Measuring device and culture system

Abstract

The objective is to continuously measure the fluorescence intensity of the target product while simultaneously measuring the turbidity of the culture medium, all within a single, stable, and accurate process. [Solution] The present invention provides a measuring device 1 comprising: at least one light irradiation unit 10 that irradiates excitation light EL toward the interface between the inner surface of a culture vessel 2 containing a culture medium W that expresses a target product and the culture medium; a first imaging unit (combined imaging unit 12) that captures an image of scattered light reflected from the excitation light at the interface and acquires it as a scattered light image; a second imaging unit (combined imaging unit 12) that captures an image of fluorescence emitted by the expressed target product due to irradiation with excitation light and acquires it as a fluorescence image; a fluorescence filter 13 that transmits light in a wavelength range including the fluorescence wavelength and restricts the transmission of light in at least the wavelength range of the excitation light; and a measuring unit 91 that measures the turbidity of the culture medium and the fluorescence intensity of the target product based on the scattered light image and the fluorescence image.

Description

Technical Field

[0001] The present invention relates to a measuring device and a culture system.

Background Art

[0002] For example, in order to analyze the structure and function of a target substance (target product substance) such as a protein, it is necessary to mass-produce the target substance. In this regard, conventionally, a method of expressing a target substance by using a microorganism such as Escherichia coli as a host has been performed. For example, in a protein expression system using Escherichia coli, a method using an expression vector whose expression is induced depending on temperature is known. The expression vector according to this method includes, for example, a target gene encoding a target substance (target protein), and a specific promoter whose expression is induced when the culture temperature of Escherichia coli is at a low temperature (for example, 15°C).

[0003] In this case, Escherichia coli containing the above-described expression vector is introduced into the culture solution contained in the culture vessel. Then, the culture vessel is shaken while maintaining a predetermined culture temperature (for example, 37°C) to culture Escherichia coli. The turbidity of the culture solution tends to increase as the culture of Escherichia coli progresses. Therefore, when a certain turbidity is reached, it can be determined (estimated) that a certain amount of Escherichia coli has been cultured. Therefore, after reaching a certain turbidity, the culture vessel is cooled and maintained at a low temperature (for example, about 15°C).

[0004] Thereby, a specific promoter can be expressed in a low-temperature state. On the other hand, the expression of proteins possessed by Escherichia coli itself can be suppressed. Therefore, it becomes possible to efficiently and highly purely express a target substance (target protein). Thereby, by performing fluorescence observation with a fluorescence microscope or the like, the fluorescence intensity of the target substance (target protein) can be measured, and it can be confirmed whether a certain amount of the target substance (target protein) has been obtained.

[0005] As a prerequisite for the expression of such target substances, it is necessary to culture a certain amount of E. coli in a culture medium. Regarding this type of culture, a method for continuously measuring the growth of cultured samples such as E. coli in a non-contact manner is known (see, for example, Patent Document 1). This method includes a light irradiation unit that irradiates infrared light onto a culture vessel while it is being shaken, and a light receiving unit that receives the light that has passed through the culture vessel. In particular, this method utilizes centrifugal force caused by shaking the culture vessel to spread the culture medium thinly and with a constant fluctuation thickness on the inner surface of the culture vessel, thereby enabling light transmission. As a result, infrared light irradiated from the light irradiation unit can pass through both the culture vessel and the culture medium (the culture medium spread on the inner wall of the culture vessel) before reaching the light receiving unit.

[0006] Furthermore, the device's calculation unit stores model data for optical density, which is used to calculate the change in optical density (OD) due to the growth of the cultured sample by comparing it with the difference between the irradiated light intensity and the transmitted light intensity. This makes it possible to calculate the optical density and measure the growth rate of the cultured sample by comparing the actually measured change in light intensity (the difference between the change in irradiated light intensity and the change in transmitted light intensity) with the model data and performing calculations. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 5774352 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The turbidity of the culture medium can be measured using the conventional method described above. However, to measure the fluorescence intensity of the target substance expressed afterward, it is necessary to take the culture medium as a sample from the culture vessel and measure it using a separate fluorescence microscope, for example. Therefore, since the measurement of the turbidity of the culture medium and the measurement of the fluorescence intensity of the target substance must be performed using separate devices, the procedure becomes complicated and there is room for improvement.

[0009] Furthermore, the conventional method described above employs a so-called light transmission type, where infrared light irradiated from the light irradiation unit is transmitted through the culture medium. In this case, it is difficult to properly transmit infrared light because it is easily affected by the film thickness of the culture medium spread on the inner surface of the culture vessel and the concentration of the culture medium. Therefore, there is room for improvement in stably and accurately measuring the turbidity of the culture medium.

[0010] The present invention has been made in consideration of these circumstances, and its purpose is to provide a measuring device and a culture system equipped with such a measuring device that can continuously measure the fluorescence intensity of a target product while simultaneously measuring the turbidity of a culture medium in a series of steps, and that can do so stably and accurately. [Means for solving the problem]

[0011] (1) The measuring device according to the present invention is characterized by comprising: at least one light irradiation unit disposed outside a culture vessel containing a culture medium that expresses a target product having the function of emitting fluorescence, and irradiating excitation light toward the interface between the inner surface of the culture vessel and the culture medium; a first imaging unit disposed outside the culture vessel that captures an image of scattered light reflected by the excitation light at the interface and acquires it as a scattered light image; a second imaging unit disposed outside the culture vessel that captures an image of fluorescence emitted by the expressed target product due to irradiation with the excitation light and acquires it as a fluorescence image; a fluorescence filter disposed between the second imaging unit and the culture vessel that transmits light in a wavelength range including the wavelength of fluorescence and restricts the transmission of light in at least the wavelength range of the excitation light; and a measuring unit that measures the turbidity of the culture medium and the fluorescence intensity of the target product based on the scattered light image and the fluorescence image.

[0012] According to the measuring device of the present invention, excitation light can be irradiated from outside the culture vessel containing the culture medium by a light irradiation unit. In this case, the light irradiation unit irradiates the excitation light toward the interface between the inner surface of the culture vessel and the culture medium. As a result, a portion of the irradiated excitation light is reflected at the interface and becomes scattered light. Using the first imaging unit located outside the culture vessel, an image of the scattered light reflected at the interface can be captured and obtained as a scattered light image. As a result, the turbidity of the culture medium can be measured by the measuring unit based on the scattered light image. This makes it possible to understand the culture state of the culture medium containing the target product based on the turbidity. Therefore, after confirming that the culture has been carried out properly, the target product can be expressed using various expression systems. Furthermore, if it is determined that the culture is insufficient, it is possible to provide feedback such as continuing the culture further.

[0013] As described above, when the target product is expressed, it emits fluorescence in response to irradiation with excitation light. Specifically, the target product absorbs the light energy of the excitation light and transitions to an excited state, then transitions to a ground state while emitting fluorescence. This allows the fluorescence emitted by the target product to be captured and obtained as a fluorescence image using the second imaging unit located outside the culture vessel. In particular, because a fluorescence filter is placed between the second imaging unit and the culture vessel, it is possible to block at least the wavelength range of the excitation light reflected by the culture vessel, preventing the excitation light from reaching the second imaging unit. Therefore, noise caused by the excitation light can be removed from the acquired fluorescence image, and a high-precision fluorescence image can be obtained. Consequently, the fluorescence intensity of the target product can be measured based on the fluorescence image using the measurement unit, and it is possible to determine whether a certain amount of the target product has been obtained.

[0014] As a result, it is possible to continuously measure the fluorescence intensity of the target product while simultaneously measuring the turbidity of the culture medium at the necessary timing within a single process. In particular, unlike conventional light transmission types, the turbidity of the culture medium can be measured based on scattered light reflected at the interface between the inner surface of the culture vessel and the culture medium, making it less susceptible to influences such as the concentration of the culture medium or foaming of the culture medium surface. Therefore, even if the turbidity of the culture medium is high and transmission is difficult, the turbidity of the culture medium can be measured stably and accurately, making it easier to accurately grasp the culture state of the culture medium containing the target product (protein, etc.). Furthermore, since the fluorescence intensity of the target product can be measured without removing the culture medium from the culture vessel, contamination of the culture medium can be prevented, and fluorescence intensity can be measured with high accuracy.

[0015] (2) The culture medium contains host cells transformed with an expression vector that expresses the target product, and the light irradiation unit may irradiate the excitation light toward the interface between the inner surface of the culture vessel and the culture medium containing the host cells.

[0016] In this case, since host cells such as E. coli transformed with an expression vector (an expression vector containing the target gene encoding the target product) are included in the culture medium, it is easy to efficiently culture E. coli in the culture vessel and easily produce a certain amount of the target product. Furthermore, if an expression method is adopted that suppresses the expression of proteins that E. coli itself possesses while efficiently expressing the target product, it is easy to obtain the target product expressed with high purity.

[0017] (3) Only one light irradiation unit is provided, and a single combined imaging unit that serves as both the first imaging unit and the second imaging unit is provided outside the culture vessel, and the fluorescence filter may be placed between the combined imaging unit and the culture vessel such that the image captured by the combined imaging unit simultaneously includes both the scattered light image and the fluorescence image.

[0018] In this case, a single combined imaging unit can be used for both the first and second imaging units, which simplifies the configuration and reduces component costs. Furthermore, since the fluorescence filter is positioned so that the image captured by the combined imaging unit simultaneously includes both a scattered light image and a fluorescence image, both scattered light images and fluorescence images can be appropriately acquired using a single combined imaging unit. Furthermore, since a single image captured by the dual-purpose imaging unit can simultaneously include both scattered light images and fluorescence images, it is possible to understand, for example, the relationship (correlation) between turbidity and fluorescence intensity.

[0019] (4) The dual-purpose imaging unit is positioned such that the optical center of the excitation light irradiated onto the interface is located at the center of the captured image, and the fluorescence filter may be positioned to block reflected light from the optical center from entering the captured image of the dual-purpose imaging unit.

[0020] In this case, since the optical center of the excitation light is located at the center of the captured image, the scattered light image and the fluorescence image can be evenly and well-balancedly included in one captured image captured by the combined imaging unit. Moreover, by using a fluorescence filter, it is possible to block the reflected light itself from the optical center from directly entering the captured image. Therefore, it is possible to suppress the entry of strong light into the captured image, and it is possible to suppress the occurrence of inconveniences such as white streaks in the scattered light image and the fluorescence image. As a result, for example, it is possible to obtain a scattered light image and a fluorescence image in which changes in color information such as RGB (red, green, blue), changes in brightness such as gradation, and changes in luminance clearly appear, and it is possible to measure turbidity and fluorescence intensity with high accuracy.

[0021] (5) The measurement unit may use, as a data acquisition region, a region located at the same distance from the optical center among the scattered light image and the fluorescence image included in the captured image of the combined imaging unit, extract partial images of the scattered light image and the fluorescence image within the data acquisition region, and measure the turbidity of the culture solution and the fluorescence intensity of the target product substance.

[0022] In this case, by extracting partial images within the data acquisition region located at the same distance from the optical center, they are acquired as scattered light images and fluorescence images, so that both images can be acquired under the condition of the same light intensity. Therefore, based on the scattered light image and the fluorescence image acquired under the same conditions, it is possible to measure turbidity and fluorescence intensity.

[0023] [[ID=!1]] (6) The light irradiation unit may be arranged to irradiate the excitation light at an acute incident angle with respect to the inner surface of the culture vessel, and the combined imaging unit may be arranged such that the imaging axis is non-coaxial with respect to the optical axis of the excitation light reflected at a reflection angle corresponding to the incident angle.

[0024] In this case, since it is possible to suppress the direct entry of the excitation light reflected at the interface into the captured image obtained by the combined imaging unit, it is possible to obtain a clear and distinct scattered light image and fluorescence image.

[0025] (7) It may also include a storage unit that associates and stores the scattered light image captured by the first imaging unit and the fluorescence image captured by the second imaging unit.

[0026] In this case, since the scattered light image and the fluorescence image can be associated and stored in the storage unit, information such as the correlation between the turbidity state and the expression state of the target product substance can be grasped. Therefore, by accumulating this information, it is also possible to make inferences such as at what turbidity level the target product substance is expressed. As a result, the operation from culturing to the expression of the target product substance can be efficiently performed.

[0027] (8) An excitation filter that is disposed between the light irradiation unit and the culture vessel and permits the transmission of light in a wavelength range different from the wavelength range permitted by the fluorescence filter may be provided.

[0028] In this case, since it has an excitation filter, excitation light in a wavelength range suitable for the emission of fluorescence can be appropriately irradiated toward the culture vessel. In particular, since the wavelength range of the excitation filter and the wavelength range of the fluorescence filter can be made different, the overlap between the excitation spectrum and the fluorescence spectrum can be prevented, and a decrease in the contrast of the fluorescence image can be suppressed.

[0029] (9) A condenser lens that is disposed between the light irradiation unit and the culture vessel and condenses the excitation light on the interface between the inner surface of the culture vessel and the culture solution may be provided.

[0030] In this case, by using the condenser lens, the excitation light irradiated from the light irradiation unit can be condensed on the interface, and the scattered light image and the fluorescence image can be obtained more clearly.

[0031] (10) The apparatus comprises a holding member for holding the light irradiation unit, a first case housing the holding member inside, and a second case which is combined with the first case and holds the dual-purpose imaging unit and the fluorescence filter, wherein the first case and the second case each have a set surface that is in close contact with the outer surface of the culture container, and the light irradiation unit may be held by the holding member so as to be able to irradiate the culture container with the excitation light so as to be in contact with the set surface.

[0032] In this case, the light irradiation unit, dual-purpose imaging unit, fluorescence filter, etc., are incorporated into a single unit formed by combining the first and second cases, making the device more compact and improving ease of handling. In particular, the relative positions of the culture container, light irradiation unit, and dual-purpose imaging unit can be set in an appropriate position by simply setting the culture container so that its outer surface contacts the setting surface of the first and second cases. Therefore, excitation light can be accurately irradiated towards the interface between the inner surface of the culture container and the culture medium without any special positional adjustments, and scattered light images and fluorescence images can be acquired using the dual-purpose imaging unit.

[0033] (11) The holding member may be held so as to be displaceable relative to the first case.

[0034] In this case, by displacing the holding member relative to the first case, the irradiation angle of the light irradiation unit to the culture vessel can be easily fine-tuned.

[0035] (12) The second case may be provided with a first holding hole for detachably holding the multi-purpose imaging unit, and a second holding hole formed between the first holding hole and the set surface for detachably holding the fluorescence filter.

[0036] In this case, the first and second retaining holes can be used to easily attach, detach, and replace the dual-purpose imaging unit and fluorescence filter in the second case. In particular, the optimal dual-purpose imaging unit, fluorescence filter, etc. can be selected and used depending on the type of target product and culture medium, culture conditions, application, etc.

[0037] (13) The light irradiation unit irradiates the excitation light toward the interface between the culture medium on which the protein is expressed as the target product and the inner surface of the culture vessel, the second imaging unit captures an image of the fluorescence emitted by the expressed protein due to the irradiation of the excitation light and acquires it as the fluorescence image, and the measurement unit may measure the turbidity of the culture medium and the fluorescence intensity of the protein based on the scattered light image and the fluorescence image.

[0038] In this case, the turbidity of the culture medium can be measured at the necessary timings, and the fluorescence intensity of the protein can be measured continuously within the same process.

[0039] (14) The culture system according to the present invention comprises the measuring device, a shaking device having a shaking platform having a mounting surface on which the culture vessel is placed, and a shaking mechanism for shaking the shaking platform in a plane parallel to the mounting surface, and a control unit for controlling the measuring device and the shaking mechanism, wherein the control unit controls the shaking mechanism so that the culture vessel moves between a culture position spaced apart from the measuring device and a measurement position where the measuring device performs the measurement, and controls the shaking mechanism to shake the shaking platform at the culture position, and further controls the shaking mechanism to change the shaking conditions of the shaking platform based on the turbidity of the culture solution measured by the measuring unit and the fluorescence intensity of the target product.

[0040] According to the culture system of the present invention, during the culture process, or after the culture is completed, the culture vessel can be moved to a measurement position and the turbidity of the culture medium and the fluorescence intensity of the target product can be measured using a measuring device. In particular, the control unit, based on the measured turbidity of the culture medium and the fluorescence intensity of the target product, provides feedback control to the shaking mechanism to change the shaking conditions of the shaking platform if it determines that, for example, the culture or expression of the target product is insufficient. This allows the shaking of the culture vessel to be restarted with a different shaking pattern, thereby promoting culture and expression. Consequently, a certain amount of the target product can be reliably and efficiently obtained. [Effects of the Invention]

[0041] According to the present invention, the turbidity of the culture medium can be measured while the fluorescence intensity of the target product can be measured continuously in a series of steps, and the turbidity and fluorescence intensity measurements can be performed stably and accurately. [Brief explanation of the drawing]

[0042] [Figure 1] This is a longitudinal cross-sectional view showing a first embodiment of the measuring device according to the present invention. [Figure 2] Figure 1 is a perspective view of a culture system equipped with the measuring device shown. [Figure 3] Figure 1 is a side view of the measuring device as seen from the left and right directions. [Figure 4] Figure 1 is a perspective view of the measuring device as seen from the front. [Figure 5] Figure 1 is a perspective view of the measuring device, taken from the front and slightly below. [Figure 6] Figure 1 is a front view of the measuring device as seen from the front. [Figure 7] Figure 1 is a perspective view of the upper case, which makes up the storage case shown in Figure 1, viewed from below. [Figure 8]Figure 1 is a perspective view of the lower case, which makes up the storage case, as seen from above. [Figure 9] This is a perspective view showing the state after the fluorescent filter unit has been removed from the state shown in Figure 8. [Figure 10] Figure 1 is a perspective view of the protein measurement process, showing the protein as it passes through the upper case. [Figure 11] Figure 10 is a perspective view showing the relationship between the holding member and the excitation light filter. [Figure 12] Figure 11 is a perspective view of the retaining member as seen from the rear. [Figure 13] Figure 1 is an exploded perspective view of the fluorescence filter unit. [Figure 14] Figure 13 is a rearward perspective view of the retaining plate shown. [Figure 15] This is an example of an image (scattered light image, fluorescence image) captured with the dual-purpose imaging unit. [Figure 16] This is a longitudinal cross-sectional view of a measuring device showing a modified example of the first embodiment. [Figure 17] This is a longitudinal cross-sectional view showing a second embodiment of the measuring device according to the present invention. [Modes for carrying out the invention]

[0043] (First Embodiment) Hereinafter, a first embodiment of the measuring device according to the present invention will be described with reference to the drawings. As shown in Figures 1 and 2, the measuring device 1 is a device that can simultaneously measure the turbidity of the culture medium W in which the target product (hereinafter simply referred to as the target product) is expressed, and measure the fluorescence intensity of the target product. The target product refers to substances produced by microorganisms through processes such as fermentation and metabolism, and includes, for example, proteins, organic compounds produced by lactic acid bacteria through fermentation, peptides, etc. In this embodiment, protein is assumed to be the target product. Therefore, the measuring device 1 is assumed to be a protein measuring device capable of simultaneously measuring the turbidity of the culture medium W in which the protein is expressed and measuring the fluorescence intensity of the protein.

[0044] The culture medium W, which expresses the target substance, is contained in the culture vessel 2. The culture vessel 2 is shaken under predetermined culture conditions by a culture apparatus including a shaking platform (not shown). As a result, the culture medium W is cultured in the culture vessel 2, and its turbidity increases as the culture progresses. The measuring device 1 of this embodiment measures the turbidity of the culture medium W to understand the progress of the culture (culture state), and also measures the fluorescence intensity of the target substance to understand that a certain amount of the target substance has been obtained.

[0045] Furthermore, as shown in Figure 2, the culture system 150 of this embodiment includes a measuring device 1, a shaking device 151, and a control unit 15 that comprehensively controls the measuring device 1 and the shaking device 151. The shaking device 151 comprises a shaking platform 152 having a mounting surface 152a on which the culture vessel 2 is placed, and a shaking mechanism 153 that shakes the shaking platform 152 in a plane parallel to the mounting surface 152a. The culture vessel 2 is held using a holding member (not shown) in a state where it is placed directly on the mounting surface 152a or indirectly via a support member (not shown).

[0046] The shaking mechanism 153 is capable of arbitrarily moving the shaking platform 152 in two dimensions within a plane parallel to the mounting surface 152a, and is also capable of shaking the shaking platform 152 in various shaking patterns. For example, the shaking mechanism 153 is capable of shaking the shaking platform 152 in a linear motion pattern, a circular motion or elliptical motion pattern, a semicircular motion that repeats back and forth, a figure-eight motion pattern, and so on.

[0047] The control unit 15 controls the shaking mechanism 153 so that the culture vessel 2 moves between the culture position P1, which is separated from the measuring device 1, and the measurement position P2, where the measuring device 1 performs the measurement. It also controls the shaking platform 152 to shake at the culture position P1 in a predetermined shaking pattern.

[0048] In this embodiment, a glass Erlenmeyer flask is used as the culture vessel 2. Therefore, the culture vessel 2 has a flat bottom 3 and a conical side wall 4. The connection between the bottom 3 and the side wall 4 is an annular curved surface 5 that extends continuously around the entire circumference of the bottom 3. However, culture vessel 2 is not limited to an Erlenmeyer flask; various types of flasks such as pear-shaped flasks, round-bottom flasks, and shaking flasks may also be used. Furthermore, test tubes, Petri dishes, or cylindrical containers such as beakers may also be used as culture vessel 2.

[0049] Furthermore, in this embodiment, we will explain using an example in which Escherichia coli is used as the host cell, an expression vector whose expression is induced in a temperature-dependent manner is used, and the target substance is expressed using the method of the Escherichia coli cold shock expression system. Therefore, the culture medium W contains E. coli transformed by an expression vector (an expression vector containing a target gene encoding the target substance).

[0050] Furthermore, the expression vector contains a specific promoter for the E. coli cold shock gene, whose expression is induced when the E. coli culture temperature is low (e.g., 15°C). Specifically, the specific promoter is the lactose operon (lac I, Lac operator). The lactose operon controls the expression of the E. coli cold shock gene promoter. Furthermore, the lactose operon can be deregulated by reagents (expression inducers) such as IPTG (Isopropyl-β-D-thiogalactopyranoside), which allows for the expression of the E. coli cold shock gene promoter. However, specific promoters such as the lactose operon are not essential and do not need to be included in the expression vector.

[0051] As shown in Figure 1, the measuring device 1 comprises a light irradiation unit 10 positioned outside the culture vessel 2 and irradiating the culture vessel 2 with excitation light EL, an excitation filter 11 positioned between the light irradiation unit 10 and the culture vessel 2, a dual-purpose imaging unit 12 positioned outside the culture vessel 2 and acquiring scattered light images 16 (see Figure 15) and fluorescence images 17 (see Figure 15), respectively, a fluorescence filter 13 positioned between the dual-purpose imaging unit 12 and the culture vessel 2, and a housing case 14 that houses these components inside.

[0052] (Storage case) First, let's explain the storage case 14. As shown in Figures 3 to 6, the housing case 14 comprises an upper case (first case according to the present invention) 20 and a lower case (second case according to the present invention) 40 which is integrally assembled with the upper case 20, and is made of, for example, synthetic resin. The light irradiation unit 10 and excitation filter 11 are housed in the upper case 20 while being held by the holding member 30. The dual-purpose imaging unit 12 and fluorescence filter 13 are housed and held in the lower case 40.

[0053] The upper case 20 and the lower case 40 are stacked vertically and assembled as a single unit. In this embodiment, as shown in Figure 1, two mutually orthogonal directions in the horizontal plane (the plane parallel to the mounting surface 152a of the shaking platform 152 shown in Figure 2) are defined as the front-to-back direction L1 and the left-to-right direction L2. Of the front-to-back direction L1, the direction toward the culture vessel 2 is defined as the front FW, and the opposite direction is defined as the rear BK.

[0054] As shown in Figures 3 to 6, the upper case 20 is formed in a rectangular shape, with a length (width) in the front-to-back direction L1 and the left-to-right direction L2 being longer than its length (height) in the vertical direction. An upper setting surface (setting surface according to the present invention) 21 is formed on the front surface 20a of the upper case 20, which is in close contact with the outer surface of the culture container 2. The upper setting surface 21 is formed in the center of the front surface 20a of the upper case 20 in the left-right direction L2, and is formed to be three-dimensionally recessed toward the rear BK, corresponding to the shape of the conical side wall portion 4. This makes it possible to bring the outer surface of the side wall portion 4 of the culture container 2 into close contact with the upper setting surface 21.

[0055] As shown in Figures 1 and 7, the upper case 20 has an upper storage chamber 22 that opens downwards. Furthermore, the lower surface 20b of the upper case 20 has a stepped portion 23 that is recessed upwards and formed along the opening of the upper storage chamber 22. The stepped portion 23 is C-shaped so that, when the upper case 20 is viewed from below, it is located in the left-right direction L2 and rearward BK relative to the opening of the upper storage chamber 22.

[0056] As shown in Figures 1 to 6, the lower case 40 is formed in a rectangular shape, with its length (width) in the front-to-back direction L1 and the left-to-right direction L2 being longer than its length (height) in the vertical direction. In the illustrated example, the lower case 40 is formed so that its length in the vertical direction is shorter than that of the upper case 20, while its length in the left-to-right direction L2 is the same as that of the upper case 20. Furthermore, the lower case 40 is formed such that its length along the front-to-back direction L1 is shorter than that of the upper case 20. The lower case 40 is assembled such that its rear surface 40c is flush with the rear surface 20c of the upper case 20. As a result, the front surface 40a of the lower case 40 is shifted towards the rear BK side compared to the front surface 20a of the upper case 20.

[0057] As shown in Figures 8 and 9, a projection 41 that bulges upward is formed on the upper surface 40b of the lower case 40. The projection 41 is positioned to face the stepped portion 23 of the upper case 20 in the vertical direction and is formed in a C-shape in plan view corresponding to the stepped portion 23. As a result, when the upper case 20 and the lower case 40 are combined, as shown in Figure 1, the lower surface 20b of the upper case 20 and the upper surface 40b of the lower case 40 can be brought into contact with each other with the protruding portion 41 in contact with the stepped portion 23 from below. In particular, since the protruding portion 41 can be fitted inside the stepped portion 23, the upper case 20 and the lower case 40 can be combined integrally with high precision.

[0058] As shown in Figures 1, 8, and 9, the lower case 40 is provided with a first holding hole 42 for removably holding the dual-purpose imaging unit 12, and a second holding hole 43 for removably holding the fluorescence filter unit 60 including the fluorescence filter 13.

[0059] The second retaining hole 43 is formed to open above the lower case 40 and, when viewed from above, is rectangular in shape, longer in the left-right direction L2 than in the front-back direction L1. Furthermore, when viewed from above, the second retaining hole 43 is located inside the protrusion 41. A measuring hole 44 is formed in the lower case 40 in the portion located forward FW beyond the second retaining hole 43, penetrating the lower case 40 in the front-to-back direction L1. The measuring hole 44 is located in the center of the front surface 40a of the lower case 40 in the left-to-right direction L2, and is formed in a rectangular shape when viewed from the front FW, and is formed to open upward. As a result, the inside of the second retaining hole 43 is largely open towards the front FW through the measuring hole 44.

[0060] The first retaining hole 42 is formed in the lower case 40 in a portion located behind the second retaining hole 43 in the BK, and is formed to penetrate the lower case 40 in the front-rear direction L1. The first retaining hole 42 is formed to be located in the center of the lower case 40 in the left-right direction L2. As a result, the inside of the second retaining hole 43 opens towards the rear BK through the first retaining hole 42.

[0061] As shown in Figures 1 and 5, an auxiliary storage chamber 45 is formed in the lower case 40 in the portion located rear BK of the second retaining hole 43, with the chamber opening downwards. The auxiliary storage chamber 45 is formed in a rectangular shape, with the left-right direction L2 being longer than the front-to-back direction L1 when viewed from below. The inside of the auxiliary storage chamber 45 is in communication with the second retaining hole 43 through the first retaining hole 42.

[0062] As shown in Figures 4-6, 8, and 9, a lower setting surface (the setting surface according to the present invention) 46 is formed on the front surface 40a of the lower case 40, which is in close contact with the outer surface of the culture container 2. As a result, the second retaining hole 43 is positioned between the first retaining hole 42 and the lower setting surface 46.

[0063] The lower setting surface 46 has a first lower setting surface 46a which is formed to be three-dimensionally recessed toward the rear BK corresponding to the shape of the conical side wall portion 4 of the culture vessel 2, and a second lower setting surface 46b which is formed to be three-dimensionally recessed toward the rear BK corresponding to the shape of the curved portion 5 of the culture vessel 2. The lower setting surface 46 (the first lower setting surface 46a and the second lower setting surface 46b) is arranged in the left-right direction L2 with the measurement hole 44 in between. This makes it possible to bring the outer surfaces of the side walls 4 and curved surfaces 5 of the culture vessel 2 into close contact with the lower setting surface 46.

[0064] As described above, the storage case 14 (upper case 20 and lower case 40) is configured such that, as shown in Figures 1 and 2, the culture container 2 can be set in such a state that its outer surface is in close contact with the upper setting surface 21 and the lower setting surface 46. The position of the culture vessel 2 when it is in close contact with the upper setting surface 21 and the lower setting surface 46 is referred to as the measurement position P2.

[0065] (Light irradiation area) As shown in Figure 1, the light irradiation unit 10 irradiates excitation light EL towards the interface between the inner surface of the culture vessel 2 and the culture medium W. For example, an LED light source can be used as the light irradiation unit 10. However, the light irradiation unit 10 is not limited to an LED light source; other light sources can be used as long as they can irradiate light that includes the wavelength range of the excitation light EL.

[0066] The light irradiation unit 10 is positioned to irradiate excitation light EL diagonally downward and to irradiate the excitation light EL with the inner surface of the conical side wall portion 4 of the culture vessel 2 at an acute incident angle θ1. Specifically, the light irradiation unit 10 is housed in the upper housing chamber 22 of the upper case 20 while being held by the holding member 30. In particular, the light irradiation unit 10 is held by the holding member 30 so as to be able to irradiate excitation light EL toward the culture container 2, which is set at the measurement position P2, so as to be in contact with the upper setting surface 21 and the lower setting surface 46. The excitation light EL irradiated from the light irradiation unit 10 is irradiated toward the culture container 2 through the measurement hole 44 of the lower case 40.

[0067] (Excitation filter) As shown in Figure 1, the excitation filter 11 is held by the holding member 30 so as to be positioned between the light irradiation unit 10 and the culture vessel 2. The excitation filter 11 allows transmission of light in a specific wavelength range used as excitation light EL from the light irradiation unit 10. As the excitation filter 11, a known optical filter (bandpass filter) having a dielectric multilayer film and capable of wavelength separation can be suitably employed. In particular, the excitation filter 11 allows transmission of light in a wavelength range different from that permitted by the fluorescence filter 13.

[0068] In the illustrated example, the excitation filter 11 is a framed filter having an excitation filter body 11a having a dielectric multilayer film or the like, and a ring-shaped frame portion 11b surrounding the excitation filter body 11a around its entire circumference. The excitation filter body 11a is formed to have a diameter at least larger than the light irradiation portion 10. The excitation filter 11 is held in place by a retaining member 30 in a replaceable manner.

[0069] (Retaining member) As shown in Figures 1, 7, and 10, the holding member 30 holds the light irradiation unit 10 and the excitation filter 11 while positioned within the upper housing chamber 22 of the upper case 20. The retaining member 30 is made of, for example, synthetic resin and is formed in a block shape. The retaining member 30 is positioned inside the upper storage chamber 22 with its side wall surface 30b, which faces in the left-right direction L2, in contact with the inner wall surface of the upper storage chamber 22, and its front wall surface 30a facing diagonally downward FW.

[0070] In particular, the retaining member 30 is held in place by the upper case 20 using fixing screws 31, and is also held so as to be displaceable relative to the upper case 20. As shown in Figures 11 and 12, the side wall surface 30b of the retaining member 30 has screw holes 32 through which two fixing screws 31 are inserted. Furthermore, the rear wall surface 30c of the retaining member 30 has a nut storage hole 33 for which a nut (not shown) is housed. As a result, as shown in Figures 7 and 10, the retaining member 30 can be held in the upper storage chamber 22 by inserting the fixing screw 31 into the screw hole 32 from the outside of the upper case 20 in the left-right direction L2 and screwing it into the nut.

[0071] Furthermore, as shown in Figure 3, a screw insertion hole 34 is formed on the side of the upper case 20, penetrating the upper case 20 in the left-right direction L2. The screw insertion hole 34 is made large enough to allow two fixing screws 31 to be inserted so as to be displaceable in the vertical and front-back directions L1. The fixing screw 31 is screwed into the screw hole 32 via a blind cover 35 positioned on the side of the upper case 20 so as to cover the screw insertion hole 34. This makes it possible to change the orientation of the retaining member 30 by loosening the fixing screw 31, thereby displacing the retaining member 30 relative to the upper case 20. Specifically, by fine-tuning the angle at which the front wall surface 30a of the retaining member 30 faces diagonally downward and forward, it is possible to change the angle of the optical axis OA of the excitation light EL emitted by the light irradiation unit 10.

[0072] As shown in Figures 1, 7, and 10-12, a filter holding hole 36 is formed in the upper wall surface 30d of the holding member 30 for removably holding the excitation filter 11. The filter holding hole 36 is sized to allow the entire excitation filter 11, including the frame portion 11b, to be removably inserted from above. The excitation filter 11 is held inside the filter holding hole 36 by being fitted into it.

[0073] Furthermore, a filter hole 37 is formed in the front wall surface 30a of the holding member 30, penetrating the front wall surface 30a in the thickness direction and communicating with the inside of the filter holding hole 36. The filter hole 37 is formed in a circular shape in plan view, with a smaller diameter than the frame portion 11b. As a result, the excitation filter 11 held in the filter holding hole 36 has its excitation filter body 11a exposed to the outside through the filter hole 37.

[0074] Furthermore, a light source holding hole 38 is formed in the rear wall surface 30c of the holding member 30, penetrating the rear wall surface 30c in the thickness direction and communicating with the inside of the filter holding hole 36. The light source holding hole 38 is formed in a circular shape in plan view, with a diameter larger than the excitation filter body 11a and a diameter smaller than the case of the light irradiation unit 10. As shown in Figures 1 and 10, the light irradiation unit 10 is held by the holding member 30 by fitting its tip into the light source holding hole 38. This makes it possible to position the light irradiation unit 10 and the excitation filter 11 on the optical axis OA of the light irradiation unit 10 using the holding member 30. Therefore, the excitation light EL emitted from the light irradiation unit 10 and transmitted through the excitation filter 11 can be irradiated toward the culture vessel 2.

[0075] The light irradiation unit 10 is combined with a heat dissipation member 39 that includes multiple heat dissipation fins. The heat dissipation member 39 is housed together with the light irradiation unit 10 in the upper housing chamber 22. This allows the heat generated by the light irradiation unit 10 to be dissipated into the upper housing chamber 22 via the heat dissipation member 39. The heat in the upper housing chamber 22 is then dissipated to the outside through the measurement hole 44 of the lower case 40.

[0076] (Multipurpose imaging unit) As shown in Figure 1, the dual-purpose imaging unit 12 has the function of capturing an image of scattered light reflected at the interface between the inner surface of the culture vessel 2 and the culture medium W from the excitation light EL irradiated from the light irradiation unit 10, and acquiring it as a scattered light image 16 (see Figure 15), and the function of capturing an image of fluorescence emitted by the expressed target substance due to irradiation with the excitation light EL, and acquiring it as a fluorescence image 17 (see Figure 15). Therefore, the dual-purpose imaging unit 12 serves as both a first imaging unit for acquiring scattered light images 16 and a second imaging unit for acquiring fluorescence images 17, enabling the acquisition of both scattered light images 16 and fluorescence images 17, respectively.

[0077] The multi-purpose imaging unit 12 includes, for example, a flexible or rigid tubular imaging guide 50 and an imaging unit 51 provided at the tip of the imaging guide 50. The imaging unit 51 includes at least an objective lens (not shown) and an image sensor (not shown) that performs imaging through the objective lens. For example, a CMOS sensor or a CCD sensor can be used as the image sensor.

[0078] The dual-purpose imaging unit 12 is held in the lower case 40 by being inserted into a first holding hole 42 formed in the lower case 40. The dual-purpose imaging unit 12 is held in the first holding hole 42 with the imaging unit 51 facing towards the second holding hole 43. The base end of the imaging guide 50 is pulled out to the outside of the lower case 40. Inside the imaging guide 50, there are wiring cables (power lines, signal lines) (not shown) that are electrically connected to the image sensor, and the two captured images (scattered light image 16, fluorescence image 17) are output to the control unit 15 that controls the measuring device 1.

[0079] (Fluorescent filter) As shown in Figure 1, the fluorescence filter 13 is held in the lower case 40 so as to be located between the dual-purpose imaging unit 12 and the culture vessel 2. The fluorescence filter 13 transmits light in a wavelength range that includes the wavelength of fluorescence emitted by the target substance, and restricts the transmission of light in at least the wavelength range of the excitation light EL. As the fluorescence filter 13, a known optical filter (bandpass filter) having a dielectric multilayer film and capable of wavelength separation can be suitably employed. In the illustrated example, the fluorescent filter 13 is a framed filter having a fluorescent filter body 13a having a dielectric multilayer film or the like, and a ring-shaped frame portion 13b that surrounds the fluorescent filter body 13a all around.

[0080] (Fluorescence filter unit) As shown in Figures 8, 13, and 14, the fluorescent filter 13 is removably fitted as a fluorescent filter unit 60 into the second retaining hole 43 of the lower case 40. The fluorescent filter unit 60 comprises a fluorescent filter 13, a filter case 70, and a retaining plate 80. The filter case 70 is made of, for example, synthetic resin and is formed in a block shape. The filter case 70 has a constant thickness in the front-to-back direction L1 and is formed in a rectangular block shape that is longer in the left-to-right direction L2 than in the up-to-down direction.

[0081] The front wall surface 70a of the filter case 70 has a first recess 71 and a second recess 72 that are recessed toward the rear BK. The first recess 71 and the second recess 72 are formed in a circular shape when viewed from the front FW and are arranged to be aligned in the left-right direction L2. In this case, the first recess 71 and the second recess 72 are arranged to be evenly aligned in the left-right direction L2 with respect to the center of the left-right direction L2 of the filter case 70.

[0082] The diameter of the first recess 71 is slightly larger than the diameter of the frame portion 13b of the fluorescent filter 13. The diameter of the second recess 72 is the same as the diameter of the first recess 71. The depth of the first recess 71 is equal to the thickness of the fluorescent filter 13. In contrast, the depth of the second recess 72 is formed to be deeper than that of the first recess 71. This makes it possible to fit the fluorescent filter 13 into the first recess 71 from the front FW.

[0083] Furthermore, the filter case 70 has a first through-hole 73 and a second through-hole 74 that penetrate the filter case 70 in the front-to-back direction L1. The first through-hole 73 is formed in a circular shape when viewed from the front FW and is formed coaxially with the first recess 71. The diameter of the first through-hole 73 is smaller than the diameter of the first recess 71. As a result, an annular first stepped wall 75 facing the front FW is formed at the connection point between the first recess 71 and the first through-hole 73. Therefore, when the fluorescent filter 13 is fitted into the first recess 71, the position of the fluorescent filter 13 can be adjusted by bringing the frame portion 13b into contact with the first stepped wall 75.

[0084] Similarly, the second through-hole 74 is formed in a circular shape when viewed from the front FW and is formed coaxially with the second recess 72. The diameter of the second through-hole 74 is smaller than the diameter of the second recess 72. As a result, an annular second stepped wall 76 facing the front FW is formed at the connection point between the second recess 72 and the second through-hole 74.

[0085] The second recess 72 is used to incorporate an auxiliary optical filter to transmit or block light in a specific wavelength range as needed when acquiring the scattered light image 16. Therefore, the second recess 72 and the second through-hole 74 are not essential and may not be present.

[0086] The retaining plate 80 is assembled to the filter case 70 from the front FW in order to hold down the fluorescent filter 13 fitted into the first recess 71. Therefore, the retaining plate 80 prevents the fluorescent filter 13 from falling out of the first recess 71. The retaining plate 80 has a constant thickness in the front-to-back direction L1 and is formed to have the same external dimensions as the filter case 70. The retaining plate 80 has a first imaging hole 81 and a second imaging hole 82 that penetrate through the retaining plate 80 in the front-to-back direction L1.

[0087] The first imaging aperture 81 is circular in shape when viewed from the front FW and is formed coaxially with the first recess 71 and the first through-hole 73. The diameter of the first imaging aperture 81 is smaller than the diameter of the first recess 71. This prevents the fluorescence filter 13 from falling out of the first recess 71. Similarly, the second imaging aperture 82 is formed in a circular shape when viewed from the front FW, and is formed coaxially with the second recess 72 and the second through-hole 74. The diameter of the second imaging aperture 82 is smaller than the diameter of the second recess 72 and is the same diameter as the first imaging aperture 81.

[0088] Furthermore, the retaining plate 80 has a fitting projection 83 that protrudes toward the rear BK and extends continuously along the periphery of the second imaging hole 82. The fitting projection 83 is designed to fit inside the second recess 72. By fitting the fitting projection 83 into the second recess 72, the retaining plate 80 can be integrally assembled with the filter case 70 without any misalignment.

[0089] As described above, the entire fluorescence filter unit 60 can be fitted into the second retaining hole 43 formed in the lower case 40, as shown in Figures 1 and 8. This allows the fluorescence filter 13 to be positioned between the dual-purpose imaging unit 12 and the culture vessel 2. In particular, the dual-purpose imaging unit 12 held in the first holding hole 42 is positioned such that its imaging axis IA is aligned along the front-to-back direction L1, as shown in Figure 1, and that its imaging axis IA intersects the interface to which the excitation light EL is irradiated. Therefore, the dual-purpose imaging unit 12 is positioned such that its imaging axis IA is non-coaxial with respect to the optical axis OA of the excitation light EL reflected at a reflection angle θ2 corresponding to the incident angle θ1.

[0090] Furthermore, as shown in Figure 6, the combined imaging unit 12 is positioned such that its imaging axis IA penetrates the central portion in the left-right direction L2 between the first imaging hole 81 and the second imaging hole 82 formed in the retaining plate 80 that constitutes the fluorescence filter unit 60, in the front-back direction L1. As a result, when the fluorescence filter unit 60 is viewed from the front FW, the imaging unit 51 of the combined imaging unit 12 is positioned to fit inside the first imaging hole 81 and the second imaging hole 82, respectively.

[0091] Therefore, as shown in Figure 15, the dual-purpose imaging unit 12 can acquire a fluorescence image 17 through the fluorescence filter 13 and the first imaging aperture 81, and can also acquire a scattered light image 16 through the second imaging aperture 82. Thus, it is possible to acquire both the scattered light image 16 and the fluorescence image 17 using a single dual-purpose imaging unit 12. Furthermore, the fluorescence image 17 and the scattered light image 16 can be included in a single image 18 captured by the dual-purpose imaging unit 12. Furthermore, as shown in Figure 1, the dual-purpose imaging unit 12 is positioned such that the imaging axis IA intersects the interface to which the excitation light EL is irradiated. Therefore, as shown in Figure 15, the optical center LO of the excitation light EL irradiated to the interface can be positioned at the center of the captured image 18.

[0092] In particular, the fluorescence filter 13 is positioned such that the center of the fluorescence filter body 13a is offset to the left and right in the L2 direction relative to the imaging axis IA, as shown in Figure 6. In addition, the fluorescence filter 13 is positioned such that the frame portion 13b is located on the imaging axis IA. As a result, as shown in Figure 15, a portion of the frame portion 13b is intentionally included in the captured image 18. In this way, the fluorescence filter 13 uses the frame portion 13b to block strong reflected light from the optical center LO from entering the captured image 18.

[0093] (Control Unit) As shown in Figure 1, the control unit 15, which comprehensively controls the measuring device 1 and the shaking device 151, is connected to the light irradiation unit 10 and the combined imaging unit 12, and controls their operation. For example, the control unit 15 controls the irradiation timing and irradiation time of the light irradiation unit 10, as well as the imaging timing of the combined imaging unit 12. Furthermore, the control unit 15 includes at least a memory unit (storage unit according to the present invention) 90 that stores the scattered light image 16 and the fluorescence image 17 captured by the combined imaging unit 12, and a measurement unit 91 that measures the turbidity of the culture medium W and the fluorescence intensity of the target substance based on the scattered light image 16 and the fluorescence image 17.

[0094] In this embodiment, since one captured image 18 includes a scattered light image 16 and a fluorescence image 17, the memory unit 90 stores the scattered light image 16 and the fluorescence image 17 in association with each other. As shown in Figure 15, the measurement unit 91 defines the region located at the same distance from the optical center LO as the data acquisition region R among the scattered light image 16 and fluorescence image 17 included in the captured image 18, extracts partial images of the scattered light image 16 and fluorescence image 17 within the data acquisition region R, and measures the turbidity of the culture medium W and the fluorescence intensity of the target substance from these partial images.

[0095] Furthermore, the control unit 15 provides feedback control to the shaking mechanism 153 to change the shaking conditions of the shaking platform 152 based on the turbidity of the culture medium W and the fluorescence intensity of the target substance measured by the measurement unit 91.

[0096] (Operation of the measuring device and culture system) Next, we will describe how to measure the turbidity of the culture medium W and the fluorescence intensity of the expressed target substance using the measuring device 1 and culture system 150 configured as described above. This will include the cultivation of E. coli and the expression of the target substance.

[0097] First, E. coli containing the expression vector is added to the culture medium W contained in the culture vessel 2. Next, cultivation is performed at the culture position P1 shown in Figure 2 using the shaking device 151. Specifically, while maintaining a predetermined culture temperature (e.g., 37°C), the shaking mechanism 153 shakes the shaking platform 152 in a predetermined shaking pattern. This agitates and stimulates the culture medium W, allowing the E. coli to be cultured. The turbidity of the culture medium W tends to increase as the E. coli culture progresses.

[0098] When the culture is performed using the shaking device 151 until certain conditions (e.g., the time spent shaking the culture vessel 2, the number of shakes, etc.) are met, the shaking of the shaking platform 152 and the culture vessel 2 is temporarily stopped. Next, the shaking mechanism 153 is used to move the culture vessel 2 from the culture position P1 and set it at the measurement position P2 shown in Figures 1 and 2. This allows the outer surface of the culture vessel 2 to be in close contact with the upper setting surface 21 of the upper case 20 and the lower setting surface 46 of the lower case 40, and the relative positional relationship between the culture vessel 2 and the light irradiation unit 10 and the combined imaging unit 12 can be set to an appropriate positional relationship.

[0099] Next, light is irradiated from the light irradiation unit 10. This allows excitation light EL to be irradiated toward the culture vessel 2 through the excitation filter 11, and also toward the interface between the inner surface of the culture vessel 2 and the culture medium W. As a result, a portion of the excitation light EL is reflected at the interface and becomes scattered light.

[0100] Therefore, by using the dual-purpose imaging unit 12, an image of the scattered light reflected at the interface can be captured and obtained as the scattered light image 16 shown in Figure 15. At this time, the dual-purpose imaging unit 12 also captures a fluorescence image 17, but since the target substance has not yet been expressed at this stage, it is different from the fluorescence image 17 that was originally intended to be obtained. The captured image 18 obtained by the dual-purpose imaging unit 12 is output to the control unit 15 and stored in the memory unit 90. Furthermore, the measurement unit 91 measures the turbidity of the culture medium W based on the acquired scattered light image 16. Specifically, the measurement unit 91 measures the turbidity based on the light intensity, including brightness and luminance, of the scattered light image 16, or on changes in the RGB color information of the scattered light image 16.

[0101] As a result, the culture status of E. coli can be determined based on the measured turbidity, and it is possible to determine (estimate) whether a certain amount of E. coli has been cultured. Therefore, after confirming that the E. coli culture has been carried out appropriately, the expression of the target substance can be induced by cold shock or other means.

[0102] On the other hand, if the turbidity measurement results indicate that the E. coli culture is insufficient, the control unit 15 performs feedback control to continue the E. coli culture. In this case, the control unit 15 uses the shaking mechanism 153 to move the culture container 2 from the measurement position P2 to the culture position P1 shown in Figure 2. Furthermore, the control unit 15 provides feedback control to the shaking mechanism 153 to change the shaking conditions of the shaking platform 152. This allows the shaking mechanism 153 to restart shaking the shaking platform 152 and the culture container 2 under different conditions than those previously used. In particular, because different shaking conditions are used, it is possible to promote culture while applying a different stimulus to the culture medium W.

[0103] After shaking the mixture again, the turbidity of the culture medium W is measured again at measurement position P2 using the measuring device 1. This process of culturing and turbidity measurement is repeated until the E. coli culture is properly established. Therefore, the culture can be carried out efficiently. If the E. coli culture is deemed sufficient based on these results, the process of expressing the target substance by inducing cold shock is performed.

[0104] In this case, the culture vessel 2 is maintained under constant low temperature conditions (approximately 15°C), and a reagent such as IPTG is added to the culture vessel 2. By adding this reagent, the control of the lactose operon contained in the expression vector can be released, and a specific promoter whose expression is induced when low temperature conditions are met can be expressed. On the other hand, the expression of proteins possessed by E. coli itself can be suppressed. As a result, the target substance can be expressed efficiently and with high purity.

[0105] Next, after the target substance has been expressed, the culture container 2 is set again to the measurement position P2 shown in Figures 1 and 2. Furthermore, excitation light EL is irradiated from the light irradiation unit 10 toward the interface between the inner surface of the culture container 2 and the culture medium W. As a result, the expressed target substance emits fluorescence due to the irradiation of the excitation light EL. Specifically, the target substance absorbs the light energy of the excitation light EL and transitions to an excited state, and then transitions to the ground state while emitting fluorescence.

[0106] Therefore, the dual-purpose imaging unit 12 can be used to capture an image of the fluorescence emitted by the target substance and obtain it as the fluorescence image 17 shown in Figure 15. The fluorescence image 17 obtained by the dual-purpose imaging unit 12 is output to the control unit 15 and stored in the memory unit 90. Furthermore, the measurement unit 91 measures the fluorescence intensity of the target substance based on the obtained fluorescence image 17. Specifically, the measurement unit 91 measures the fluorescence intensity based on the light intensity including brightness and luminance of the fluorescence image 17, or changes in the color information of the RGB colors of the fluorescence image 17.

[0107] In particular, since the fluorescent filter 13 is placed between the dual-purpose imaging unit 12 and the culture vessel 2, it is possible to block at least the wavelength range of the excitation light EL reflected by the culture vessel 2, thereby preventing the excitation light EL from reaching the dual-purpose imaging unit 12. As a result, noise caused by the excitation light EL can be removed from the acquired fluorescence image 17, and a high-precision fluorescence image 17 can be obtained. Consequently, the fluorescence intensity of the target substance can be measured by the measurement unit 91 based on the fluorescence image 17, and it is possible to determine whether a certain amount of the target substance has been obtained.

[0108] Furthermore, when capturing a fluorescence image 17 using the dual-purpose imaging unit 12, a scattered light image 16 can also be captured, as shown in Figure 15. Therefore, a single captured image 18 can include both the scattered light image 16 and the fluorescence image 17. Accordingly, the memory unit 90 can store the scattered light image 16 and the fluorescence image 17 in association.

[0109] Furthermore, if the measurement of fluorescence intensity indicates that the expression of the target substance is insufficient, the control unit 15 provides feedback control to the shaking mechanism 153 to restart shaking of the culture vessel 2 in order to further promote expression. In this case, the control unit 15 uses the shaking mechanism 153 to move the culture vessel 2 from the measurement position P2 to the culture position P1 shown in Figure 2. Furthermore, the control unit 15 provides feedback control to the shaking mechanism 153 to change the shaking conditions of the shaking platform 152. This allows the shaking of the culture vessel 2 to be restarted under different conditions than those previously used. In particular, because different shaking conditions are used, a different stimulus can be applied to the culture medium W. This can promote the expression of the target substance.

[0110] After shaking the culture medium again, the fluorescence intensity of the target substance is measured again at measurement position P2 using the measuring device 1. In this way, the shaking of the culture medium W and the measurement of fluorescence intensity are repeated until a suitable amount of the expressed target substance is obtained. Therefore, a certain amount of the target substance can be obtained efficiently.

[0111] As a result, it is possible to measure the turbidity of the culture medium W at the necessary timing while simultaneously measuring the fluorescence intensity of the target substance in a continuous sequence. Unlike conventional light-transmitting types, this method measures the turbidity of the culture medium W based on scattered light reflected at the interface between the inner surface of the culture vessel 2 and the culture medium W. Therefore, it is less susceptible to influences such as the concentration of the culture medium W or foaming at the surface of the culture medium W. Consequently, the turbidity of the culture medium W can be measured stably and accurately, making it easier to accurately understand the culture state of the culture medium W containing the target substance. Furthermore, since the fluorescence intensity of the target substance can be measured without removing the culture medium W from the culture vessel 2, contamination of the culture medium W can be prevented, and fluorescence intensity can be measured with high accuracy.

[0112] Therefore, according to the measuring device 1 of this embodiment, it is possible to continuously measure the fluorescence intensity of the target substance while simultaneously measuring the turbidity of the culture medium W in a single process, and to measure turbidity and fluorescence intensity stably and accurately.

[0113] Furthermore, according to the measuring device 1 of this embodiment, since scattered light images 16 and fluorescence images 17 are captured using a single multi-purpose imaging unit 12, there is no need to use two imaging units. Therefore, the configuration can be simplified and component costs can be reduced. Moreover, since the scattered light image 16 and fluorescence image 17 can be simultaneously included in a single image 18 captured by the multi-purpose imaging unit 12, it is possible to understand, for example, the relationship (correlation) between turbidity and fluorescence intensity.

[0114] Furthermore, as shown in Figure 15, since the optical center LO of the excitation light EL is located at the center of the captured image 18, the scattered light image 16 and the fluorescence image 17 can be included evenly and in a balanced manner in a single captured image 18 captured by the combined imaging unit 12. Furthermore, by utilizing the frame portion 13b of the fluorescence filter 13, it is possible to block the reflected light (excitation light EL) from the optical center LO from directly entering the captured image 18. Therefore, it is possible to suppress strong light from entering the captured image 18, and to suppress problems such as overexposure in the scattered light image 16 and fluorescence image 17. As a result, it is possible to obtain scattered light image 16 and fluorescence image 17 in which changes in color information of RGB colors, changes in brightness such as gradients, changes in luminance, etc. are clearly displayed, and turbidity measurement and fluorescence intensity measurement can be performed with high accuracy.

[0115] Furthermore, the measurement unit 91 extracts a partial image within the data acquisition region R located at the same distance from the optical center LO, thereby acquiring it as a scattered light image 16 and a fluorescence image 17. This allows both images to be acquired under equivalent light intensity conditions. Consequently, turbidity and fluorescence intensity can be measured based on the scattered light image 16 and fluorescence image 17 acquired under equivalent conditions. Furthermore, as shown in Figure 1, the dual-purpose imaging unit 12 is positioned such that the imaging axis IA is non-coaxial with respect to the optical axis OA of the excitation light EL reflected at a reflection angle θ2 corresponding to the incident angle θ1. Therefore, it is possible to suppress the direct inclusion of the excitation light EL reflected at the interface into the captured image 18 shown in Figure 15, thereby enabling the acquisition of a clear and sharp scattered light image 16 and fluorescence image 17.

[0116] Furthermore, since the scattered light image 16 and the fluorescence image 17 can be associated and stored in the memory unit 90, information such as the correlation between turbidity levels and the expression state of the target substance can be grasped. Therefore, by accumulating this information, it is possible to make predictions, for example, about what level of turbidity will cause the target substance to be expressed. As a result, the process from culturing to the expression of the target substance can be carried out efficiently.

[0117] Furthermore, since it has an excitation filter 11, excitation light EL in a wavelength range suitable for fluorescence emission can be appropriately irradiated toward the culture vessel 2. In particular, since the wavelength range of the excitation filter 11 and the wavelength range of the fluorescence filter 13 can be made different, overlap between the excitation spectrum and the fluorescence spectrum can be prevented, and a decrease in the contrast of the fluorescence image 17 can be suppressed.

[0118] Furthermore, since the light irradiation unit 10, excitation filter 11, dual-purpose imaging unit 12, fluorescence filter 13, etc. are incorporated into a single housing case 14 formed by combining the upper case 20 and the lower case 40, the device can be made more compact and its handling can be improved. In particular, by simply setting the culture container 2 so that its outer surface contacts the upper setting surface 21 of the upper case 20 and the lower setting surface 46 of the lower case 40, the relative positional relationship of the culture container 2, the light irradiation unit 10, and the combined imaging unit 12 can be set to an appropriate position. Therefore, without performing any special positional adjustments, excitation light EL can be accurately irradiated toward the interface between the inner surface of the culture container 2 and the culture medium W, and scattered light images 16 and fluorescence images 17 can be acquired using the combined imaging unit 12.

[0119] Furthermore, by loosening the fixing screw 31, the holding member 30 can be displaced relative to the upper case 20, making it easy to fine-tune the irradiation angle of the light irradiation unit 10 to the culture container 2.

[0120] Furthermore, the first retaining holes 42 and the second retaining holes 43 allow for easy attachment, detachment, and replacement of the dual-purpose imaging unit 12 and the fluorescent filter 13 to the lower case 40. In particular, the optimal dual-purpose imaging unit 12, fluorescent filter 13, etc., can be selected and used depending on the type of target substance and culture medium W, culture conditions, application, etc. Furthermore, since the fluorescent filter 13 can also be replaced using the filter holding holes 36, the optimal fluorescent filter 13 can be selected and used.

[0121] (Modified version of the first embodiment) In the first embodiment described above, for example, as shown in Figure 16, a focusing lens 100 for focusing the excitation light EL may be placed between the light irradiation unit 10 and the culture vessel 2. In the illustrated example, the focusing lens 100 is provided on the holding member 30 so as to be positioned on the optical axis OA of the excitation light EL between the excitation filter 11 and the culture vessel 2. In this case, by using the focusing lens 100, the excitation light EL irradiated from the light irradiation unit 10 can be focused onto the interface between the inner surface of the culture vessel 2 and the culture medium W, making it possible to acquire the scattered light image 16 and the fluorescence image 17 more clearly.

[0122] (Second Embodiment) Next, a second embodiment of the measuring device according to the present invention will be described with reference to the drawings. In the second embodiment, the same reference numerals are used for parts that are the same as those in the first embodiment, and their descriptions are omitted.

[0123] In the first embodiment, a single multi-purpose imaging unit 12 was used to acquire both a scattered light image 16 and a fluorescence image 17. However, in this embodiment, the scattered light image 16 is acquired by the first imaging unit, and the fluorescence image 17 is acquired by the second imaging unit.

[0124] As shown in Figure 17, the measuring device 110 of this embodiment includes two storage cases. Specifically, it includes a first storage case 120 and a second storage case 130, which are configured similarly to the storage case 14 of the first embodiment. Each of the first and second containment cases 120 and 130 is equipped with a light irradiation unit 10 and an excitation filter 11 held by a holding member 30. Therefore, the measuring device 110 is equipped with two light irradiation units 10, and is capable of irradiating excitation light EL from each light irradiation unit 10 toward the interface between the inner surface of the culture container 2 and the culture medium W.

[0125] In the lower case 40 that constitutes the first housing case 120, a first imaging unit 121 for acquiring scattered light images 16 is held via a first holding hole 42, instead of the multi-purpose imaging unit 12. The first imaging unit 121 acquires scattered light images 16 only through the second imaging hole 82 of the retaining plate 80 that constitutes the fluorescence filter unit 60, which is held in the second holding hole 43 of the lower case 40.

[0126] In contrast, the lower case 40 constituting the second housing case 130 holds a second imaging unit 131 for acquiring fluorescence images 17 via a first holding hole 42, instead of the multi-purpose imaging unit 12. The second imaging unit 131 acquires fluorescence images 17 only through the fluorescence filter 13 of the fluorescence filter unit 60 held in the second holding hole 43 of the lower case 40 and the first imaging hole 81.

[0127] Even with the measuring device 110 configured in this way, a scattered light image 16 can be acquired using the first imaging unit 121, and a fluorescence image 17 can be acquired using the second imaging unit 131. Therefore, the same effects and advantages as those of the measuring device 1 of the first embodiment can be achieved.

[0128] In the second embodiment, the system may be configured to include one light irradiation unit 10, a first imaging unit 121, and a second imaging unit 131. The first imaging unit 121 may acquire a scattered light image 16 due to excitation light EL irradiated from a common light irradiation unit 10, and the second imaging unit 131 may acquire a fluorescence image 17 caused by the excitation light EL.

[0129] Although embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. Embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. Embodiments and their modifications include, for example, those that can be easily imagined by those skilled in the art, those that are substantially the same, and those that are equivalent.

[0130] For example, in each of the above embodiments, if the fluorescence intensity of the target substance cannot be determined based on the fluorescence image 17, it is possible that the E. coli culture was insufficient. In this case, the E. coli culture may be repeated, and the same procedure may be repeated to obtain the scattered light image 16 and fluorescence image 17. In particular, since the fluorescence intensity can be measured without removing the culture medium W from the culture vessel 2, it is also possible to perform feedback control such as repeating the culture as needed.

[0131] Furthermore, in the above embodiment, the expression vector does not need to include a specific promoter such as a lactose operon. In this case, the target substance can be expressed without adding reagents when the culture temperature of E. coli is set to a low temperature (approximately 15°C). Furthermore, in the above embodiment, we have described an example where an expression vector whose expression is induced in a temperature-dependent manner is used, and the target substance is expressed using the E. coli cold shock expression system method, but the invention is not limited to this case. Other expression vectors can be used as long as the target substance can be expressed.

[0132] Furthermore, the target substance can be anything that at least emits fluorescence and is subject to fluorescent staining; it is not limited to any specific substance. For example, a typical protein that is subject to fluorescent staining can be used as the target substance. In addition, other biomolecules that are subject to fluorescent staining, such as nucleic acids, cations, chitin, cellulose, and AT regions, can also be used as target substances.

[0133] Furthermore, although the above embodiments have described the case in which a triangular flask is used as the culture vessel 2, as mentioned earlier, other shapes of flasks, test tubes, petri dishes, beakers, etc. may be used as the culture vessel 2. When the culture vessel is changed, the relative positional relationship between the light irradiation unit 10 and the combined imaging unit 12 (first imaging unit, second imaging unit) with respect to the culture vessel may be appropriately changed in accordance with the shape of the culture vessel. For example, when a cylindrical beaker is used as the culture vessel, the light irradiation unit and the combined imaging unit may be arranged so that they are horizontal to the peripheral wall surface of the beaker. Thus, the relative positions of the light irradiation unit and the combined imaging unit (first imaging unit, second imaging unit) may be appropriately changed depending on the type, shape, and intended use of the culture vessel.

[0134] Furthermore, the present invention includes the following embodiments. <1> A light irradiation unit is positioned outside a culture vessel containing a culture medium that expresses a target product having at least the function of emitting fluorescence, and irradiates excitation light toward the interface between the inner surface of the culture vessel and the culture medium, A first imaging unit is positioned outside the culture vessel and captures an image of the scattered light reflected by the excitation light at the interface, and acquires it as a scattered light image. A second imaging unit is positioned outside the culture vessel and captures an image of the fluorescence emitted by the expressed target product due to irradiation with the excitation light, and acquires it as a fluorescence image. A fluorescence filter is placed between the second imaging unit and the culture vessel, which transmits light in a wavelength range including the wavelength of the fluorescence and restricts the transmission of light in at least the wavelength range of the excitation light, A measuring device characterized by comprising a measuring unit that measures the turbidity of the culture medium and the fluorescence intensity of the target product based on the scattered light image and the fluorescence image. <2> <1> In the measuring device described above, The culture medium contains host cells transformed with an expression vector that expresses the target product. The light irradiation unit is a measuring device that irradiates the excitation light toward the interface between the inner surface of the culture vessel and the culture medium containing the host cells. <3> <1> or <2> In the measuring device described above, Only one light irradiation unit is provided. Outside the culture vessel, a single combined imaging unit is provided that serves as both the first imaging unit and the second imaging unit. The fluorescent filter is positioned between the dual-purpose imaging unit and the culture vessel such that the image captured by the dual-purpose imaging unit simultaneously includes both the scattered light image and the fluorescent image. <4> <3> In the measuring device described above, The multi-purpose imaging unit is positioned such that the optical center of the excitation light irradiated onto the interface is located at the center of the captured image. The fluorescent filter is arranged to block reflected light from the optical center from entering the captured image of the combined imaging unit, in the measuring device. <5> <4> In the measuring device described above, The measuring unit is a measuring device that, among the scattered light image and the fluorescence image included in the captured image of the combined imaging unit, defines the region located at the same distance from the optical center as the data acquisition region, extracts a partial image of the scattered light image and a partial image of the fluorescence image within the data acquisition region, and measures the turbidity of the culture medium and the fluorescence intensity of the target product. <6> <3> from <5> In any one of the measuring devices described above, The light irradiation unit is positioned to irradiate the inner surface of the culture vessel with the excitation light at an acute angle of incidence. The combined imaging unit is a measuring device in which the imaging axis is arranged such that it is non-coaxial with respect to the optical axis of the excitation light reflected at a reflection angle corresponding to the incidence angle. <7> <1> from <6> In any one of the measuring devices described above, A measuring device comprising a storage unit that stores the scattered light image captured by the first imaging unit and the fluorescence image captured by the second imaging unit in association with each other. <8> <1> from <7> In any one of the measuring devices described above, A measuring device comprising an excitation filter disposed between the light irradiation unit and the culture vessel, which allows the transmission of light in a wavelength range different from the wavelength range that the fluorescence filter allows to transmit. <9> <1> from <8> In any one of the measuring devices described above, A measuring device disposed between the light irradiation unit and the culture vessel, and equipped with a focusing lens that focuses the excitation light at the interface between the inner surface of the culture vessel and the culture medium. <10> <3> In the measuring device described above, A holding member for holding the light irradiation section, A first case that houses the aforementioned retaining member inside, The system comprises a second case which is combined with the first case and holds the multi-purpose imaging unit and the fluorescence filter, The first case and the second case each have a set surface formed therein that is in close contact with the outer surface of the culture vessel. The measuring device is such that the light irradiation unit is held by the holding member so as to be able to irradiate the excitation light toward the culture vessel which is set in contact with the set surface. <11> <10> In the measuring device described above, The holding member is held in a displaceable manner relative to the first case in the measuring device. <12> <10> or <11> In the measuring device described above, The measuring device is provided with a second case having a first holding hole for removably holding the multi-purpose imaging unit, and a second holding hole formed between the first holding hole and the set surface for removably holding the fluorescence filter. <13> <1> from <12> In any one of the measuring devices described above, The light irradiation unit irradiates the excitation light toward the interface between the culture medium, which expresses the protein as the target product, and the inner surface of the culture vessel. The second imaging unit captures an image of the fluorescence emitted by the expressed protein in response to the irradiation of the excitation light, and acquires it as the fluorescence image. The measurement unit is a measuring device that measures the turbidity of the culture medium and the fluorescence intensity of the protein based on the scattered light image and the fluorescence image. <14> <1> from <13> A measuring device described in any one of the following, A shaking device comprising: a shaking platform having a mounting surface on which the culture vessel is placed; and a shaking mechanism for shaking the shaking platform in a plane parallel to the mounting surface; The system comprises the measuring device and the control unit for controlling the shaking mechanism, The control unit controls the shaking mechanism so that the culture vessel moves between a culture position separated from the measuring device and a measurement position where the measuring device performs the measurement, and also controls the shaking mechanism so that the shaking platform shakes at the culture position. Furthermore, the control unit is characterized by feedback-controlling the shaking mechanism to change the shaking conditions of the shaking platform based on the turbidity of the culture medium measured by the measuring unit and the fluorescence intensity of the target product. [Explanation of Symbols]

[0135] R...Data acquisition area W…Culture solution θ1…Incidence angle θ2…reflection angle P1…Culture position P2…Measurement position EL…excitation light OA... Optical axis of excitation light 1, 110... Measuring device 2…Culture container 10...Light-irradiating section 11…Excitation filter 12…Multipurpose imaging unit 13…Fluorescent filter 15…Control Unit 16…Scattered light image 17…Fluorescence images 18… Captured image 20…Upper case (first case) 21…Setting surface of the upper case 30…Retaining member 40…Lower case (second case) 42...1st holding hole 43…Second holding hole 46…Setting surface of the lower case 90...Memory section (storage section) 91...Measuring part 100... Focusing lens 121...First Imaging Unit 131...Second Imaging Unit 150...Culture system 151... Shaking device 152... Shaking platform 152a... Mounting surface of the shaking platform 153...Shaking mechanism

Claims

1. It comprises at least one light irradiation unit positioned outside a culture vessel containing a culture medium that expresses a target product having the function of emitting fluorescence, and which irradiates excitation light toward the interface between the inner surface of the culture vessel and the culture medium, A first imaging unit is positioned outside the culture vessel and captures an image of the scattered light reflected by the excitation light at the interface, and acquires it as a scattered light image. A second imaging unit is positioned outside the culture vessel and captures an image of the fluorescence emitted by the expressed target product due to irradiation with the excitation light, and acquires it as a fluorescence image. A fluorescence filter is placed between the second imaging unit and the culture vessel, which transmits light in a wavelength range including the wavelength of the fluorescence and restricts the transmission of light in at least the wavelength range of the excitation light, A measuring device characterized by comprising a measuring unit that measures the turbidity of the culture medium and the fluorescence intensity of the target product based on the scattered light image and the fluorescence image.

2. In the measuring device according to claim 1, The culture medium contains host cells transformed with an expression vector that expresses the target product. The light irradiation unit is a measuring device that irradiates the excitation light toward the interface between the inner surface of the culture vessel and the culture medium containing the host cells.

3. In the measuring device according to claim 1 or 2, Only one light irradiation unit is provided. Outside the culture vessel, a single combined imaging unit is provided that serves both as the first imaging unit and the second imaging unit. The fluorescent filter is positioned between the dual-purpose imaging unit and the culture vessel such that the captured image of the dual-purpose imaging unit simultaneously includes both the scattered light image and the fluorescent image.

4. In the measuring device according to claim 3, The multi-purpose imaging unit is positioned such that the optical center of the excitation light irradiated onto the interface is located at the center of the captured image. The fluorescent filter is arranged to block reflected light from the optical center from entering the captured image of the combined imaging unit, in the measuring device.

5. In the measuring device according to claim 4, The measuring unit is a measuring device that, among the scattered light image and the fluorescence image included in the captured image of the combined imaging unit, defines the region located at the same distance from the optical center as the data acquisition region, extracts a partial image of the scattered light image and a partial image of the fluorescence image within the data acquisition region, and measures the turbidity of the culture medium and the fluorescence intensity of the target product.

6. In the measuring device according to claim 3, The light irradiation unit is positioned to irradiate the inner surface of the culture vessel with the excitation light at an acute angle of incidence. The combined imaging unit is a measuring device in which the imaging axis is arranged such that it is non-coaxial with respect to the optical axis of the excitation light reflected at a reflection angle corresponding to the incidence angle.

7. In the measuring device according to claim 1, A measuring device comprising a storage unit that stores the scattered light image captured by the first imaging unit and the fluorescence image captured by the second imaging unit in association with each other.

8. In the measuring device according to claim 1, A measuring device comprising an excitation filter disposed between the light irradiation unit and the culture vessel, which allows the transmission of light in a wavelength range different from the wavelength range that the fluorescence filter allows to transmit.

9. In the measuring device according to claim 1, A measuring device disposed between the light irradiation unit and the culture vessel, and equipped with a focusing lens that focuses the excitation light at the interface between the inner surface of the culture vessel and the culture medium.

10. In the measuring device according to claim 3, A holding member for holding the light irradiation section, A first case that houses the aforementioned retaining member inside, The system comprises a second case which is combined with the first case and holds the multi-purpose imaging unit and the fluorescence filter, The first case and the second case each have a set surface formed therein that is in close contact with the outer surface of the culture vessel. The measuring device is such that the light irradiation unit is held by the holding member so as to be able to irradiate the excitation light toward the culture vessel which is set in contact with the set surface.

11. In the measuring device according to claim 10, The holding member is held in a displaceable manner relative to the first case in the measuring device.

12. In the measuring device according to claim 10, The measuring device is provided with a second case having a first holding hole for removably holding the multi-purpose imaging unit, and a second holding hole formed between the first holding hole and the set surface for removably holding the fluorescence filter.

13. In the measuring device according to claim 1, The light irradiation unit irradiates the excitation light toward the interface between the culture medium, which expresses the protein as the target product, and the inner surface of the culture vessel. The second imaging unit captures an image of the fluorescence emitted by the expressed protein due to irradiation with the excitation light, and acquires it as the fluorescence image. The measurement unit is a measuring device that measures the turbidity of the culture medium and the fluorescence intensity of the protein based on the scattered light image and the fluorescence image.

14. The measuring device according to claim 1, A shaking device comprising: a shaking platform having a mounting surface on which the culture vessel is placed; and a shaking mechanism for shaking the shaking platform in a plane parallel to the mounting surface; The system comprises the measuring device and the control unit for controlling the shaking mechanism, The control unit controls the shaking mechanism so that the culture vessel moves between a culture position separated from the measuring device and a measurement position where the measuring device performs the measurement, and also controls the shaking mechanism so that the shaking platform shakes at the culture position. Furthermore, the control unit is characterized by feedback-controlling the shaking mechanism to change the shaking conditions of the shaking platform based on the turbidity of the culture medium measured by the measuring unit and the fluorescence intensity of the target product.

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