JP2012215609A - Culture medium observation microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture medium observation microscope device for objectively determining a deterioration level of a culture medium.SOLUTION: Illumination means radiates illumination light to the culture medium. Detection means detects transmission light, of the illumination light from the illumination means, from the culture medium, or reflection light, of the illumination light, from the culture medium. Calculation means calculates a ratio of the intensity of the transmission light of the culture medium or the intensity of the reflection light from the culture medium to light of a plurality of wavelength bands based on the transmission light or the reflection light detected by the detection means.

Description

  The present invention relates to a microscope apparatus for observing cultured cells for observing cells cultured in a medium.

  In the field of cell research, it is practiced to observe phenomena in cells while culturing the cells for a long time. Here, in order to culture the cells for a long period of time, the cells are cultured in a liquid or gel that gives the cells a growth environment called a medium. And since this culture medium deteriorates with the proliferation of a cell, it is necessary to replace | exchange a culture medium suitably. The following Patent Document 1 discloses that the color tone of a medium changes due to deterioration of a medium in which cells are cultured together with a culture apparatus for culturing living cells for a long period of time.

JP 2004-236547 A

  In order to appropriately manage the replacement time of the medium when the cells are cultured for a long time, it is necessary to objectively determine the degree of deterioration of the medium. However, conventionally, no device has been developed that can objectively detect the state of the medium based on the color of the medium. In addition, the state of the culture medium is not objectively detected by a microscope apparatus that performs fluorescence observation of living cells.

  An object of the present invention is to provide a microscope apparatus for observing cultured cells that can objectively determine the degree of deterioration of a culture medium.

The microscope apparatus for observing cultured cells of the present invention is a microscope apparatus for observing cells cultured in a culture medium, wherein the illumination means irradiates illumination light to the culture medium, and the illumination light from the illumination means. Detecting means for detecting the transmitted light of the culture medium based on or the reflected light from the culture medium based on the illumination light, and light in a plurality of wavelength bands based on the transmitted light or the reflected light detected by the detecting means And calculating means for calculating a ratio of transmitted light intensity of the medium or reflected light intensity from the medium.
According to this cultured cell observation microscope apparatus, the ratio of the transmitted light intensity of the culture medium or the reflected light intensity from the culture medium with respect to light in a plurality of wavelength bands is calculated, so the degree of deterioration of the culture medium is objectively determined. be able to.

  The illumination unit may be a transmitted light observation light source for observing the cell, and the calculation unit may calculate a ratio of transmitted light intensity of the culture medium based on the transmitted light.

  The detection means may be an image sensor for observing transmitted light that receives the transmitted light.

  The illumination means may be a fluorescence excitation light source that emits fluorescence excitation light for observing the cell, and the calculation means may calculate the ratio of the emitted light intensity based on the reflected light.

  The detection means may be an image sensor for fluorescence observation that receives fluorescence excited by the fluorescence excitation light.

  One of the plurality of wavelength bands may be a wavelength band included in a range of 550 nm to 600 nm, and another one of the plurality of wavelength bands may be a wavelength band included in a range of 600 nm or more.

  According to the cultured cell observation microscope apparatus of the present invention, the ratio of the transmitted light intensity of the culture medium or the reflected light intensity from the culture medium for light in a plurality of wavelength bands is calculated, so the degree of deterioration of the culture medium can be objectively determined. Can be determined.

The top view which shows the structure of the microscope apparatus for cultured cell observation of one Embodiment. The front view seen from the II-II line direction of FIG. The perspective view which shows the function of a disc unit. The figure which shows the structure of a laser unit. The top view of the confocal microscope which shows the state at the time of setting a sample. The top view which shows the structural example in the case of detecting the state of a culture medium based on the reflected light from a culture medium.

  Hereinafter, embodiments of the microscope apparatus for observing cultured cells according to the present invention will be described.

  FIG. 1 is a top view showing the configuration of a microscope apparatus for observing cultured cells of one embodiment, and FIG. 2 is a front view seen from the direction of II-II in FIG. FIG. 3 is a perspective view showing the function of the disk unit.

  As shown in FIG. 1 and FIG. 2, the cultured cell observation microscope apparatus of this embodiment controls the confocal microscope 100 for observing cultured cells, the confocal microscope 100, and the confocal microscope 100. And a control arithmetic device 200 that executes a calculation based on the image signal of the observation image obtained by the above.

  As shown in FIGS. 1 and 2, the confocal microscope 100 includes a protective housing 1 that protects the inside of the apparatus, a temperature control chamber 2 that is housed in the protective housing 1, and a laser unit 5 that outputs laser light. And a high-sensitivity CCD 6 (FIG. 1) for obtaining an observation image.

  The temperature control chamber 2 includes a disk unit 21 that receives the laser light from the laser unit 5, a tube lens 22 that converts the light passing through the disk unit 21 into parallel light, a mirror 23 that bends the optical path, and an objective lens 24 ( 2), a drive unit 25 (FIG. 2) for driving the objective lens 24 in the Z direction, a sample cassette 32 for storing a sample 31 such as a glass bottom dish set above the objective lens 24, and a sample cassette 32. Includes a drive unit 33a that can be attached and detached, and an XY drive mechanism 33 that drives the sample 31 in the XY directions via the drive unit 33a, and a halogen for transmission observation provided above the XY drive mechanism 33. A white light source 27 (FIG. 2) such as a light source, a condenser lens 28 (FIG. 2) for guiding the light from the white light source 27 to the sample 31, and a disk drive. The dichroic mirror 41 (FIG. 1) provided in the base 21, the relay lens 42 (FIG. 1) that receives the light bent by the dichroic mirror 41, and the mirror 43 (FIG. 1) that bends the light that has passed through the relay lens 42. ), A filter wheel 44 (FIG. 1) to which a plurality of filters having different transmission characteristics are attached, a motor 45 (FIG. 1) for rotating the filter wheel 44, and light passing through the filter of the filter wheel 44 The relay lens 46 (FIG. 1) is accommodated.

  As shown in FIGS. 1 to 3, the disk unit 21 includes a disk-shaped microlens disk 21a in which a number of microlenses 21L (FIG. 3) are arranged in a spiral pattern, and the same spiral shape as the microlens 21L. In this pattern, a disk-shaped pinhole disk 21b having the same number of pinholes 21h (FIG. 3) as the microlens 21L, a hub 21c for connecting the microlens disk 21a and the pinhole disk 21b, and a microlens disk 21a and a motor 21d for rotating the pinhole disk 21b.

  The microlens 21L disposed on the microlens disk 21a and the pinhole 21h formed on the pinhole disk 21b have a one-to-one correspondence, and the light passing through the microlens 21L is focused on the corresponding pinhole 21h. Is configured to tie.

  A dichroic mirror 41 is fixed between the microlens disk 21a and the pinhole disk 21b by a fixing member (not shown). The dichroic mirror 41 uses a laser beam that transmits laser light (described later) from the laser unit 5 and reflects fluorescence from the laser light. For example, light having a wavelength around 488 nm, 561 nm, and 635 nm is transmitted, and light having wavelengths of 500 nm to 540 nm, 570 nm to 620 nm, and 650 nm to 700 nm, and light corresponding to transmitted illumination light described later is selected. Is done.

  FIG. 4 is a diagram showing the configuration of the laser unit 5.

  As shown in FIG. 4, the laser unit 5 is provided with three types of lasers 51, 52 and 53 having different wavelengths, and these lasers are individually controlled to be turned on / off by the control arithmetic device 200. These lasers are adjusted so as to be parallel light inside. As these lasers, semiconductor lasers and solid-state lasers can be used.

  As shown in FIG. 4, a mirror 51 a reflects the laser light having a wavelength emitted from the laser 52 in front of the laser 51, and transmits the laser light having a wavelength emitted from the laser 51. A dichroic mirror 52 a reflects laser light having a wavelength emitted from the laser 53 in front of the laser 53 and transmits laser light having a wavelength emitted from the laser 51 and laser light having a wavelength emitted from the laser 52. Each of the mirrors 53a is arranged. A mirror 54 is provided on the optical axes of the laser 51, the laser 52, and the laser 53.

  The laser 51, the laser 52, and the laser 53 are collimated, respectively, and are adjusted by the mirror 54 and the dichroic mirrors 51a, 52a, and 53a so that the optical axes are coaxial. The light from these three lasers 51, 52 and 53 is guided to the disk unit 21 by the mirror 54. The wavelengths of the laser beams of the laser 51, the laser 52, and the laser 53 can be set to, for example, 488 nm, 561 nm, and 635 nm, respectively.

  Next, the operation of the cultured cell observation microscope apparatus of this embodiment will be described.

  FIG. 5 is a top view of a confocal microscope showing a state when a sample is set.

  As shown in FIG. 5, when setting the sample, the XY drive mechanism 33 moves the sample cassette 32 to a predetermined position in the Y direction, thereby opening the temperature control chamber 2 and the opening of the protective housing 1 (not shown). ), The sample cassette 32 is taken out, the sample cassette 32 is taken out, and a sample 31 such as a glass bottom dish in which a medium and cells are placed is set therein. Thereafter, the sample 31 is pulled into the apparatus by the XY drive mechanism 33. The same procedure applies when changing the medium.

  The sample 31 accommodated in the temperature control chamber 2 is supplied with moisture, carbon dioxide and the like that are optimal for cell culture, and the temperature control chamber 2 has an optimal temperature (for example, 30 ° C.). Maintained. In general, in order to culture cells for a long period of time, for example, a liquid called a culture medium that forms a suitable environment is sealed inside a glass bottom dish. This medium is generally added with a dye called phenol red, and is configured such that its color changes depending on the pH of the medium environment. As a result, the pH of the culture medium becomes almost neutral when the environment is suitable. In this state, the color of the culture medium is generally kept red. However, when the environment of the medium deteriorates due to cell growth or the like, the pH of the medium tends to be acidic, and the color of the medium changes to yellow accordingly. Here, in a state where it looks red, the medium mainly absorbs light having a wavelength of 400 nm to 580 nm, so that the complementary red color is reflected and transmitted and is observed as red. Moreover, in the state which looks yellow, since the culture medium mainly absorbs light having a wavelength of 400 nm to 520 nm, the complementary yellow color is reflected and transmitted and observed as yellow.

  Next, the optical path of light observed through the objective lens 24 will be described.

  The light from the laser unit 5 is collected by the microlenses 21L, 21L,... (FIG. 3) on the microlens disk 21a of the disk unit 21, passes through the dichroic mirror 41, and corresponds to the pinhole disk 21b. Focus on the pinholes 21h, 21h,... (FIG. 3). Here, in order to accurately position the focal point on the pinholes 21h, 21h,..., It is necessary to accurately adjust the position and inclination of the laser beam.

  The light passing through the pinhole disk 21b becomes parallel light having an inclination corresponding to the position of the pinhole 21h by the tube lens 22. The parallel light is bent upward by the mirror 23 and focused by the objective lens 24. When there is a fluorescently stained sample at the focal position, a part of this fluorescence is incident on the objective lens 24 again, returns through the same optical path as the above laser beam, is bent by the mirror 23, and is further handled by the tube lens 22 respectively. Focus again on the pinholes 21h, 21h,. Here, the light passing through the pinhole 21 h is reflected by the dichroic mirror 41, passes through the relay lens 42, is reflected by the mirror 43, passes through the filter wheel 44, and is guided to the relay lens 46.

  Here, the respective focal points on the pinhole 21 h are transferred to corresponding positions on the high-sensitivity CCD 6 by the two relay lenses 42 and 46.

  In addition, when one of the laser 51, laser 52, and laser 53 of the laser unit 5 is selected, the control arithmetic device 200 sets an optimum filter that transmits only the fluorescence corresponding to the wavelength of the laser in the filter wheel 44. Select from.

  In this state, the operation when the micro lens disk 21a and the pinhole disk 21b of the disk unit 21 are rotated by the motor 21d will be described below with reference to FIG.

  The light that has passed through the microlens disk 21a as described above is condensed in the corresponding pinhole 21h of the pinhole disk 21b, and the light that has passed through the pinhole 21h is objective by the action of the tube lens 22 and the objective lens 24. The lens 24 is focused at a corresponding position on the focal plane 24A (FIG. 3). In FIG. 3, the actions of the tube lens 22 and the objective lens 24 are schematically expressed as the action of a single lens.

  The fluorescence from the focal point on the focal plane 24A passes through the same optical path, passes through the same pinhole 21h again, is reflected by the dichroic mirror 41, and is imaged on the high-sensitivity CCD 6 by the relay lenses 42 and 46. In FIG. 3, the action of the relay lenses 42 and 46 is schematically expressed as the action of one lens.

  Here, when the micro lens disk 21a and the pinhole disk 21b are integrally rotated by the motor 21d, the focus on the corresponding focal plane 24A is also moved, and the fluorescence imaged on the high sensitivity CCD 6 is also moved. .

  Thus, the focal points of the plurality of laser beams scan the focal plane 24A at high speed, and the fluorescence from the focal plane 24A is imaged on the high-sensitivity CCD 6. This image can be displayed on a monitor (not shown) by the control arithmetic device 200 or stored in a storage device (not shown).

  Here, the fluorescence caused by the light deviating from the focal plane 24A does not pass through the same optical path as the laser light, and therefore hardly enters the corresponding pinhole 21h. Therefore, the image observed by the high-sensitivity CCD 6 is a confocal image very close to the focal plane 24A.

  Further, by driving the Z drive unit 25 by the control arithmetic device 200, it is possible to focus on an optimum position on the sample or to capture tomographic images having different depths. Further, the desired position of the sample 31 can be observed by the XY drive mechanism 33.

  In addition to switching the laser to be used by the control arithmetic device 200 (any one of the lasers 51, 52, 53) and selecting an optimum filter in the filter wheel 44, it is possible to acquire transmitted images of other wavelengths. It is.

  Furthermore, the transmitted light can be observed by illumination with light from the white light source 27 with the laser light turned off. At this time, the transmitted light passes through the same optical path as fluorescence, and light from the vicinity of the focal plane 24A is selected by the pinhole disk 21b of the disk unit 21. Further, a part of the transmitted light is reflected by the dichroic mirror 41. For example, light having a wavelength of 500 nm to 540 nm, 570 nm to 620 nm, and 650 nm to 700 nm is reflected by the dichroic mirror 41, and then passes through an optical path similar to that of fluorescence and is imaged on the high sensitivity CCD 6. Here, by selecting an optimum filter from the filter wheel 44, a transmission image corresponding to each wavelength can be obtained.

  Next, a procedure for detecting the state of the medium will be described.

  When detecting the state of the culture medium, the transmitted light observation is performed by illumination with light from the white light source 27 with the laser light turned off.

  Next, a filter that transmits a yellow band in the wavelength range of 570 nm to 590 nm is selected by the filter wheel 44, and an image in this wavelength range is captured and acquired. Next, a filter that transmits a red band in the wavelength range of 650 nm to 680 nm is selected by the filter wheel 44, and an image in this wavelength range is captured and acquired. Both images are images of the same region that differ only in the wavelength region. The image data of these images is taken into the control arithmetic device 200.

  Next, the control arithmetic device 200 calculates the average light intensity Avy of each pixel of the image obtained in the yellow wavelength region and the average light intensity Avr of each pixel of the image obtained in the red wavelength region. .

  Next, the control arithmetic device 200 calculates a ratio Avy / Avr between the average Avy and the average Avr.

  When the state of the medium is good, the medium shows red, absorbs light in the yellow wavelength band, and transmits light in the red band, and thus the Avy / Avr value is relatively small. However, when the state of the medium has deteriorated, the medium shows yellow, and mainly transmits light in the yellow band, so the value of Avy / Avr becomes a relatively large value.

  Therefore, when the value of Avy / Avr becomes equal to or greater than a certain value, it is possible to display a prompt for medium replacement or to start an automatic medium replacement operation in the confocal microscope 100.

  Further, instead of using the value of Avy / Avr as an index value, the transmitted light intensity in the yellow band where the difference in the state of the medium is most significant is used as an index value, and the deterioration of the medium is detected by measuring this index value. Can do.

  In the above embodiment, the filter wheel 44 is arranged in the vicinity of the high-sensitivity CCD, but the position of the filter is arbitrary. Further, a transmission image may be acquired by changing the wavelength band of the illumination light. In this case, for example, by arranging a filter wheel below the white light source 27, the illumination light in the yellow wavelength band and the red wavelength band are arranged. Illumination light can be obtained.

  Further, instead of the white light source, a light source capable of obtaining yellow wavelength band illumination light and red wavelength band illumination light may be used. For example, multiple color LEDs may be used.

  In the above embodiment, the average of the light intensity of all the pixels is obtained when calculating the average intensity of the transmitted light in the yellow and red wavelength bands, but the light intensity histogram of each pixel has a certain frequency or more. An average value of the distribution may be calculated, and the state of the culture medium may be detected based on the average value. In this case, since the amount of light necessary for detecting the state of the medium can be suppressed, the influence on the cells can be reduced.

  Moreover, in the said embodiment, although the state of a culture medium is detected based on the color tone of the light which permeate | transmits a culture medium, illumination light is irradiated to a culture medium from the objective lens side, and based on the reflected light from a culture medium, A state may be detected.

  As described above, according to the microscope apparatus for observing cultured cells of the above embodiment, since the change in the color tone of the medium can be detected while culturing the cells, it is possible to easily grasp the deterioration state of the medium.

  In addition, according to the cultured cell observation microscope apparatus of the above-described embodiment, the color tone of the culture medium is obtained by using the white light source for transmission observation as the illumination means, so that the configuration of the apparatus can be simplified. In addition, since the confocal optical system for transmitted light observation and fluorescence observation is used as the detection means for acquiring the color tone of the culture medium, the configuration of the apparatus can be simplified. Furthermore, since the CCD for transmitted light observation and fluorescence observation is used as the detection means for acquiring the color tone of the culture medium, the configuration of the apparatus can be simplified.

  FIG. 6 is a top view showing a configuration example in the case of detecting the state of the culture medium based on the reflected light from the culture medium. In FIG. 6, the same elements as those in FIG.

  As shown in FIG. 6, the confocal microscope 100A includes a light source 27A including a green LED having a wavelength of 580 nm and a red LED having a wavelength of 630 nm, a beam splitter 61 provided below the condenser lens 28, and a beam splitter 61. And a photodiode 62 that receives the reflected light.

  When detecting the state of the culture medium, the green LED is turned on to apply green illumination light from the light source 27A to the culture medium, and only the red LED is turned on to apply red illumination light from the light source 27A to the culture medium. Part of the reflected light from the medium is reflected by the beam splitter 61 and received by the photodiode 62.

  The reflected light intensity detected by the photodiode 62 is input to the control arithmetic device 200, and the control arithmetic device 200 compares the ratio of the reflected light intensity in the yellow band to the reflected light intensity in the red band (the reflected light intensity in the yellow band). / Reflected light intensity in the red band) is calculated.

  When the medium is in good condition, the medium shows red, absorbs light in the yellow wavelength band, and the reflection of light in the red band increases, so the above ratio value is relatively small. . However, when the state of the culture medium has deteriorated, the culture medium exhibits a yellow color, and mainly reflects the light in the yellow band, so that the value of the above ratio is a relatively large value.

  Therefore, when the value of the above ratio is equal to or greater than a certain value, it is possible to display a prompt for medium replacement, or to start an automatic medium replacement operation in the confocal microscope 100A.

  In addition, instead of using the ratio value as an index value, the reflected light intensity in the yellow band where the difference is most significant in the state of the medium is used as an index value, and the deterioration of the medium is detected by measuring this index value. Can do.

  Although the configuration example using the white light source 27 (FIG. 2) or the light source 27 </ b> A (FIG. 6) has been shown as the illumination unit when detecting the state of the culture medium, the laser light from the laser unit 5 can also be used. In this case, a single color LED may be used as a light source for transmission observation instead of the light source 27A.

  When detecting the state of the culture medium, a laser beam is emitted from a yellow (561 nm) laser provided in the laser unit 5, a part of the light transmitted through the culture medium is received by the photodiode 62, and then the laser unit. Laser light is emitted from a red (635 nm) laser provided in 5, and a part of the light transmitted through the culture medium is received by the photodiode 62.

  Next, in the control arithmetic device 200, based on the reflected light intensity detected by the photodiode 62, the ratio between the transmitted light intensity in the yellow band and the transmitted light intensity in the red band (transmitted light intensity in the yellow band / red color). (Transmitted light intensity in the band).

  When the state of the medium is good, the medium shows red, absorbs light in the yellow wavelength band, and transmits light in the red band, and thus the value of the above ratio is relatively small. However, when the state of the culture medium has deteriorated, the culture medium shows a yellow color, and mainly transmits light in the yellow band, so the value of the above ratio becomes a relatively large value.

  Therefore, when the value of the above ratio is equal to or greater than a certain value, it is possible to display a prompt for medium replacement, or to start an automatic medium replacement operation in the confocal microscope 100A.

  In addition, instead of using the ratio value as an index value, the transmitted light intensity in the yellow band where the difference is most significant in the state of the medium is used as an index value, and the deterioration of the medium is detected by measuring this index value. Can do.

  In this case, since the laser unit 5 can be used as illumination means when detecting the state of the culture medium, the configuration of the apparatus can be simplified.

  In each of the above embodiments, the configuration in which the state of the culture medium is detected based on the light intensity in the yellow band and the light intensity in the red band is shown, but the light band is not limited to this. For example, the state of the medium is determined based on the light intensity of the transmitted or reflected light in the wavelength band included in the range of 550 nm to 600 nm and the light intensity of the transmitted or reflected light in the wavelength band included in the range of 600 nm or more. Can be detected.

  The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied to a cultured cell observation microscope apparatus for observing cells cultured in a medium.

6 High sensitivity CCD (detection means)
27 White light source (illumination means, transmission)
27A Light source (illumination means)
200 Control arithmetic device (calculation means)

Claims (6)

In the microscope apparatus for observing cells cultured in a culture medium,
Illumination means for irradiating the medium with illumination light;
Detecting means for detecting transmitted light of the medium based on the illumination light from the illumination means or reflected light from the medium based on the illumination light;
Based on the transmitted light or the reflected light detected by the detection means, a calculating means for calculating a ratio of the transmitted light intensity of the medium or the reflected light intensity from the medium for light of a plurality of wavelength bands;
A microscope apparatus for observing cultured cells, comprising: The illumination means is a transmitted light observation light source for observing the cells,
2. The cultured cell observation microscope apparatus according to claim 1, wherein the calculating means calculates a ratio of transmitted light intensity of the culture medium based on the transmitted light.   The culture cell observation microscope apparatus according to claim 2, wherein the detection means is an image sensor for observation of transmitted light that receives the transmitted light. The illumination means is a fluorescence excitation light source that emits fluorescence excitation light for observing the cells,
2. The cultured cell observation microscope apparatus according to claim 1, wherein the calculating means calculates the ratio of the emitted light intensity based on the reflected light.   5. The cultured cell observation microscope apparatus according to claim 4, wherein the detection means is a fluorescence observation image pickup element that receives fluorescence excited by the fluorescence excitation light.   The one of the plurality of wavelength bands is a wavelength band included in a range of 550 nm to 600 nm, and the other one of the plurality of wavelength bands is a wavelength band included in a range of 600 nm or more. Item 6. The microscope device for observing cultured cells according to any one of Items 1 to 5.

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