JP3813730B2 - Fluorescence measuring device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fluorescence measurement equipment, and more particularly to fluorescence measurement apparatus for measuring the fluorescence from such cells contained in a plurality of wells of a microplate.
[0002]
[Prior art]
As a conventional fluorescence measurement device, a fluorescence measurement device is known in which a sample is accommodated in a plurality of wells of a microplate and fluorescence from each sample is measured. For example, Japanese Patent Laid-Open No. 60-40955 discloses a plurality of branched optical fibers that branch from the main optical fiber in the middle and transmit light from a light source to a plurality of wells corresponding to a plurality of wells of a microplate, Fluorescence detection optical fibers bundled with a plurality of branched optical fibers, respectively, and the ends of the branch optical fibers and the fluorescence detection optical fibers correspond to the plurality of wells on the bottom surface side of the microplate. A fixed fluorescence measurement device is disclosed.
[0003]
[Problems to be solved by the invention]
However, in the fluorescence measuring apparatus disclosed in Japanese Patent Application Laid-Open No. 60-40955, when detecting fluorescence from a cell or the like, the fluorescence may not be sufficiently detected depending on the cell.
[0004]
In view of the above circumstances, an object of the present invention is to provide a fluorescence measuring apparatus that can sufficiently detect fluorescence from a sample regardless of the type of the sample.
[0005]
[Means for Solving the Problems]
As a result of intensive studies on the conventional fluorescence measuring apparatus, the present inventors have found that cells include adherent cells that adhere to the bottom of the well and suspension cells that float in the well. The present inventors completed the present invention by finding that there are cases where the fluorescence intensity of the fluorescent material cannot be detected sufficiently.
[0006]
That is, the invention according to claim 1 is a fluorescence measurement apparatus that irradiates a sample in a plurality of wells of a microplate from the bottom side of the microplate and detects fluorescence from the sample on the bottom side of the microplate. An excitation light generator that generates excitation light, a plurality of excitation light irradiation optical fibers that distribute excitation light incident from the excitation light generator according to a plurality of wells, and a sample in the plurality of wells. A fluorescence acquisition optical fiber for transmitting fluorescence, a fluorescence detection device for detecting fluorescence transmitted by the fluorescence acquisition optical fiber, and a plurality of wells provided on the bottom side of the microplate, each of which is excitation light A plurality of optical fiber bundle parts configured by bundling the emission end part of the irradiation optical fiber and the incident end part of the fluorescence acquisition optical fiber are arranged in the optical fiber band. Characterized in that it comprises a fiber moving mechanism for moving to increase or decrease the distance between the bottom surface of the part end face microplate.
[0007]
According to the present invention, when the excitation light from the excitation light generator is transmitted through the excitation light irradiation optical fiber and irradiated onto the samples in the plurality of wells of the microplate, the light from each sample passes through the fluorescence acquisition optical fiber. The fluorescence from the sample is detected by the fluorescence detection device. Here, when using a liquid containing adherent cells or floating cells as a sample, the end face of the optical fiber bundle portion is moved closer to or away from the bottom surface of the microplate in advance by a fiber moving mechanism according to the properties of these cells. In other words, fluorescence having an appropriate intensity is detected regardless of the properties of the cells.
[0008]
Moreover, it is preferable that the said fluorescence measuring apparatus is further provided with the reagent dispensing apparatus which dispenses a reagent in a some well, and the microplate vibration mechanism which vibrates a microplate.
[0009]
According to the present invention, when the reagent is dispensed into the well of the microplate by the reagent dispensing device, the fluorescence intensity may change due to the change in cell distribution in the sample due to the fluctuation of the liquid level, etc. If the microplate is vibrated by the microplate vibration mechanism before and after, the fluorescence intensity hardly changes even when the reagent is dispensed.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the fluorescence measuring apparatus of the present invention will be described with reference to the accompanying drawings. In all drawings, the same or corresponding components are denoted by the same reference numerals.
[0011]
FIG. 1 is a schematic diagram showing a basic configuration of a fluorescence measuring apparatus according to the present invention. As shown in FIG. 1, the fluorescence measuring device 1 uses a microplate 4. A plurality of wells 3 are formed on the surface 4b side of the microplate 4, for example, 96 wells 3 are formed, and these are arranged in 8 columns and 12 rows. The microplate 4 is made of an optically transparent material or a transparent material only at the bottom of the well 3 so that light incident from the bottom surface 4a can be transmitted. As the sample S, cells loaded with a fluorescent indicator (for example, Fura2) are used, and the cells are immersed in a cell culture medium or the like. The fluorescence measuring apparatus 1 irradiates the plurality of samples S with excitation light from the bottom surface 4a side of the microplate 4 in the dark box 2, and simultaneously measures the fluorescence emitted from the plurality of samples S.
[0012]
The fluorescence measurement device 1 includes an excitation light generator 5 that generates excitation light. The excitation light generator 5 extracts the excitation light having a desired wavelength by allowing the light emitted from the light source 7 to pass through the wavelength selection device 6 through the condenser lens 8. The wavelength selection device 6 includes a disc 11 in which a plurality (for example, two) of excitation light selection filters (for example, transmission wavelengths 340 nm and 380 nm) 9 and 10 having different transmission wavelength ranges are fitted along the circumference. . When the disk 11 is rotated around the center by the rotary motor 12, the light emitted from the light source 7 is collected by the condenser lens 8 and then passes through the excitation light selection filter 9 or 10 to be excited at a desired wavelength. Light is extracted. The wavelength selection device 6 is provided with a sensor system for identifying the positions of the excitation light selection filters 9 and 10. Based on a signal from the sensor system, a control signal CN1 is sent from the personal computer 24 to the controller 50, and the controller 50 controls the rotation of the rotary motor 33.
[0013]
From this pumping light generator 5, 96 pumping light irradiation optical fibers 13 for transmitting pumping light extend. The incident end portion 13 a of the excitation light irradiation optical fiber 13 is accommodated in the cylindrical tube 14 and connected to the excitation light generator 5. Also, as shown in FIGS. 2 and 3, the emission end portions 13b are distributed one by one in, for example, 8 columns and 12 rows corresponding to the wells 3 of the microplate 4, and the optical axes of these emission end portions 13b are: The direction is orthogonal to the bottom surface 4 a of the microplate 4. Around the emission end portions 13b of the plurality of excitation light irradiation optical fibers 13, there are six incident end portions 15a of the fluorescence acquisition optical fibers 15 for transmitting the light emitted from each sample S. Has been placed. One emission end 13 b corresponding to each of the plurality of wells 3 and the incident ends 15 a of the six fluorescence acquisition optical fibers 15 surrounding the wells 3 are accommodated in the cylindrical tube 16. Thus, the optical fiber bundle part 17 is comprised.
[0014]
As shown in FIG. 1, when the light emitted from the emission end 15b of the fluorescence acquisition optical fiber 15 passes through a fluorescence selection filter 18 (for example, an interference filter that transmits light of 400 nm or more), fluorescence is extracted. The fluorescence is detected by the photomultiplier tube 19. The fluorescence selection filter 18 and the photomultiplier tube 19 constitute a fluorescence detection device 20. The photomultiplier tube 19 is connected to a high voltage power source 21 and an independent amplifier circuit 22, and each amplifier circuit 22 is connected to a personal computer 24 via an analog data processing device 23. Therefore, the signal output from each photomultiplier tube 19 is amplified by the amplifier circuit 22, each amplified signal is converted into a digital value by the analog data processing device 23, and each data is collected and analyzed by the personal computer 24. Etc. are performed.
[0015]
With the above configuration, when the microplate 4 is disposed above the plurality of optical fiber bundle portions 17, the excitation light emitted from the plurality of optical fiber bundle portions 17 is irradiated to the plurality of wells 3 of the microplate 4, respectively. The light emitted from the plurality of samples S is incident on the incident end 15a of the fluorescence acquisition optical fiber 15 of the optical fiber bundle portion 17, and the light emitted from the fluorescence acquisition optical fiber 15 passes through the fluorescence selection filter 18. After that, fluorescence is extracted, and the fluorescence is detected by the photomultiplier tube 19.
[0016]
As shown in FIG. 2, the optical fiber bundle portion 17 is fixed to a flat fiber holder 25. The fiber holder 25 is fixed by penetrating through 8 rows and 12 rows of openings 25 a formed corresponding to the wells 3 of the microplate 4. As shown in FIG. 4, the fiber holder 25 is supported by a U-shaped guide member 26. As shown in FIG. 5, the guide member 26 includes a support plate 27 and a pair of support arms 28, 28 provided orthogonal to both sides of the support plate 27, and the support plate 27 and the pair of support arms. The fiber holder 25 is supported by 28 and 28. A ball screw 29 is screw-engaged with the support plate 27, and this ball screw 29 is connected to an elevation stepping motor 30. In this way, the fiber moving mechanism is configured.
[0017]
When the elevating stepping motor 30 is operated, the ball screw 29 is rotated and the guide member 26 is raised. Therefore, when the microplate 4 is disposed above the optical fiber bundle portion 17, the end surface 17 a of the optical fiber bundle portion 17 approaches the bottom surface 4 a of the microplate 4. Further, when the ball screw 29 is rotated in the reverse direction, the guide member 26 is lowered, and the end surface 17 a of the optical fiber bundle portion 17 is moved away from the bottom surface 4 a of the microplate 4.
[0018]
By the way, the cells used as the sample S include adherent cells attached to the bottom surface of the well 3 of the microplate 4 (see FIG. 6A) and floating cells suspended in the cell culture medium in the well 3 (see FIG. 6). 6 (b)). Since the adherent cells are in a fixed position in the well 3, there are cases where sufficient fluorescence cannot be obtained when the excitation light is irradiated from a distant position. On the other hand, since floating cells are floating in the cell culture solution, there are cases where sufficient fluorescence cannot be obtained when excitation light is irradiated from a close position.
[0019]
Therefore, when the adherent cells are irradiated with excitation light, sufficient fluorescence can be obtained by bringing the end surface 17a of the optical fiber bundle portion 17 close to the bottom surface 4a of the microplate 4 as shown in FIG. When irradiating the suspension cells with excitation light, sufficient fluorescence can be obtained by moving the end surface 17a of the optical fiber bundle portion 17 away from the bottom surface 4a of the microplate 4 as shown in FIG. For example, for adherent cells, a sufficient fluorescence intensity can be obtained when the distance between the end surface 17a of the optical fiber bundle portion 17 and the bottom surface 4a of the microplate 4 is 1 mm, but when the distance is 4 mm, the fluorescence intensity is 1 mm. Is approximately half of the fluorescence intensity obtained. Thus, by adjusting the distance between the end surface 17a of the optical fiber bundle portion 17 and the bottom surface 4a of the microplate 4 in advance according to the properties of the cells, sufficient intensity of fluorescence can be reliably obtained regardless of the properties of the cells. It is done. The position of the fiber holder 25 is controlled by sending a pulse input signal from the personal computer 24 to the lifting stepping motor 30 according to the properties of the cells.
[0020]
FIG. 8 is a schematic diagram showing the configuration of the fluorescence measuring apparatus 1 according to the present embodiment in more detail. As shown in FIG. 8, the fluorescence measuring device 1 is further provided with a reagent dispensing device 31 that dispenses a reagent to the plurality of wells 3 of the microplate 4 in the dark box 2, and the reagent dispensing device 31 includes: A reagent dispenser 33 is provided above the guide rails 32 in the dark box 2. The reagent dispenser 33 dispenses a reagent from a storage tank 34 that stores the reagent through a plurality of dispensing nozzles 35, and the dispensing nozzle 35 corresponds to the plurality of wells 3 of the microplate 4. They are arranged in 8 columns and 12 rows. Therefore, the reagent is simultaneously injected into each well 3 through these dispensing nozzles 35. The reagent dispenser 33 is suspended in the dark box 2 by a pair of guide rails 36 and 36 extending in parallel with the guide rails 32 and 32 above the guide rails 32 and 32. A reagent dispenser 37 having substantially the same configuration as the reagent dispenser 33 is suspended from the guide rails 36 and 36. The reagent dispensers 33 and 37 are controlled by the dispensing instruction signals B1 and B2 sent from the drive control unit 38 based on the input instruction signal CB sent from the personal computer 24, respectively. And dispensing timing are controlled.
[0021]
FIG. 9 shows changes in the distribution state of the adherent cells in the cell culture solution before and after dispensing the reagent from the dispensing nozzle 35 to the plurality of samples S of the microplate 4 using the reagent dispensing device 31 described above. FIG. 10 is a diagram of a change in the distribution state of adherent cells before and after reagent dispensing as viewed from the surface side of the microplate 4. As shown in these figures, a sufficient number of cells are irradiated with excitation light before reagent dispensing, whereas after reagent dispensing, the liquid level fluctuates and cells rise due to reagent dispensing. The number of cells irradiated with the excitation light decreases. Therefore, the fluorescence intensity may decrease after reagent dispensing. FIG. 11 shows an example of changes in fluorescence intensity when the sample S is irradiated with two or more wavelengths of excitation light of 340 nm and 380 nm on the adherent cell before and after the reagent dispensing, and fluorescence is emitted when the reagent is dispensed. It can be seen that the intensity decreases sharply. If the fluorescence intensity changes in this way, it is not preferable whether the change in fluorescence intensity is due to a change in the cell itself due to reagent dispensing, and the dynamics of the cell cannot be accurately grasped.
[0022]
Therefore, the fluorescence measuring apparatus 1 is provided with a microplate vibration mechanism so that the cells in the well 3 of the microplate 4 that vibrates the microplate 4 maintain a state of movement before and after reagent dispensing.
[0023]
FIG. 12 is a plan view showing an example of the microplate vibration mechanism, and FIG. 13 is a cross-sectional view taken along line XIII-XIII in FIG. As shown in these drawings, the microplate vibration mechanism 39 includes, for example, a rectangular movable frame 40 that can reciprocate along the guide rails 32, 32, and a pair of support columns 41 on the movable frame 40. , 41, the microplate holder 42 is supported. The microplate holder 42 is for supporting the microplate 4, and a rectangular opening 42 a is formed so that excitation light can be irradiated from the bottom surface 4 a side of the microplate 4. In addition, one through hole 43 is formed in the microplate holder 42, and a disk 44 is rotatably fitted in the through hole 43, and the rotary shaft 45 is spaced from the center C of the disk 44. , And the rotating shaft 45 is connected to the vibration motor 46. The vibration motor 46 is fixed on the movable frame 40.
[0024]
In operating such a microplate vibration mechanism 39, first, the end surfaces 17a of the plurality of optical fiber bundle portions 17 are arranged so as to face the corresponding wells 3 of the microplate 4, respectively. And the space | interval between the end surface 17a of the optical fiber bundle part 17 and the bottom face 4a of the microplate 4 is adjusted beforehand according to the property of a cell. When the vibration motor 46 is operated in such a state, the center C of the disk 44 rotates around the rotation shaft 45, and the microplate holder 42 vibrates accordingly. The microplate holder 42 vibrates in a direction along the guide rails 32 and 32 and also vibrates in a direction orthogonal to the guide rails 32 and 32.
[0025]
Here, the microplate holder 24 is vibrated so that light emitted from the sample S in the well 3 is incident on the end surface 17a of the same optical fiber bundle portion 17 during vibration. At this time, the state in which the cells in the well 3 of the microplate 4 have moved is maintained. For this reason, even if the reagent is dispensed from the reagent dispensers 33 and 36, the cells in the well 3 are moved before and after the reagent dispensing, so that the stationary cells in the well 3 start to move by the reagent dispensing. The change in the fluorescence intensity caused by is sufficiently prevented. For this reason, the change in fluorescence intensity due to reagent dispensing is due to changes in the cells themselves, and the dynamics of the cells immediately after reagent dispensing can be accurately grasped.
[0026]
A rotation speed control circuit 47 is connected to the vibration motor 46, and the rotation speed control circuit 47 controls the rotation speed of the rotary shaft 45. The rotation speed control circuit 47 is used to turn the vibration motor 46 ON / OFF. The switch to control is connected.
[0027]
Further, as shown in FIG. 8, a storage box 48 for storing a plurality of microplates 4 is provided on the side surface 2a of the dark box 2, and these microplates 4 are stored in a storage box by a microplate delivery mechanism (not shown). 48 is sequentially fed onto the guide rails 32 and 32. Further, on the side surface 2b opposite to the side surface 2a of the dark box 2, the microplate 4 fed from the guide rails 32 and 32 is accommodated in the storage box 49, and the microplate 4 is sequentially moved by a lift mechanism (not shown). Moved upwards. The microplate delivery mechanism and the lift mechanism are controlled by instruction signals CL and CS sent from the drive control unit 38 based on the input instruction signal CB sent from the personal computer 24, respectively, and along the guide rails 32 and 32. The movable frame 40 that can reciprocate is controlled by an instruction signal CC sent from the drive control unit 38 based on an input instruction signal CB sent from the personal computer 24.
[0028]
Next, an example of how to use the fluorescence measuring apparatus 1 having the above configuration will be briefly described.
[0029]
First, for example, ten microplates 4 are stored in the storage box 48 in advance. Then, the cell properties and measurement conditions such as whether the cells to be measured in advance are adherent cells or suspension cells, and whether the cells are easy to move by reagent dispensing are input to the personal computer 24.
[0030]
When the measurement start instruction is given in this way, the microplate delivery mechanism is operated by the control signal CL from the drive control unit 38, and the microplate 4 is moved to one end (initial position) of the guide rails 32, 32 in the dark box 2. Install in. Next, the microplate 4 is disposed above the optical fiber bundle portion 17 by moving the movable frame 40 according to the control signal CC from the drive control portion 38.
[0031]
Here, in accordance with the cell properties inputted in advance, the elevating stepping motor 30 is controlled so as to be about 4 mm for adherent cells and about 1 mm for floating cells, for example, and the bottom surface 4a of the microplate 4 and the optical fiber bundle. An interval between the end surface 17a of the portion 17 is set. For floating cells or adherent cells that are easily moved by reagent dispensing, the vibration motor 46 of the microplate vibration mechanism 39 is operated.
[0032]
Next, the reagent dispenser 36 is moved to a position facing the microplate 4 by a dispensing control signal CB from the personal computer 24. Next, by outputting a dispensing instruction signal B1 to the reagent dispensing device 36, a predetermined amount of the first reagent according to the dispensing instruction signal B1 is transferred from the reagent dispensing device 36 into the plurality of wells 3. Dispense all at once.
[0033]
When dispensing with the reagent dispenser 36 is completed, the reagent dispenser 36 is returned to the original position, and then the reagent dispenser 33 is moved to a position facing the microplate 4. Then, the second reagent is dispensed simultaneously into the plurality of wells 3 in the same manner as the dispensing of the first reagent.
[0034]
The microplates 4 that have been measured are moved one by one by moving the movable frame 40 by the control signal CC from the drive control unit 38 and by operating the lift mechanism by the control signal CS from the drive control unit 38. It is stored in the storage box 49. Hereinafter, the measurement is performed in the same manner for the unmeasured microplate 4.
[0035]
In addition, this invention is not limited to embodiment mentioned above. For example, the fiber moving mechanism is not limited to the configuration of the above embodiment as long as it is a mechanism that moves the end face 17a of the optical fiber holder 17 in a direction perpendicular to the bottom face 4a of the microplate 4.
[0036]
Moreover, as long as the microplate vibration mechanism 39 is a mechanism that vibrates the microplate 4, it may be a mechanism that moves the microplate 4 in the vertical direction or a mechanism that moves it only in one direction.
[0037]
【The invention's effect】
As described above, according to the present invention, when the excitation light from the excitation light generation device is transmitted through the optical fiber for excitation light irradiation and irradiated to the samples in the plurality of wells of the microplate, the light from each sample is fluorescent. The light is transmitted through the acquisition optical fiber, and the fluorescence from the sample is detected by the fluorescence detection device. Here, when using a liquid containing adherent cells or floating cells as a sample, the end face of the optical fiber bundle portion is moved closer to or away from the bottom surface of the microplate in advance by a fiber moving mechanism according to the properties of these cells. In other words, fluorescence having an appropriate intensity is sufficiently detected according to the properties of the cells. As a result, sufficient intensity of fluorescence can be detected from the sample regardless of the cell properties.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view showing a basic configuration of an embodiment of a fluorescence measuring apparatus according to the present invention.
FIG. 2 is a side view showing a positional relationship between an optical fiber bundle portion and a microplate.
3 is a cross-sectional view taken along line III-III in FIG.
FIG. 4 is a side view showing an example of a fiber moving mechanism.
5 is a plan view showing the fiber moving mechanism of FIG. 4; FIG.
6A is a cross-sectional view showing adherent cells adhering to the bottom surface of a well, and FIG. 6B is a cross-sectional view showing floating cells floating in a cell culture medium.
7A is a cross-sectional view showing an appropriate position of an optical fiber bundle portion when irradiating an adherent cell with excitation light, and FIG. 7B is a light when irradiating the suspension cell with excitation light. It is sectional drawing which shows the suitable position of a fiber bundle part.
FIG. 8 is a schematic diagram showing the configuration of the fluorescence measuring apparatus in FIG. 1 in more detail.
FIG. 9 shows changes in cell distribution before and after reagent dispensing.
10 is a diagram showing a change in cell distribution due to reagent dispensing in FIG. 9 as seen from the surface side of the microplate.
FIG. 11 is a graph showing changes in fluorescence intensity when an adherent cell is irradiated with excitation light of two types of wavelengths before and after reagent dispensing, respectively.
FIG. 12 is a plan view showing an example of a microplate vibration mechanism.
13 is a cross-sectional view taken along line XIII-XIII in FIG.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Fluorescence measuring apparatus, 3 ... Well, 4 ... Microplate, 4a ... Bottom surface, 5 ... Excitation light generator, 7 ... Light source (excitation light generator), 8 ... Condensing lens, 9,10 ... Excitation light selection filter (Excitation light generator), 13 ... Excitation light irradiation optical fiber, 13b ... Emission end, 15 ... Fluorescence acquisition optical fiber, 15a ... Incoming end, 17 ... Optical fiber bundle, 17a ... End face, 18 ... Fluorescence Selection filter (fluorescence detection device), 19 ... photomultiplier tube (fluorescence detection device), 25 ... fiber holder (fiber movement mechanism), 26 ... guide member (fiber movement mechanism), 27 ... support plate (fiber movement mechanism), 28 ... support arm (fiber movement mechanism), 29 ... ball screw (fiber movement mechanism), 30 ... elevating stepping motor (fiber movement mechanism), 33, 36 ... reagent dispenser (reagent dispensing device), 5 ... dispensing nozzle (reagent dispensing device), 36 ... guide rail (reagent dispensing device), 40 ... movable frame (microplate vibration mechanism), 41 ... support (microplate vibration mechanism), 42 ... microplate holder ( Microplate vibration mechanism), 44 ... Disk (microplate vibration mechanism), 45 ... Rotating shaft (microplate vibration mechanism), 46 ... Vibration motor (microplate vibration mechanism), 47 ... Rotational speed control circuit (microplate vibration) mechanism).

Claims (2)

In the fluorescence measurement device that irradiates the sample in the plurality of wells of the microplate from the bottom side of the microplate with excitation light, and detects fluorescence from the sample on the bottom side of the microplate,
An excitation light generator for generating the excitation light;
A plurality of excitation light irradiating optical fibers that distribute the excitation light incident from the excitation light generator according to the plurality of wells;
A fluorescence acquisition optical fiber that transmits fluorescence incident from a sample in the plurality of wells;
A fluorescence detection device that detects fluorescence transmitted by the fluorescence acquisition optical fiber;
A plurality of wells provided corresponding to the plurality of wells on the bottom surface side of the microplate and each configured by bundling an emission end of the excitation light irradiation optical fiber and an incidence end of the fluorescence acquisition optical fiber; A fiber moving mechanism for moving the optical fiber bundle part so as to increase or decrease the distance between the end face of the optical fiber bundle part and the bottom surface of the microplate;
A fluorescence measuring apparatus comprising:   The fluorescence measuring apparatus according to claim 1, further comprising: a reagent dispensing device that dispenses a reagent into the plurality of wells; and a microplate vibration mechanism that vibrates the microplate.

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