculated from the number of rows situated adjacent to one another, the rows situated adjacent to one another being from among the identified rows of pixels.

When the width of the stripe has been calculated, the light sheet microscope apparatus determines the subordinate ray angle (Step S350), sets the determined angle to be the subordinate ray angle (Step S360), and terminates the illumination setting processing. Step S350and Step S360are similar to Step S230and Step S240ofFIG. 11. After that, the light sheet microscope apparatus radiates a light sheet onto the sample S in the setting performed in Step S350and acquires an image of the sample S, an observer observes the sample S.

The illumination setting processing illustrated inFIG. 12also permits obtaining of an effect similar to the illumination setting processing illustrated inFIG. 11. In other words, it is possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance without making the subordinate ray angle too large.

Third Embodiment

A light sheet microscope apparatus according to a third embodiment is different from the light sheet microscope apparatus1in that it includes a controller80instead of the controller40. It is similar to the light sheet microscope apparatus1in regard to the other points.

FIG. 14illustrates a functional configuration of the controller80. The controller80includes an image acquisition unit80a, a stripe identification unit80b, a width calculation unit80c, an angle determination unit80d, and an output unit80e. The hardware configuration of the controller80is similar to that of the controller40. At least one of the units described above may be configured on the memory42by the processor41loading a program into the memory42and executing the loaded program, and it may be configured by hardware such as an integrated circuit such as an FPGA or an ASIC.

The image acquisition unit80a, the angle determination unit80d, and the output unit80eare similar to the image acquisition unit70a, the angle determination unit70c, and the output unit70daccording to the controller70according to the second embodiment. On the basis of the image of the sample S that has been acquired by the imaging device30, the stripe identification unit80bidentifies a stripe that appears in the image of the sample S. On the basis of the image of the sample S that has been acquired by the imaging device30, in particular, on the basis of stripe information output from the stripe identification unit80b, the width calculation unit80ccalculates the width of the stripe that appears in the image of the sample S.

FIG. 15is a flowchart that illustrates a procedure of illumination setting processing.FIG. 16is a flowchart that illustrates a procedure of stripe identification processing. Referring toFIGS. 15 and 16, the illumination setting processing performed in the light sheet microscope apparatus according to the present embodiment is described below, focusing on the difference from the illumination setting processing illustrated inFIG. 11.

First, the light sheet microscope apparatus performs an initial setting of a subordinate ray angle of a light sheet emitted from the illumination optical system10(Step S400). The angle set here is an angle at which a stripe occurs behind a light-blocking portion, which is similar to Step S200of FIG.11. However, it is set to an angle other than zero degrees. After that, the light sheet microscope apparatus acquires an image of the sample S in the initial setting (Step S410). Step S410is similar to Step S210ofFIG. 11.

When the image has been acquired, the light sheet microscope apparatus identifies a stripe (Step S420). Here, on the basis of the image acquired in Step S410, the controller80identifies a stripe that appears in the image. In this stripe identification processing, as illustrated inFIG. 16, first, on the basis of the image, the controller80identifies an area, in the image, in which a pixel value (that is, an intensity of image signal) is not greater than a predetermined value (Step S421). Further, a stripe is identified on the basis of the area identified in Step S421(Step S422). In Step S422, for example, an area having a tapered shape from among the identified area may be identified as a stripe, the tapered shape having a width that becomes narrower in a direction in which the light sheet travels.

When the stripe has been identified, the light sheet microscope apparatus calculates the width of the stripe (Step S430). Here, the controller80calculates the width of the stripe by measuring, on the image, the width of the stripe identified in Step S420.

When the width of the stripe has been calculated, the light sheet microscope apparatus determines the subordinate ray angle (Step S440), sets the determined angle to be the subordinate ray angle (Step S450), and terminates the illumination setting processing. Step S440and Step S450are similar to Step S230and Step S240ofFIG. 11. After that, an observer radiates a light sheet onto the sample S in the setting performed in Step S450and acquires an image of the sample S, so as to observe the sample S.

When the light sheet microscope apparatus performs the illumination setting processing ofFIG. 15, the width of a stripe is calculated from an image and a subordinate ray angle is determined on the basis of the width of the stripe. As in the first embodiment, this makes it possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance without making the subordinate ray angle too large. Thus, it is possible to easily perform an appropriate illumination setting for a light sheet illumination. Further, only one image is sufficient, so it is possible to perform an illumination setting in a shorter time and to suppress damage caused to a sample due to an illumination setting, as in the second embodiment.

The stripe identification processing illustrated inFIG. 16has been described as an example of a method for identifying a stripe, but the stripe identification processing illustrated inFIG. 17may be performed. In other words, the controller80may perform pattern matching processing on an image on the basis of a preset stripe pattern (Step S423), so as to identify a stripe on the basis of a pattern matching result (Step S424).

Fourth Embodiment

A light sheet microscope apparatus according to a fourth embodiment is different from the light sheet microscope apparatus1in that it includes a controller90instead of the controller40. It is similar to the light sheet microscope apparatus1in regard to the other points.

FIG. 18illustrates a functional configuration of the controller90. The controller90includes an image acquisition unit90a, a stripe identification unit90b, an image comparison unit90c, an angle determination unit90d, and an output unit90e. The hardware configuration of the controller90is similar to that of the controller40. At least one of the units described above may be configured on the memory42by the processor41loading a program into the memory42and executing the loaded program, and it may be configured by hardware such as an integrated circuit such as an FPGA or an ASIC.

The image acquisition unit90aand the output unit90eare similar to the image acquisition unit40aand the output unit40dof the controller40according to the first embodiment. On the basis of the image of the sample S that has been acquired by the imaging device30, the stripe identification unit90bidentifies a stripe that appears in the image of the sample S. The image comparison unit90ccompares a plurality of images of the sample that have been acquired by the imaging device30, in particular, small regions, in the plurality of images, that each include an identified stripe. The small region is not the entirety of an image, but a region that is a portion of the image. The angle determination unit90ddetermines a subordinate ray angle with respect to the width direction of a light sheet on the basis of a result of the comparison performed by the image comparison unit90c, in particular, on the basis of a result of comparing the above-described small regions.

FIG. 19is a flowchart that illustrates a procedure of illumination setting processing. Referring toFIG. 19, the illumination setting processing performed in the light sheet microscope apparatus according to the present embodiment is described below, focusing on the difference from the illumination setting processing illustrated inFIG. 8.

First, the light sheet microscope apparatus performs an initial setting of a subordinate ray angle of a light sheet emitted from the illumination optical system10(Step S500), and acquires an image of the sample S in the initial setting (Step S510). Step S500and Step S510are similar to Step S100and Step S110ofFIG. 8.

When the image has been acquired, the light sheet microscope apparatus identifies a stripe (Step S520). Step S520is similar to Step S420ofFIG. 15. After that, the light sheet microscope apparatus changes the setting of the subordinate ray angle of the light sheet (Step S530), and acquires an image of the sample S in a setting after the change (Step S540). Step S530and Step S540are similar to Step S120and Step S130ofFIG. 8.

Next, the light sheet microscope apparatus compares a plurality of images of the sample S (Step S550). Here, the controller90compares small regions, in a plurality of images, that each include the stripe identified in Step S520, and evaluates a change in a small region in an image, the plurality of images being images of the sample S onto which light sheets with different subordinate ray angles have been radiated.

After that, the light sheet microscope apparatus determines whether the change in image is small (Step S560). Here, on the basis of a result of comparing the small regions in Step S550, the controller90determines whether a value representative of a change in a small region is smaller than a predetermined value.

When the change in image (the change in small region between images) has been determined to not be small, the light sheet microscope apparatus performs the processes of Step S530to Step S560again. The light sheet microscope apparatus repeats the processes until the change in image is determined to be small in Step S560.

When the change in image (the change in small region between images) has been determined to be small, the light sheet microscope apparatus determines the subordinate ray angle (Step S570), sets the determined angle to be the subordinate ray angle (Step S580), and terminates the illumination setting processing. Step S570and Step S580are similar to Step S160and Step S170ofFIG. 8. After that, the light sheet microscope apparatus radiates a light sheet onto the sample S in the setting performed in Step S580and acquires an image of the sample S, an observer observes the sample S.

When the light sheet microscope apparatus performs the illumination setting processing ofFIG. 19, it is possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance without making the subordinate ray angle too large, as in the first embodiment. Thus, it is possible to easily perform an appropriate illumination setting for a light sheet illumination. Further, in the illumination setting processing illustrated inFIG. 19, small regions that each include a stripe are compared, so it is possible to detect a change in image due to a change in the stripe with a high sensitivity. Further, it is also possible to suppress an amount of calculation compared to when a comparison is performed on the entirety of an image.

Fifth Embodiment

A light sheet microscope apparatus according to a fifth embodiment is different from the light sheet microscope apparatus1in that it includes a controller100instead of the controller40. It is similar to the light sheet microscope apparatus1in regard to the other points.

FIG. 20illustrates a functional configuration of the controller100. The controller100includes an image acquisition unit100a, a stripe identification unit100b, a display control unit100c, a stripe designation unit100d, a width calculation unit100e, an angle determination unit100f, and an output unit100g. The hardware configuration of the controller100is similar to that of the controller40. At least one of the units described above may be configured on the memory42by the processor41loading a program into the memory42and executing the loaded program, and it may be configured by hardware such as an integrated circuit such as an FPGA or an ASIC.

The image acquisition unit100aand the output unit100gare similar to the image acquisition unit40aand the output unit40dof the controller40. On the basis of the image of the sample S that has been acquired by the imaging device30, the stripe identification unit100bidentifies a stripe that appears in the image of the sample S. The display control unit100cdisplays, on the display device51, an image of the sample in which a portion that is the stripe identified by the stripe identification unit100bhas been marked. The stripe designation unit100ddesignates a stripe to be eliminated according to an input from an observer. The width calculation unit100ecalculates the width of the stripe designated by the stripe designation unit100d. The angle determination unit100fis similar to the angle determination unit80dof the controller80.

FIG. 21is a flowchart that illustrates a procedure of illumination setting processing. Referring toFIG. 21, the illumination setting processing performed in the light sheet microscope apparatus according to the present embodiment is described below, focusing on the difference from the illumination setting processing illustrated inFIG. 15.

First, the light sheet microscope apparatus performs an initial setting of a subordinate ray angle of a light sheet emitted from the illumination optical system10(Step S600), and acquires an image of the sample S in the initial setting (Step S610). Further, the light sheet microscope apparatus identifies a stripe on the basis of the acquired image (Step S620). Step S600to Step S620are similar to Step S400to Step S420ofFIG. 15.

When the stripe has been identified, the light sheet microscope apparatus displays an image in which a portion that is the stripe identified in Step S620has been marked (Step S630). Here, the controller100displays, on the display device51, an image of the sample in which a portion that is the identified stripe has been marked. In other words, the light sheet microscope apparatus displays, on the display device51, a position of the identified stripe together with the image of the sample. For example, as illustrated inFIG. 22, the controller100updates the image that is being displayed on the display device51from an image51aof the sample in which stripe portions have not been marked to an image51bof the sample in which the stripe portion have been marked (a mark M1, a mark M2, and a mark M3).

After that, while viewing the image that is being displayed on the display device51, the observer selects, on a screen, a stripe to be eliminated using the input devices (the keyboard52and the mouse53). The observer may select all of the stripes to be eliminated or may only select a largest stripe among the stripes to be eliminated.

During the image being displayed, the light sheet microscope apparatus determines whether a stripe has been designated by the observer (Step S640). Here, the controller100determines whether a stripe has been designated on the basis of a signal from the input devices (the keyboard52and the mouse53).

When a stripe has been determined to be designated, the light sheet microscope apparatus calculates the width of the stripe (Step S650). Here, the controller100measures, on the screen, the width of the stripe designated in Step S640so as to calculate the width of the stripe. When a plurality of stripes have been selected, the width of each of the stripes is calculated.

When the width of the stripe has been calculated, the light sheet microscope apparatus determines the subordinate ray angle (Step S660). Here, the controller100determines the subordinate ray angle on the basis of the width of the stripe that has been calculated in Step S650. When the widths of the plurality of stripes have been calculated, it is preferable that the subordinate ray angle be determined on the basis of the width of a largest stripe.

Finally, the light sheet microscope apparatus sets the determined angle to be the subordinate ray angle (Step S670), and terminates the illumination setting processing. Step S670is similar to Step S450ofFIG. 15. After that, the light sheet microscope apparatus radiates a light sheet onto the sample S in the setting performed in Step S670and acquires an image of the sample S, the observer observes the sample S.

When the light sheet microscope apparatus performs the illumination setting processing ofFIG. 21, the width of a stripe is calculated from an image and a subordinate ray angle is determined on the basis of the width of the stripe. As in the first embodiment, this makes it possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance without making the subordinate ray angle too large. Thus, it is possible to easily perform an appropriate illumination setting for a light sheet illumination. Further, only one image is sufficient, so it is possible to perform an illumination setting in a shorter time and to suppress damage caused to a sample due to an illumination setting, as in the second embodiment. Furthermore, in the present embodiment, a subordinate ray angle is determined such that a stripe eliminating effect is obtained for at least a stripe selected by an observer, and this results in being able to further suppress a reduction in illumination performance while providing a stripe eliminating effect that satisfies the requirements of the observer.

Sixth Embodiment

FIGS. 23A and 23Billustrate a configuration of a light sheet microscope apparatus2. Like the light sheet microscope apparatus1, the light sheet microscope apparatus2is, for example, a fluorescence microscope that detects a fluorescence from the sample S such as a biological sample, and is configured to illuminate the sample S with a light sheet.

The light sheet microscope apparatus2is different from the light sheet microscope apparatus1in that it includes an illumination optical system200instead of the illumination optical system10, a controller110instead of the controller40, and a setting device65instead of the setting device60.

The illumination optical system200is configured to form a light sheet having a sheet shape substantially perpendicular to the optical axis of the detection optical system20and to radiate the light sheet onto the sample S from a direction substantially perpendicular to the optical axis of the detection optical system20. Compared with the illumination optical system10, the illumination optical system200forms a wider light sheet so that it is possible to illuminate the observation range R at one time.

The illumination optical system200includes a laser201. The laser201is a light source that emits a laser beam (illumination light) that will be converted into a light sheet. The illumination optical system200further includes, in order from the side of the laser201, a lens202, a lens203, a cylindrical lens204, a mirror205, a cylindrical lens206, and a cylindrical lens207.

The cylindrical lens204and the cylindrical lens206are arranged to have a refractive power in the xy plane and to not have a refractive power in the xz plane. The cylindrical lens207is arranged to have a refractive power in the xz plane and to not have a refractive power in the xy plane.

The mirror205is a rotation mirror that can change the angle with respect to incident light by rotating about the z axis, and the angle of the mirror205is changed according to the setting device65. It is preferable that the mirror205be arranged within a pupil plane of the illumination optical system200.

The controller110is a microscope controller that controls the light sheet microscope apparatus2. The controller110is configured to output a control signal to various electrical mechanisms provided in a microscope body of the light sheet microscope apparatus2, and has, for example, a hardware configuration similar to the controller40.

The setting device65is a device that performs a setting of the illumination optical system200, and is one of the electrical mechanisms, in the microscope body, which operate according to the control signal from the controller110. Specifically, the setting device65is a driving device, such as a motor, that changes the angle of the mirror205. The angle of a principal ray of a light sheet emitted from the illumination optical system200is changed by the setting device65changing the angle of the mirror205.

In the light sheet microscope apparatus2having the configuration described above, the cylindrical lens204and the cylindrical lens206do not substantially act on a laser beam in the xz plane because they do not have a refractive power in the xz plane. Further, the mirror205that rotates about the z axis also does not substantially act on a laser beam in the xz plane. Thus, as illustrated inFIG. 23B, a laser beam is collected into a certain position by the cylindrical lens207independent of the angle of the mirror205, as viewed from the width direction (y-axis direction).

Further, the cylindrical lens204and the cylindrical lens206have a refractive power in the xy plane. Thus, as illustrated inFIG. 23A, the width of a laser beam is adjusted with a combination of the lens202and the lens203and is further adjusted with a combination of the cylindrical lens204and the cylindrical lens206, as viewed from the thickness direction (z-axis direction). The cylindrical lens207does not have a refractive power in the xy plane, so a laser beam emitted from the cylindrical lens206is radiated onto a sample with an unchanged width. However, the direction of the principal ray of the laser beam depends on the angle of the mirror205.

Thus, according to the light sheet microscope apparatus2, it is possible to change the direction of a principal ray of a light sheet emitted from the illumination optical system200according to the angle of the mirror205. Therefore, the change in the angle of the mirror205makes it possible to change an incident angle at which a principal ray of the light sheet enters a sample. Then, the incident angle is changed during the exposure time period of the imaging device30so as to illuminate the sample from various directions, which permits obtaining of a stripe eliminating effect.

FIG. 24is a flowchart that illustrates a procedure of illumination setting processing.FIG. 25is a flowchart that illustrates a procedure of incident angle determination processing. Referring toFIGS. 24 and 25, the illumination setting processing performed in the light sheet microscope apparatus2is specifically described below.

First, the light sheet microscope apparatus2acquires an image of the sample S onto which a light sheet emitted from the illumination optical system200has been radiated (Step S700). Here, the illumination optical system200radiates a light sheet onto the sample S, and the imaging device30captures an image of the sample S and generates image data of the sample S. The generated image data of the sample S is output to the controller110.

Next, on the basis of the acquired image, the light sheet microscope apparatus2determines an incident angle at which a principal ray of the light sheet enters the sample (Step S710). Here, the controller110performs the incident angle setting processing illustrated inFIG. 25, and determines, on the basis of the image acquired from the imaging device30, an incident angle at which a principal ray of the light sheet emitted from the illumination optical system200enters the sample. Step S711to Step S714are similar to Step S620to Step650ofFIG. 21. The controller110determines the incident angle on the basis of the calculated width of the stripe (Step S715). The incident angle may be geometrically determined, for example, on the basis of the width of the stripe that has been calculated in Step S714and a preset acceptable length of the stripe.

When the incident angle has been determined, the light sheet microscope apparatus2performs a setting of the illumination optical system200according to the determined incident angle (Step S720), and radiates a light sheet onto the sample (Step S730). Here, in the light sheet microscope apparatus2, the setting device65radiates a light sheet onto the sample while repeatedly performing a setting of the illumination optical system200according to a control signal output from the controller110. Specifically, the light sheet microscope apparatus2radiates a light sheet while changing the incident angle from zero degrees up to the angle determined in Step S710.

Also when the light sheet microscope apparatus2performs the illumination setting processing ofFIG. 24, it is possible to perform a setting that permits obtaining of a sufficient stripe eliminating effect while suppressing a reduction in illumination performance without making the subordinate ray angle too large. Thus, it is possible to easily perform an appropriate illumination setting for a light sheet illumination.

In the illumination setting processing illustrated inFIG. 24, the example in which the setting of the illumination optical system200is performed according to the determined incident angle has been described, but it is sufficient if the incident angle is controlled. Thus, instead of performing the setting of the illumination optical system200, a setting of a direction of a sample may be performed by rotating, for example, a stage on which the sample is placed. Further, it is sufficient if at least one of these settings is performed, so both the setting of the illumination optical system200and the setting of a direction of a sample may be performed.

The embodiments described above are just examples to facilitate understanding of the present invention, and the embodiment of the present invention is not limited to these examples. Various modifications and alterations may be made to an illumination setting method, a light sheet microscope apparatus, and a recording medium without departing from the scope of the invention specified in the claims.