Observation device and observation method

Abstract

Provided are an observation device and an observation method with which it is possible to selectively observe, from among two or more types of particles contained in a suspension, a target particle to be observed. This observation device is provided with: a holding unit that holds a suspension containing two or more types of particles having different size relationships with respect to the specific gravity of the liquid, said suspension being separated into a target layer containing the target particles to be observed from among the two or more types of particles contained in the suspension, and a non-target layer containing the target particles to be observed from among the two or more types of particles contained in the suspension; the non-target layer contains particles other than target particles among the two or more types of particles; an observation light source that causes observation light to enter from the object layer side; an objective lens which is provided on the object layer side and amplifies an image of the object particles; and an imaging unit that captures an image of the target particle magnified by the objective lens. The depth of field of the optical system including the objective lens is smaller than the thickness of the object layer.

Description

Technical Field

[0001] The present invention relates to an observation device and an observation method. Background Technology

[0002] As a technique related to cell observation, the following techniques are known. For example, Patent Document 1 describes a cell detection method characterized by pouring a cell suspension containing cells into a storage tank in such a way that a second liquid with a specific gravity greater than that of the cell suspension and the cells forms the bottom layer, photographing the interface between the cell suspension and the second liquid from above the storage tank, and detecting the presence or number of rare cells, including those in the cells, from the image obtained by photographing.

[0003] Patent document 2 describes a cell observation device that continuously observes a capture wafer with through-holes for capturing cells in a cell suspension. The cell observation device also includes an image processing unit with a light source and an objective lens.

[0004] Patent document 3 describes an apparatus comprising: a fluid system for delivering a suspension containing cells to a light-irradiated area; a fluorescence photometric optical system for irradiating each cell descending within the fluid system to measure fluorescence intensity; and an information processing system for detecting signals from the fluorescence photometric optical system and issuing signals for sorting and collecting cells.

[0005] Patent Document 1: Japanese Patent Application Publication No. 2012-021828

[0006] Patent Document 2: Japanese Patent Application Publication No. 2006-094783

[0007] Patent Document 3: Japanese Patent Application Publication No. 63-233371

[0008] Various treatment methods have been developed in recent years along with advancements in regenerative medicine research. One treatment method in regenerative medicine involves the following process: breaking down living tissue collected from a patient through enzymatic treatment, thereby extracting cells, counting the number of extracted cells, and seeding / culturing the cells at a desired concentration. Cell counting is typically performed under a bright-field microscope using white light. However, when living tissue contains fat, it is difficult to identify cells and granular fat particles under a bright-field microscope, making accurate cell counting challenging. Centrifugation can also be considered to separate cells and fat and remove the fat, but this carries the risk of cell damage during centrifugation or cell loss during fat removal. Summary of the Invention

[0009] The technology of the present invention was made in view of the above-mentioned problems, and its purpose is to provide an observation device and observation method that can selectively observe the target particles among two or more types of particles contained in a suspension.

[0010] The observation apparatus according to the present invention comprises: a holding unit for holding a suspension separated into an object layer and a non-object layer, the suspension containing two or more types of particles with different specific gravities relative to the liquid; the object layer containing an object particle, which is the object of observation, from the two or more types of particles contained in the suspension; the non-object layer containing particles other than the object particle from the two or more types of particles; an observation light source for incident observation light from the object layer side; an objective lens disposed on the object layer side for magnifying the image of the object particle; and an imaging unit for capturing the image of the object particle magnified by the objective lens. The depth of field of the optical system including the objective lens is smaller than the thickness of the object layer.

[0011] The observation light source can output excitation light that excites the phosphor introduced into the target particle. The observation device can have a filter positioned between the objective lens and the camera unit to shield the wavelength component of the excitation light while allowing the wavelength component of the fluorescence emitted from the target particle to pass through. The angle between the optical axis of the observation light and the optical axis of the objective lens can be greater than 0° and less than 90°.

[0012] The holding part may have a pair of parallel plates that are light-transmitting and whose main surfaces extend in a horizontal direction, and can hold the suspension between the pair of parallel plates.

[0013] The observation device may have an imaging lens with a magnification of less than 1x, which images the image of the object particle magnified by the objective lens onto the imaging surface of the imaging unit.

[0014] The observation device may have a counting unit that counts the number of object particles captured by the camera unit.

[0015] The object particles can be cells.

[0016] The observation method involved in this invention includes the following steps: maintaining a suspension in a state separated into an object layer and a non-object layer, the suspension containing two or more types of particles with different specific gravities relative to the liquid; the object layer containing the object particles, which are the objects of observation, from the two or more types of particles contained in the suspension; the non-object layer containing particles other than the object particles from the two or more types of particles; incident observation light from the object layer side; magnifying the image of the object particles through an objective lens disposed on the object layer side; and capturing the magnified image of the object particles through the objective lens. The depth of field of the optical system including the objective lens is smaller than the thickness of the object layer.

[0017] The object particles can be cells.

[0018] Invention Effects

[0019] According to the technology of the present invention, an observation device and observation method are provided that can selectively observe the target particles among two or more types of particles contained in a suspension. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating an example of the structure of an observation device according to an embodiment of the technology of the present invention.

[0021] Figure 2 This is a cross-sectional view illustrating an example of the structure of the retaining part involved in an embodiment of the present invention.

[0022] Figure 3 This is a diagram illustrating an example of the frequency characteristics of a multi-band filter according to an embodiment of the present invention.

[0023] Figure 4 This is a graph showing the relationship between the depth of field and the thickness of the object layer in the observation optical system 40.

[0024] Figure 5A These are images of cells contained in a cell suspension, obtained using the observation apparatus described in the comparative example.

[0025] Figure 5B These are images of cells contained in a cell suspension, acquired using an observation device according to an embodiment of the technology of the present invention.

[0026] Figure 6 This is a diagram illustrating an example of the structure of an observation device according to another embodiment of the technology of the present invention. Detailed Implementation

[0027] Hereinafter, an example of an embodiment of the present invention will be described with reference to the accompanying drawings. Furthermore, in each drawing, the same or equivalent components and parts are labeled with the same reference numerals, and repeated descriptions are omitted.

[0028] Figure 1 This diagram schematically illustrates an example of the structure of an observation device 10 according to an embodiment of the present invention. The observation device 10 includes a holding section 11, an observation light source 13, a reflector 14, a filter 15, an imaging lens 16, and an imaging unit 17. The observation light source 13, reflector 14, filter 15, imaging lens 16, and imaging unit 17 constitute an observation optical system 40. The observation device 10 is used to observe particles contained in a suspension 20 held by the holding section 11. The particles to be observed are, for example, cells. The cells can be single cells, or colonies or spheroids formed by the aggregation of multiple cells.

[0029] Figure 2 This is a cross-sectional view showing an example of the structure of the holding section 11. The holding section 11 is a component for holding a suspension 20 containing particles that are the object of observation (hereinafter referred to as object particles 31). The suspension 20 also contains particles other than object particles 31 (hereinafter referred to as non-object particles 32). The specific gravities of object particles 31 and non-object particles 32 relative to the liquid (medium) 30 contained in the suspension 20 are different. That is, the specific gravity of one of object particles 31 and non-object particles 32 is less than that of liquid 30, and the specific gravity of the other is greater than that of liquid 30. Hereinafter, the case where the specific gravity of non-object particles 32 is less than that of liquid 30 and the specific gravity of object particles 31 is greater than that of liquid 30 will be described as an example. The suspension 20 is, for example, a cell suspension, the object particles 31 are, for example, cells, the non-object particles 32 are, for example, fat (oil droplets), and the liquid 30 is, for example, a culture medium.

[0030] The suspension 20 is held in the holding section 11 in a state separated into an object layer 21 containing target particles 31 and a non-target layer 22 containing non-target particles 32. After the suspension 20 is contained in the holding section 11, it is allowed to stand, whereby the non-target particles 32 float due to buoyancy, and the target particles 31 settle due to gravity. Thus, the suspension 20 is separated into an object layer 21 containing target particles 31 and a non-target layer 22 containing non-target particles 32. The object layer 21 is disposed on the lower side in the vertical direction, and the non-target layer 22 is disposed on the upper side in the vertical direction. The holding section 11 is configured to include a pair of parallel plates 18, which are light-transmitting and whose main surfaces extend in the horizontal direction. The suspension 20 is held between the pair of parallel plates 18. When the distance between the parallel plates is defined as T, the thicknesses of the object layer 21 and the non-target layer 22 are each defined as T / 2.

[0031] An observation optical system 40, including an observation light source 13, a reflector 14, a filter 15, an imaging lens 16, and an imaging unit 17, is disposed on the object layer 21 side. The objective lens 12 is configured to focus on the object layer 21 and magnify the image of the object particle 31. The observation light used by the observation light source 13 to observe the object particle 31 is incident from the object layer 21 side. The observation light source 13 is configured such that the angle between the optical axis of the observation light and the optical axis of the objective lens 12 is greater than 0° and less than 90°. This indicates that the optical axis of the observation light is not coaxial with the optical axis of the objective lens 12, which faces the vertical direction, and that the observation light is incident from the object layer 21 side. By setting the optical axis of the observation light to be coaxial with the optical axis of the objective lens 12, the configuration freedom of the observation light source 13 can be increased. For example, as... Figure 1 As shown, observation light can be shone onto the object layer 21 from close range. As a result, a clearer image of the object particles 31 can be obtained.

[0032] The observation light shines on the object particle 31 from the object layer 21 side. Depending on the observation light, reflected light, fluorescence, or phosphorescence is emitted from the object particle 31. The reflected light, fluorescence, or phosphorescence emitted from the object particle 31 is incident on the objective lens 12. In the case of fluorescence observation of the object particle 31, the object particle 31 is subjected to fluorescent staining using a fluorescent reagent. In this case, the observation light source 13 is a light source that outputs excitation light that excites the phosphor introduced into the object particle 31.

[0033] When the target particle 31 is a cell, the fluorescent reagent used is one that selectively fluoresces the cell. The fluorescent reagent may contain, for example, acridine orange (AO) and 4',6-diamidino-2-phenylindole (DAPI). AO is used to stain all cells, and DAPI is used to stain dead cells. Fats (oil droplets), which are not the target particle 32, are not stained by the fluorescent reagent. For the cell stained with AO, a blue light source with a wavelength around 470 nm can be used as the excitation light. For the cell stained with DAPI, ultraviolet light with a wavelength around 365 nm can be used as the excitation light. The observation device 10 may have two or more observation light sources 13 that output excitation light of different wavelengths.

[0034] Light from the transmission objective lens 12 is bent 90° in its direction of travel by the reflecting mirror 14 and then incident on the filter 15. The filter 15 has the function of selectively passing the wavelength component of the fluorescence emitted from the target particle 31. That is, the filter 15 has the characteristic of shielding the wavelength component of the excitation light and allowing the wavelength component of the fluorescence emitted from the target particle 31 to pass through. When the observation device 10 has two or more observation light sources 13 that output excitation light of different wavelengths, and two or more types of fluorescence of different wavelengths are emitted from the target particle 31, a multi-band filter with multiple transmission bands is used as the filter 15.

[0035] Here, Figure 3 The figure above shows the state of cells stained with AO fluorescence irradiated with excitation light with a peak wavelength of about 500 nm[1], thereby emitting fluorescence with a peak wavelength of about 526 nm[1]. Figure 3 The mid-section diagram shows the state in which cells stained with DAPI fluorescence emit fluorescence with a peak wavelength of about 360 nm [2] after being irradiated with excitation light [2] at a peak wavelength of about 460 nm. In the observation device 10, when it is desired to observe both fluorescence [1] and fluorescence [2], a filter 15 can be used, for example, a filter with a [missing information - likely a specific material or function]. Figure 3The multi-band filter with frequency characteristics shown in the lower figure has a first transmission band of 500 nm-550 nm that allows fluorescence [1] to pass through while shielding excitation light [1], and a second transmission band of 400 nm-450 nm that allows fluorescence [2] to pass through while shielding excitation light [2]. Furthermore, filter 15 is not required if fluorescence observation of the target particle 31 is not performed.

[0036] Light after the transmission filter 15 is incident on the imaging lens 16. The imaging lens 16 images the image of the object particle 31 magnified by the objective lens 12 onto the imaging surface of the imaging unit 17. The imaging unit 17 captures the image of the object particle 31 magnified by the objective lens 12 and imaged by the imaging lens 16, and outputs the image of the object particle 31. The imaging unit 17 can be, for example, a digital camera capable of color photography equipped with an imaging element such as a CMOS (Complementary Metal-Oxide-Semiconductor) image sensor.

[0037] Figure 4 This represents the depth of field (DoF) in the observation optical system 40. i A graph showing the relationship between the thickness of object layer 21 and the thickness of the object layer. (See figure.) Figure 4 As shown, observe the depth of field (DoF) of optical system 40. i The thickness is less than T / 2 of the object layer 21. Therefore, it is possible to avoid including information from the non-object layer 22 in the image acquired by the observed optical system 40. Thus, it is possible to obtain an image that includes the object particles 31 but not the non-object particles 32.

[0038] To satisfy DoF i <T / 2, objective lens 12 is preferably high magnification. The higher the magnification of objective lens 12, the greater the reduction in depth of field (DoF). i On the other hand, the lower the magnification of the objective lens 12 and the imaging lens 16, the wider the field of view for observing the object particles 31. When counting the number of object particles 31 using the observation device 10 by sampling, a lower magnification of the objective lens 12 and the imaging lens 16 increases the number of object particles included in the field of view, thus enabling statistically accurate count values ​​to be obtained. This is to balance reducing the depth of field (DoF). i To expand the field of view, the objective lens 12 is preferably high magnification (1x or more) and the imaging lens 16 is low magnification (less than 1x). For example, the magnification of the objective lens 12 can be set to 4x and the magnification of the imaging lens 16 can be set to 0.5x.

[0039] As described above, the observation apparatus 10 according to the embodiments of the present invention includes: a holding unit 11 for holding the suspension 20 separated into an object layer 21 containing object particles 31 and a non-object layer 22 containing non-object particles 32; an observation light source 13 for incident observation light from the object layer 21 side; an objective lens 12 disposed on the object layer 21 side for magnifying the image of the object particles 31; and an imaging unit 17 for capturing the image of the object particles 31 magnified by the objective lens 12. In the observation apparatus 10, the depth of field (DoF) of the observation optical system 40 including the objective lens 12 is... i Less than the thickness T / 2 of object layer 21.

[0040] Here, Depth of Field (DoF) i It is represented by the following formula (1).

[0041] [Formula 1]

[0042]

[0043] In equation (1), DoF o The focal depth is denoted by α, the vertical magnification is β, the horizontal magnification is CoC, and the allowable scattering circle diameter is F. eff This is the effective F-value. For example, in the observation optical system 40, at F... eff =3.85, CoC = 7.04μm, β = 2, according to equation (1), DoF i =13.5μm. In this case, if the thickness T / 2 of the object layer 21 is 50μm, then DoF i <T / 2, so it is possible to focus on and observe only the object layer 21 in the object layer 21 and the non-object layer 22.

[0044] According to the observation apparatus 10 of this embodiment, observation light is irradiated onto the object particle 31 from the object layer 21 side, and correspondingly, reflected light, fluorescence, or phosphorescence emitted from the object particle 31 is captured by the observation optical system 40. On the other hand, transmitted light after passing through the non-object layer 22 is not captured by the observation light source 13. Therefore, it is possible to avoid including information about the non-object layer 22 in the image acquired by the observation optical system 40. Thus, an image containing the object particle 31 but not the non-object particle 32 can be obtained. Furthermore, according to the observation apparatus 10 of this embodiment, the depth of field (DoF) of the observation optical system 40 is... i The thickness is less than that of the object layer 21. This improves the effect of excluding information from the non-object layer 22. Consequently, it prevents non-object particles 32 from being reflected in the image output from the camera unit 17.

[0045] Thus, according to the observation apparatus 10 of this embodiment, it is possible to selectively observe the target particles among two or more types of particles contained in a suspension. For example, for a cell suspension containing cells and fat (oil droplets), an image containing cells but not fat can be obtained. By using the image containing cells but not fat to count the number of cells contained in the cell suspension, an accurate count value can be obtained. Furthermore, according to the observation apparatus 10 of this embodiment, centrifugation separation processing, which carries the risk of cell damage and loss, is not required.

[0046] In the observation apparatus 10 of this embodiment, the observation light source 13 can output excitation light that excites the phosphor introduced into the target particle 31. As a result, fluorescence observation of the target particle 31 can be performed, and a clearer image of the target particle 31 can be obtained.

[0047] The observation apparatus 10 according to this embodiment may include a filter 15 disposed between the objective lens 12 and the imaging unit 17, which shields the wavelength component of the excitation light and allows the wavelength component of the fluorescence emitted from the object particle 31 to pass through. Thus, the image projection caused by the excitation light can be avoided.

[0048] In the observation apparatus 10 of this embodiment, the angle between the optical axis of the observation light and the optical axis of the objective lens is greater than 0° and less than 90°. That is, the optical axis of the observation light is not coaxial with the optical axis of the objective lens 12, and the observation light is incident from the object layer 21 side. As a result, the degree of freedom in the arrangement of the observation light source 13 is increased. For example, the observation light can be irradiated onto the object layer 21 from close range, and thus a clearer image of the object particles 31 can be obtained.

[0049] In the observation device 10 according to this embodiment, the holding part 11 has a pair of parallel plates that are light-transmitting and whose main surfaces extend in the horizontal direction, and the suspension 20 is held between the pair of parallel plates. Normally, the gas-liquid interface becomes a curved surface due to surface tension. When the surface of the suspension is curved, it is difficult to obtain a clear image of the object particles throughout the entire field of view. By having a pair of parallel plates in the holding part 11 that are light-transmitting and whose main surfaces extend in the horizontal direction, the surface of the suspension becomes flat, thus enabling the acquisition of a clear image of the object particles throughout the entire field of view. Furthermore, it allows for a uniform thickness of the object layer 21.

[0050] In the observation apparatus 10 according to this embodiment, the imaging lens 16 has a magnification of 1x or less. Therefore, a high-magnification objective lens 12 can be used while expanding the field of view. By using the high-magnification objective lens 12, the depth of field (DoF) of the observation optical system 40 can be reduced. i This can prevent the image acquired by the observed optical system 40 from containing information from the non-object layer 22.

[0051] Figure 5A This is an image of cells contained in a cell suspension, acquired using the observation apparatus (not shown) involved in the comparative example. In the comparative example, no process was performed to separate the cells and lipids contained in the cell suspension. Furthermore, the observation apparatus involved in the comparative example has a structure in which an excitation light source is arranged on one side of the cell suspension sandwiched in the middle, and an objective lens is arranged on the other side. That is, the excitation light passes through the cell suspension containing cells and lipids and enters the objective lens. Fluorescent staining was performed on the cells. The image acquired using the observation apparatus involved in the comparative example shows multiple lipid particles (indicated by arrows in the figure). When using the image showing multiple lipid particles to count the number of cells, an accurate count value cannot be obtained.

[0052] Figure 5B This is an image of cells contained in a cell suspension, acquired using the observation device 10 according to an embodiment of the present invention. The cells were subjected to fluorescent staining. An observation light source 13 outputs excitation light to excite the fluorophores introduced into the cells. After the cell suspension is contained in the holding section 11, it is allowed to stand, thereby separating the cell suspension into a target layer containing cells and a non-target layer containing fat. Fat is not reflected in the image acquired by the observation device 10 according to an embodiment of the present invention. By using images of particles other than cells that are not reflected, accurate cell counts can be obtained.

[0053] Furthermore, the objective lens 12 is preferably a variable focus lens. The objective lens 12 can be configured to include an objective lens body and a variable focus lens. The depth of field (DoF) of the observation optical system 40 is... i In small objects, it is difficult to focus on the target particle. If the focal position of the objective lens 12 is fixed relative to the target particle, it may be impossible to focus on the target particle due to the tolerance of the distance T between the parallel plates in the holding part 11. Therefore, it is preferable that the focal position of the objective lens 12 and the relative position of the target particle are variable. As a method to make the focal position of the objective lens 12 and the relative position of the target particle variable, a movable stage can be considered. However, if a movable stage is introduced, the device will become larger. By using a variable focal point objective lens, the focal position of the objective lens 12 and the relative position of the target particle can be variable without increasing the size of the device.

[0054] In the above description, the case where the target particle is a cell and the non-target particle is fat is illustrated, but the technology of the present invention can be applied to any suspension containing two or more types of particles with different specific gravities relative to the liquid.

[0055] [Second Implementation]

[0056] Figure 6This is a diagram illustrating an example of the structure of the observation device 10A according to the second embodiment of the present invention. The observation device 10A according to the second embodiment differs from the observation device 10 according to the first embodiment in that it includes a counting unit 19. The counting unit 19 counts the number of object particles captured in the imaging unit 17. Specifically, the counting unit 19 acquires an image output from the imaging unit 17, extracts granular objects from the acquired image, and outputs a count value of the number of extracted objects. The counting unit 19 can output the particle density D (number per unit volume) by performing the operation represented by the following formula (1). In formula (1), S is the size of the image containing the particles of the detected object, T is the distance between the parallel plates in the holding unit 11, and C is the count value of the number of particles. Furthermore, the counting unit 19 can output the total number of particles A by performing the operation represented by the following formula (2). In formula (2), D is the density calculated according to formula (1), and V is the volume of the suspension 20 held in the holding unit 11. The counting unit 19 is configured to include a computer that performs the above series of processes. The counting unit 19 counts the number of target particles based on the image of the non-target particles output from the camera unit 17, thus obtaining an accurate count value.

[0057] D=C / (S×T) ··· (1)

[0058] A=D×V··· (2)

[0059] Regarding the first and second embodiments described above, the following notes are further disclosed.

[0060] (Postscript 1)

[0061] An observation device having:

[0062] The holding section holds a suspension separated into an object layer and a non-object layer. The suspension contains two or more types of particles with different specific gravities relative to the liquid. The object layer contains an object particle, which is the object of observation, among the two or more types of particles contained in the suspension. The non-object layer contains particles other than the object particle among the two or more types of particles.

[0063] Observe the light source so that the observation light is incident from the object layer side;

[0064] An objective lens, positioned on the object layer side, magnifies the image of the object particles; and

[0065] The camera unit captures an image of the object particle, magnified by the objective lens.

[0066] The depth of field of the optical system including the objective lens is less than the thickness of the object layer.

[0067] (Postscript 2)

[0068] According to the observation apparatus described in Appendix 1, wherein,

[0069] The observation light source outputs excitation light that is introduced into the phosphor in the target particle.

[0070] (Note 3)

[0071] The observation apparatus according to Appendix 2 has a filter.

[0072] The filter is disposed between the objective lens and the imaging unit, shielding the wavelength component of the excitation light and allowing the wavelength component of the fluorescence emitted from the object particle to pass through.

[0073] (Postscript 4)

[0074] The observation apparatus according to any one of Annexes 1 to 3, wherein,

[0075] The angle between the optical axis of the observation light and the optical axis of the objective lens is greater than 0° and less than 90°.

[0076] (Note 5)

[0077] The observation apparatus according to any one of Annexes 1 to 4, wherein,

[0078] The retaining part has a pair of parallel plates, which are light-transmitting and whose main surfaces extend in a horizontal direction.

[0079] The suspension is maintained between the pair of parallel plates.

[0080] (Note 6)

[0081] The observation apparatus according to any one of Appendices 1 to 5 has an imaging lens with a magnification of 1x or less.

[0082] The imaging lens images the object particle magnified by the objective lens onto the imaging surface of the camera unit.

[0083] (Note 7)

[0084] The observation device according to any one of Annexes 1 to 6 has a counting unit.

[0085] The counting unit counts the number of object particles captured by the camera unit.

[0086] (Note 8)

[0087] The observation apparatus according to any one of Annexes 1 to 7, wherein,

[0088] The object particle is a cell.

[0089] (Note 9)

[0090] An observation method comprising the following steps:

[0091] The suspension is maintained in a state of separation into an object layer and a non-object layer. The suspension contains two or more types of particles with different specific gravities relative to the liquid. The object layer contains the object particles among the two or more types of particles contained in the suspension, which are the objects of observation. The non-object layer contains particles other than the object particles among the two or more types of particles.

[0092] The observation light is incident from the object layer side;

[0093] The image of the object particles is magnified by an objective lens positioned on the object layer side; and

[0094] To capture an image of the object particle magnified by the objective lens.

[0095] The depth of field of the optical system including the objective lens is less than the thickness of the object layer.

[0096] (Postscript 10)

[0097] According to the observation method described in Appendix 9, wherein,

[0098] The object particle is a cell.

[0099] Symbol Explanation

[0100] 10, 10A - Observation apparatus; 11 - Holding part; 12 - Objective lens; 13 - Observation light source; 14 - Reflector; 15 - Filter; 16 - Imaging lens; 17 - Imaging unit; 18 - Parallel plate; 19 - Counting unit; 20 - Suspension; 21 - Object layer; 22 - Non-object layer; 30 - Liquid; 31 - Object particles; 32 - Non-object particles; 40 - Observation optical system.

Claims

1. An observation device comprising: The holding section holds a suspension separated into an object layer and a non-object layer. The suspension contains two or more types of particles with different specific gravities relative to the liquid. The object layer contains an object particle, which is the object of observation, among the two or more types of particles contained in the suspension. The non-object layer contains particles other than the object particle among the two or more types of particles. Observe the light source so that the observation light is incident from the object layer side; An objective lens, positioned on the object layer side, magnifies the image of the object particles; and The camera unit captures an image of the object particle, magnified by the objective lens. The depth of field of the optical system including the objective lens is less than the thickness of the object layer.

2. The observation device according to claim 1, wherein, The observation light source outputs excitation light that is introduced into the phosphor in the target particle.

3. The observation device according to claim 2, comprising a filter, The filter is disposed between the objective lens and the imaging unit, shielding the wavelength component of the excitation light and allowing the wavelength component of the fluorescence emitted from the object particle to pass through.

4. The observation device according to claim 1, wherein, The angle between the optical axis of the observation light and the optical axis of the objective lens is greater than 0° and less than 90°.

5. The observation device according to claim 1, wherein, The retaining part has a pair of parallel plates, which are light-transmitting and whose main surfaces extend in a horizontal direction. The suspension is maintained between the pair of parallel plates.

6. The observation device according to claim 1, wherein it has an imaging lens with a magnification of 1x or less. The imaging lens images the object particle magnified by the objective lens onto the imaging surface of the imaging unit.

7. The observation device according to claim 1, further comprising a counting unit. The counting unit counts the number of object particles captured by the camera unit.

8. The observation device according to any one of claims 1 to 7, wherein, The object particle is a cell.

9. An observation method, comprising the following steps: The suspension is maintained in a state of separation into an object layer and a non-object layer. The suspension contains two or more types of particles with different specific gravities relative to the liquid. The object layer contains the object particles among the two or more types of particles contained in the suspension, which are the objects of observation. The non-object layer contains particles other than the object particles among the two or more types of particles. The observation light is incident from the object layer side; The image of the object particles is magnified by an objective lens located on the object layer side; and To capture an image of the object particle magnified by the objective lens. The depth of field of the optical system including the objective lens is less than the thickness of the object layer.

10. The observation method according to claim 9, wherein, The object particle is a cell.

Images