Fluorescence observation device

Abstract

This fluorescence observation device comprises: an excitation light source that irradiates an observation target with excitation light; an imaging unit that captures an observation image using fluorescence emitted by the observation target excited by the excitation light; a light guide optical system that guides the fluorescence to the imaging unit; a fluorescence filter that is disposed on the light incident side of the light guide optical system and transmits light in a wavelength range including the wavelength of the fluorescence; and a light shielding member disposed on the light incident side of the fluorescence filter. The light shielding member shields light incident on the fluorescence filter from a direction intersecting the normal of the fluorescence filter.

Description

Fluorescence observation device

[0001] This application claims priority to Japanese Patent Application No. 2024-051018, filed March 27, 2024, the contents of which are incorporated herein by reference.

[0002] A technique known as "fluorescence imaging" is known in the art, in which an object to be observed, such as a living cell, emits fluorescence and the resulting fluorescence is detected. Fluorescence imaging is utilized, for example, in the diagnosis of various infectious diseases and the observation of cellular activity. This technique employs a fluorescence imaging device that magnifies and observes minute objects with high resolution (see, for example, Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2023-003158

[0004] In the above technical fields, it is necessary to create an image using weak fluorescence emitted by an object to be observed. Therefore, a fluorescence imaging device is required to suppress stray light, which hinders the formation of a clear observation image. The device described in Patent Document 1 has room for improvement in terms of suppressing stray light.

[0005] The present invention has been made in view of the above circumstances, and has as its object to provide a fluorescence observation device capable of capturing a clear image using fluorescence emitted from an object to be observed.

[0006] In an imaging device, if light other than light emitted from the object being observed reaches the imaging device, it adversely affects the image obtained. In the following description, "light other than light emitted from the object being observed" is referred to as "stray light." When stray light reaches the imaging device, an image of the stray light is formed in the image obtained. As described above, in fluorescence imaging, images are created using weak fluorescence, so the adverse effects of stray light are likely to be significant.

[0007] In Patent Document 1, the numerical aperture NA of the lens through which the fluorescence emitted from the measurement object is incident is taken into consideration, and by irradiating excitation light (illumination light) from outside the NA at a larger angle, it is possible to achieve good imaging without the illumination light affecting the captured image. However, after investigations by the inventors, it was found that simply adjusting the illumination light irradiation angle is insufficient as a countermeasure, and that the illumination light may adversely affect the captured image. Such adverse effects are more likely to become apparent when the device is miniaturized and the physical distance between the object and the lens is reduced.

[0008] As a result of intensive research into the above-mentioned problems, the inventors discovered that the problems could be solved effectively by taking into account the characteristics of the fluorescence filter (bandpass filter), which is essential for fluorescence imaging, and adding a configuration, thereby completing the invention.

[0009] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0010] [1] A fluorescence observation device comprising: an excitation light source that irradiates an observation object with excitation light; an imaging unit that captures an observation image using fluorescence emitted by the observation object excited by the excitation light; a light-guiding optical system that guides the fluorescence to the imaging unit; a fluorescence filter that is arranged on a light incident side of the light-guiding optical system and that transmits light in a wavelength range that includes the wavelength of the fluorescence; and a light-shielding member that is arranged on the light incident side of the fluorescence filter, wherein the light-shielding member blocks light that is incident on the fluorescence filter in a direction that intersects with a normal to the fluorescence filter.

[0011] [2] The fluorescence observation device according to [1], wherein the light-guiding optical system has a telecentric lens system and a lens barrel that holds the telecentric lens system, the fluorescence filter is disposed on the incident side of the lens barrel, and the light-shielding member is provided on the incident side and extends in the axial direction of the lens barrel.

[0012] [3] The fluorescence observation device according to [2], wherein the light-shielding member is a cylindrical member.

[0013] [4] The fluorescence observation device according to [3], wherein the light-shielding member is a truncated cone-shaped member whose diameter gradually decreases in a direction away from the lens barrel.

[0014] [5] The fluorescence observation device according to any one of [2] to [4], further comprising: a mounting table on which the observation object is placed; and a reflecting mirror, disposed between the mounting table and the lens barrel, that guides the fluorescence to the telecentric lens system.

[0015] [6] The fluorescence observation device according to [5], wherein, in a plan view, the central axis of the lens barrel intersects with an imaginary line connecting the excitation light source and the stage.

[0016] According to the present invention, it is possible to provide a fluorescence observation device that can capture a clear image using fluorescence emitted from an observation object.

[0017] FIG. 1 is a schematic perspective view showing a fluorescence observation device 1 of this embodiment. FIG. 2 is an explanatory diagram illustrating a fluorescence observation device 1X that does not have the above-mentioned light-shielding member 40. FIG. 3 is an explanatory diagram illustrating the relationship between the transmission wavelength range of the band-pass filter 12 and the transmission wavelength range of the fluorescence filter 50. FIG. 4 is an explanatory diagram illustrating the relationship between the transmission wavelength range of the band-pass filter 12 and the transmission wavelength range of the fluorescence filter 50. FIG. 5 is an explanatory diagram illustrating a fluorescence observation device 1 that has a light-shielding member 40. FIG. 6 is an explanatory diagram showing the positional relationship between the light-guiding optical system 60 and the excitation light source 10.

[0018] The fluorescence observation device according to this embodiment will be described below with reference to Figures 1 to 6. Note that in all of the following figures, the dimensions and proportions of the components have been changed as appropriate to make the drawings easier to understand.

[0019] In the following explanation, an xyz Cartesian coordinate system is set, and the positional relationships of the components are explained with reference to this xyz Cartesian coordinate system. Here, a predetermined direction in a horizontal plane is defined as the x-axis direction, a direction perpendicular to the x-axis direction in the horizontal plane is defined as the y-axis direction, and a direction perpendicular to both the x-axis and y-axis directions (i.e., the vertical direction) is defined as the z-axis direction.

[0020] Furthermore, "up" refers to the +z direction, which is vertically upward, and "down" refers to the -z direction, which is vertically downward.

[0021] In the following description, "planar view" refers to viewing an object from vertically above (+z side) downward (-z direction).

[0022] Fig. 1 is a schematic perspective view showing a fluorescence observation device 1 of this embodiment. As shown in Fig. 1, the fluorescence observation device 1 includes an excitation light source 10, a light-shielding member 40, a fluorescence filter (bandpass filter) 50, a light-guiding optical system 60, an imaging unit 70, and a control unit 80. The fluorescence observation device 1 further includes a mounting unit 20 and a reflecting mirror 30. In addition, the fluorescence observation device 1 includes a housing (not shown) that houses these components.

[0023] The fluorescence observation device 1 can excite an observation object placed on a sample plate P by irradiating the object with excitation light EL, causing the object to emit fluorescence, and can form and capture an observation image using the emitted fluorescence F. The formed observation image can be used for various types of analysis using image analysis.

[0024] [Excitation Light Source] The excitation light source 10 is a light source that irradiates an object to be observed with excitation light EL. The excitation light source 10 includes a light source 11 and a bandpass filter 12 that transmits light in a specific wavelength range to be used as the excitation light EL from the light source 11 and blocks light in other wavelength ranges.

[0025] There are no particular limitations on the light source 11 as long as it can emit light L that includes the wavelength range of the excitation light EL. As the light source 11, an LED light source that emits light L can be used.

[0026] An optical filter having a known configuration that includes a dielectric multilayer film and is capable of wavelength separation can be used as the bandpass filter 12. The bandpass filter 12 is preferably replaceable.

[0027] 1 has an excitation light source 10A that emits excitation light EL upward from the +y direction, and an excitation light source 10B that emits excitation light EL upward from the −y direction. It is preferable that the excitation light sources 10A and 10B are designed to emit excitation light EL with different wavelengths, since this widens the wavelength range of the emitted excitation light EL. The fluorescence observation device 1 may also have only one excitation light source 10.

[0028] [Placement Section] The placement section 20 has a placement table 21 for placing a sample plate P on which an observation target object is arranged, and a position adjustment section 22 for adjusting the position of the placement table 21.

[0029] The mounting table 21 has a frame 211 formed in a ring shape in a plan view, and a window portion 212 arranged inside the frame 211 in a plan view. The mounting table 21 supports the sample plate P by placing the sample plate P on an upper surface 21 a thereof.

[0030] The window 212 is a transparent member that is transparent to the excitation light EL and the fluorescence F. The excitation light EL emitted from the excitation light source 10 is irradiated onto the sample plate P through the window 212, and the fluorescence F emitted from the observation object on the sample plate P is emitted downward (in the −z direction) through the window 212.

[0031] There are no limitations on the material of the window portion 212 as long as it can transmit the excitation light EL and the fluorescence F. Examples of materials that can be used for the window portion 212 include optically transparent resin materials such as PMMA, and inorganic materials such as glass and quartz.

[0032] The mounting table 21 may be configured with only the frame 211, with no object in the window 212. In such a mounting table 21, the frame 211 supports the sample plate P. The excitation light EL is incident through the window 212 that opens in the z direction, and the fluorescence F is similarly emitted through the window 212. When the mounting table 21 is configured with only the frame 211, the excitation light is not reflected on the surface of the window, thereby reducing the total amount of stray light.

[0033] The position adjustment unit 22 supports the mounting table 21 and can adjust the spatial position of the mounting table 21. The position adjustment unit 22 has a known XY stage 221 that can move the mounting table 21 in the x and y directions, and an elevator unit 222 that can move the XY stage 221 in the z direction. A known configuration can be adopted as the position adjustment unit 22.

[0034] [Reflecting Mirror] The reflecting mirror 30 is provided on the optical path of the fluorescence F between the mounting table 21 and the light-guiding optical system 60, and guides the fluorescence F to the light-guiding optical system 60. The observation object excited by the excitation light EL isotropically emits fluorescence F. The reflecting mirror 30 reflects a portion of the fluorescence F thus isotropically emitted toward the light-guiding optical system 60.

[0035] The reflecting mirror 30 is arranged, for example, at a position overlapping with the mounting table 21 in a planar view, at an inclination angle of 45° with respect to the normal (z-axis) to the surface of the mounting table 21, and is configured to reflect the fluorescence F emitted in the -z direction from the object to be observed on the mounting table 21 by 90° and guide it in the -x direction.

[0036] The inclination angle of the reflecting mirror 30 does not have to be 45°. When the fluorescence observation device 1 has the reflecting mirror 30, the degree of freedom in the relative positions of the mounting table 21 and the light-guiding optical system 60 increases.

[0037] [Light Shielding Member] The light shielding member 40 is provided on the optical path of the fluorescence F between the fluorescence filter 50 and the mounting table 21, and shields stray light. The configuration and function of the light shielding member 40 will be described in detail later.

[0038] [Fluorescence Filter] The fluorescence filter 50 is an optical filter arranged on the light incident side of the light-guiding optical system 60 (on the side of the light-blocking member 40 on the optical path of the fluorescence F). The fluorescence filter 50 is designed to transmit light in a wavelength range that includes the wavelength of the fluorescence F and to block light in other wavelength ranges. This allows the fluorescence F used for observation to be guided to the light-guiding optical system 60, enabling a clear image to be captured by the imaging unit 70, which will be described later.

[0039] The fluorescence filter 50 can be an optical filter (bandpass filter) having a known configuration that includes a dielectric multilayer film and is capable of wavelength separation.

[0040] [Light-guiding optical system] The light-guiding optical system 60 guides the light (fluorescence F) that has passed through the fluorescence filter 50 to the imaging unit 70. The light-guiding optical system 60 is a telecentric optical system that includes a telecentric lens 61 and a lens barrel 62 that extends in the x direction and holds the telecentric lens 61. Because the light-guiding optical system 60 is a telecentric optical system, it is possible to suppress the occurrence of aberrations in the peripheral parts of the field of view of the light-guiding optical system 60, and to obtain a clear image. The light-guiding optical system 60 can be either a double-telecentric optical system or a single-telecentric optical system (on the object side) as a telecentric optical system.

[0041] In the drawing, for the sake of simplicity, the telecentric lens 61 is depicted as a pair of lenses, but the telecentric lens 61 may be composed of a plurality of optical elements.

[0042] [Image Capturing Unit] The image capturing unit 70 receives the fluorescence F guided through the light guiding optical system 60 and captures an observation image. The image capturing unit 70 can be a digital camera having a CMOS sensor or a CCD sensor as an image capturing element. Either a consumer or industrial image capturing unit can be used as the image capturing unit 70.

[0043] The control unit 80 is connected to each part of the fluorescence observation device 1, such as the mounting unit 20 and the image capturing unit 70, and controls the operation of each part. For example, the control unit 80 controls the spatial position of the sample plate P by the mounting unit 20, and captures and saves the observation image by the image capturing unit 70.

[0044] The control unit 80 can also be connected to other analytical devices and control the operation of the other analytical devices. For example, the control unit 80 may also control the operation of an external device that adjusts the sample plate P and transports the adjusted sample plate P to the mounting table 21, and synchronize the operation control of such an external device with the fluorescence observation device 1. With this configuration, a system can be constructed that can automatically perform everything from adjusting the sample plate P to fluorescent observation of an observation target placed on the sample plate P.

[0045] The above-described fluorescence observation device 1 has the light-shielding member 40, which makes it possible to capture clear images using the fluorescence F emitted by the observation object. Below, using Figures 2 to 4, we will explain the issues that arise with a fluorescence observation device that does not have the light-shielding member 40, and then using Figure 5, we will explain the functions and effects of the fluorescence observation device 1.

[0046] FIG. 2 is an explanatory diagram for explaining a fluorescence observation device 1X that does not have the above-described light-shielding member 40, and is a schematic cross-sectional view on a virtual plane that includes the optical axis OA of the light-guiding optical system 60 and is parallel to the xz plane.

[0047] As described above, the excitation light EL emitted from the excitation light source 10 toward the mounting table 21 (the sample plate P on the mounting table 21) excites the observation object placed on the sample plate P, generating fluorescence F. Of the fluorescence F isotropically emitted from the observation object, the fluorescence F emitted toward the reflecting mirror 30 is reflected by the reflecting mirror 30 and then guided to the light-guiding optical system 60 via the fluorescence filter 50, and is used to form an observation image in the imaging unit 70.

[0048] At this time, part of the excitation light EL may be reflected by the lower surface of the mounting table 21 or the lower surface of the sample plate P, becoming stray light SL. Furthermore, because the excitation light EL is emitted from the excitation light source 10 at a constant light distribution angle according to the device characteristics of the excitation light source 10, it is thought that part of the generated stray light SL will be incident on the fluorescence filter 50 from a direction that intersects with the normal NL of the fluorescence filter 50 at an angle θ.

[0049] In this case, the following problem occurs with the fluorescence filter 50. Figures 3 and 4 are explanatory diagrams illustrating the relationship between the transmission wavelength range of the bandpass filter 12 and that of the fluorescence filter 50. The horizontal axis of Figures 3 and 4 represents wavelength WL (unit: nm), and the vertical axis represents transmittance T (unit: %). The transmission wavelength range of the bandpass filter 12 is equal to the wavelength range of the excitation light EL.

[0050] 3 is a graph showing the relationship between the transmission wavelength range of the bandpass filter 12 and the transmission wavelength range of the fluorescence filter 50 for light incident from the normal direction of the fluorescence filter 50. As shown in FIG. 3, the bandpass filter 12 has a transmission wavelength range W1 that transmits light of wavelengths WL1 to WL2, and the fluorescence filter 50 has a transmission wavelength range W2 that transmits light of wavelengths WL3 to WL4.

[0051] 3, if wavelength WL3 is greater than wavelength WL2, transmission wavelength range W1 and transmission wavelength range W2 do not overlap with each other. Therefore, even if the excitation light EL emitted through band-pass filter 12 becomes stray light, it is blocked by fluorescence filter 50 when it is incident on fluorescence filter 50 from the normal direction.

[0052] 4 is a graph showing the relationship between the transmission wavelength range of the bandpass filter 12 and the transmission wavelength range of the fluorescence filter 50 for light incident obliquely with respect to the normal to the fluorescence filter 50. The transmission wavelength range of the fluorescence filter 50, which has a dielectric multilayer film, exhibits a low wavelength shift (indicated by the symbol α in the figure) for light incident obliquely with respect to the normal. In FIG. 4, the fluorescence filter 50 is shown as having a transmission wavelength range W3 that transmits light of wavelengths WL5 to WL6 for light incident at an angle θ with respect to the normal.

[0053] At this time, when the wavelength WL5 becomes smaller than the wavelength WL2 due to the downshift in the transmission wavelength range, the transmission wavelength range W3 of the fluorescence filter 50 partially overlaps with the wavelength range of the excitation light EL (indicated by the symbol A in FIG. 4 ). Therefore, if the excitation light EL emitted through the bandpass filter 12 becomes stray light, and if it is incident obliquely (at an angle θ) on the fluorescence filter 50, it is not blocked by the fluorescence filter 50, and the stray light indicated by the symbol A passes through the fluorescence filter 50. The stray light that passes through the fluorescence filter 50 in this manner is often more intense than the weak fluorescence emitted by the object being observed, and therefore adversely affects the observation image captured by the imaging unit 70.

[0054] On the other hand, the fluorescence observation device 1 solves the above problem by using the light-shielding member 40. Fig. 5 is an explanatory diagram for explaining the fluorescence observation device 1 having the light-shielding member 40, and is a schematic cross-sectional view in the same field of view as Fig. 2 .

[0055] As shown in FIG. 5, in the fluorescence observation device 1, the light blocking member 40 is provided at a position where it blocks stray light SL that is incident on the fluorescence filter 50 from a direction intersecting the normal line NL of the fluorescence filter 50.

[0056] In detail, the fluorescence filter 50 is provided on the incident side (incident side end 62a) of the lens barrel 62 that constitutes the light-guiding optical system 60, on which the fluorescence F enters, and the light-blocking member 40 is provided on the end 62a of the lens barrel 62 and extends in the axial direction of the lens barrel 62. Note that the fluorescence filter 50 and the light-blocking member 40 may be spaced apart from the end 62a of the lens barrel 62 as long as the effects of the invention are not impaired.

[0057] 5 is a cylindrical member, and is a truncated cone-shaped member whose diameter gradually decreases in the direction away from the lens barrel 62. The light blocking member 40 functions as a diaphragm that transmits the fluorescence F reflected by the reflecting mirror 30 to the inner surface 40a and blocks stray light SL with the outer surface 40b.

[0058] The light blocking member 40 can have various shapes as long as it can block stray light SL. For example, while the light blocking member 40 is shown in Fig. 5 as having a truncated cone shape, it is not limited to this and may also have a cylindrical shape. Furthermore, when the light blocking member 40 has a cylindrical shape, it may have a square cylindrical shape.

[0059] Since the light-shielding member 40 is cylindrical and is provided around the entire circumference of the optical axis OA of the light-guiding optical system 60, the generated stray light SL is reflected or diffracted inside the device, thereby effectively blocking the light even if it enters the fluorescent filter 50 from various directions.

[0060] Furthermore, when the incident direction of the stray light SL onto the fluorescent filter 50 is known, it is possible to provide a canopy-shaped light-shielding member only on the incident side of the stray light SL onto the fluorescent filter 50, without providing the light-shielding member 40 around the entire circumference of the optical axis OA of the light-guiding optical system 60.

[0061] Furthermore, the effects of the invention are achieved as long as the light-shielding member 40 is provided in a position that blocks stray light SL that is incident on the fluorescence filter 50 from a direction that intersects with the normal line NL of the fluorescence filter 50. In this sense, the light-shielding member 40 does not have to be provided at the end 62 a of the lens barrel 62.

[0062] The surface of the light-shielding member 40 may be subjected to an anti-reflection treatment. This makes it difficult for stray light SL that reaches the light-shielding member 40 to be reflected by the surface of the light-shielding member 40, thereby effectively reducing stray light. As the anti-reflection treatment, a known anti-reflection film may be formed on the surface of the light-shielding member 40, or an anti-reflection paint may be applied.

[0063] The size and shape of the light-shielding member 40 may be appropriately designed taking into account various conditions, such as the size of the fluorescence observation device 1, the numerical aperture (NA) of the lens in the light-guiding optical system 60, the incident angle of the excitation light EL with respect to the mounting table 21, and the expected incident direction of the stray light SL with respect to the fluorescence filter 50. In this case, the incident direction of the stray light SL with respect to the fluorescence filter 50 may be confirmed by a preliminary experiment using a specific device, or may be calculated by optical simulation.

[0064] Furthermore, in the fluorescence observation device 1, the generation of stray light is suppressed by the arrangement of the light-guiding optical system 60 and the excitation light source 10. FIG. 6 is an explanatory diagram showing the positional relationship between the light-guiding optical system 60 and the excitation light source 10.

[0065] 6, in the fluorescence observation device 1, the central axis C of the lens barrel 62 is arranged to intersect (orthogonal in FIG. 2) with an imaginary line HL connecting the excitation light source 10 and the mounting table 21. Due to this arrangement, even if the excitation light EL is specularly reflected by the underside of the mounting table 21 and becomes stray light SL, the resulting stray light travels in the direction along the imaginary line HL (y direction) and is unlikely to travel toward the light-guiding optical system 60, which is arranged in a direction intersecting the imaginary line HL.

[0066] As a result, in the fluorescence observation device 1, the amount of stray light SL that is generated and heads toward the light-guiding optical system 60 is reduced. Furthermore, even if the stray light SL heads toward the light-guiding optical system 60, it is blocked by the light-blocking member 40.

[0067] According to the fluorescence observation device 1 configured as above, the influence of stray light can be reduced by the light blocking member 40, and a clear image can be captured using the fluorescence emitted by the observation object.

[0068] In this embodiment, the light-guiding optical system 60 of the fluorescence observation device 1 is described as being a telecentric optical system, but this is not limiting. Even if the light-guiding optical system 60 is not a telecentric optical system, the effect of the invention that a clear image can be captured can be achieved by blocking stray light with the light-blocking member 40.

[0069] Furthermore, in this embodiment, the fluorescence observation device 1 has a reflecting mirror 30, and the lens barrel 62 constituting the light-guiding optical system 60 extends in the x direction, but this is not limiting. For example, the lens barrel 62 may extend in the z direction, and the lens (telecentric lens 61) of the light-guiding optical system 60 may be configured to directly face the underside of the mounting table 21. In this case, it is preferable that the excitation light EL is irradiated onto the sample plate P from outside the numerical aperture (NA) of the telecentric lens 61 at a larger angle. Even in a fluorescence observation device configured in this way, the effect of the invention of being able to capture clear images can be achieved by blocking the resulting stray light with the light-shielding member 40.

[0070] Furthermore, the above-described fluorescence observation device 1 can appropriately employ optically equivalent optical members depending on the design.

[0071] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on the design, specifications, etc., without departing from the spirit of the present invention.

[0072] 1, 1X... fluorescence observation device, 10, 10A, 10B... excitation light source, 11... light source, 21... mounting table, 30... reflecting mirror, 40... light blocking member, 50... fluorescence filter, 60... light guiding optical system, 61... telecentric lens, 62... lens barrel, 62a... end portion, 70... imaging unit, C... central axis, EL... excitation light, F... fluorescence, HL... virtual line, L... light, NL, NS... normal line, WL1, WL2, WL3, WL4, WL5, WL6... wavelength

Claims

1. A fluorescence observation device comprising: an excitation light source that irradiates an observation object with excitation light; an imaging unit that captures an observation image using fluorescence emitted by the observation object excited by the excitation light; a light-guiding optical system that guides the fluorescence to the imaging unit; a fluorescence filter that is arranged on the light incident side of the light-guiding optical system and that transmits light in a wavelength range that includes the wavelength of the fluorescence; and a light-shielding member that is arranged on the light incident side of the fluorescence filter, wherein the light-shielding member blocks light that is incident on the fluorescence filter from a direction that intersects with the normal to the fluorescence filter.

2. A fluorescence observation device according to claim 1, wherein the light-guiding optical system comprises a telecentric lens system and a lens barrel that holds the telecentric lens system, the fluorescence filter is disposed on the incident side of the lens barrel for the fluorescence, and the light-shielding member is provided on the incident side and extends in the axial direction of the lens barrel.

3. A fluorescence observation device according to claim 2, wherein the light blocking member is a cylindrical member.

4. A fluorescence observation device according to claim 3, wherein the light-shielding member is a truncated cone-shaped member whose diameter gradually decreases in the direction away from the lens barrel.

5. A fluorescence observation device according to any one of claims 2 to 4, comprising: a mounting table for mounting the object to be observed; and a reflecting mirror between the mounting table and the lens barrel for guiding the fluorescence to the telecentric lens system.

6. A fluorescence observation device according to claim 5, wherein, in a plan view, the central axis of the lens barrel intersects with an imaginary line connecting the excitation light source and the stage.

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