JP4868879B2 - Cell state detector - Google Patents

Description

  The present invention relates to a device for detecting the state of a human cell, and more particularly to a cell state detection device used when performing regenerative medicine or the like.

  In recent years, with the development of tissue engineering, stem cells have been cultivated and applied to the surface of medical devices to be introduced into the body. Has progressed to. In such a cell culture technique, it is necessary to evaluate the state of the cultured cells and then introduce it into the body. However, cell state detection methods therefor are also being developed along with advances in tissue engineering. As one of such cell state detection methods, a method is known in which specific cells are collected, cultured on a filter, and then observed with a reagent (fluorescent dye) using a fluorescence microscope. It has been.

Here, the cell state detection method by the fluorescence observation will be described. Light (excitation light) in a specific wavelength range is irradiated on a cell on the filter that is made to act on a reagent (fluorescent dye). Thereby, fluorescence is emitted from the fluorescent dye that has acted on the cells. Then, the fluorescence is passed through an absorption filter, and the light cut in a specific wavelength region is observed through an eyepiece. At this time, the fluorescence-stained cells emit different light depending on the life and death state, the differentiation state, or the cell type, so that the state and type of the cell can be immediately discriminated (see, for example, Patent Document 1).
Japanese Patent No. 2592114

  However, in the conventional method described above, in order to detect the state of the cell, the fluorescent dye must be allowed to act on the cell as described above. Therefore, it is not suitable for evaluating the state of the cell to be returned to the human body after culture such as regenerative medicine. Met. It was also impossible to continuously monitor the same cells.

  In view of such a problem, the present invention is a device that can accurately detect the state of a cell in a non-invasive manner without causing a reagent such as a fluorescent dye to act on the cell, or a state of a cell. It provides a way to

The cell state detection apparatus of the present invention includes an excitation light irradiation unit that irradiates a human cell with excitation light (G excitation) having a wavelength of 530 nm, and a wavelength of 600 nm to 700 nm emitted from the human cell by the excitation light from the excitation light irradiation unit. Autofluorescence detecting means for detecting the autofluorescence of the cell, and cell state output means for outputting a state relating to the life or death of the human cell based on the detection result of the autofluorescence detecting means. Any one of the emitted autofluorescence intensity or the autofluorescence spectrum pattern, or a combination thereof is output, or information obtained by processing the output is output.

According to a second aspect of the present invention, in the cell state detection apparatus according to the first aspect, the human cell is a human mesenchymal stem cell.

  As described above, a human cell state (for example, a cell) can be obtained by snooping a device that can accurately detect the state of a cell without causing a reagent such as a fluorescent dye to act on the human cell. Experiments have confirmed that the intensity of autofluorescence varies depending on the life and death of the cells, the viability of the cells, the cell type, and the degree of cell differentiation.

Therefore, according to the cell state detection device of the present invention, the excitation light irradiation means for irradiating the human cell with excitation light (G excitation) having a wavelength of 530 nm, and the excitation light from the excitation light irradiation means emits from the human cell. An autofluorescence detecting means for detecting autofluorescence with a wavelength of 600 nm to 700 nm, and a cell state output means for outputting a state relating to the life or death of a human cell based on the detection result of the autofluorescence detecting means, Since any one of the autofluorescence intensity emitted from human cells or the autofluorescence spectrum pattern, or a combination thereof is output, or information obtained by processing the output is output. Based on the output information, the state of the human cell can be determined.

  Thus, using the cell state detection device of the present invention, autofluorescence emitted from human cells is detected by the autofluorescence detection means, and the state of the human cell is output by the cell state output means based on the result. Thus, the state and type of the cell can be determined. Thereby, it becomes possible to discriminate | determine the state and kind of a cell, without making a reagent act on a human cell.

  In particular, if the cell state detection device of the present invention is used, cells are irradiated with excitation light excluding wavelengths in the ultraviolet region and infrared region, so that the state and type can be easily maintained without damaging human cells. It becomes useful for evaluating the state of cells to be returned to the human body after culturing such as regenerative medicine.

  In addition, since the autofluorescence detection means detects autofluorescence emitted from human cells and having a wavelength of 600 nm to 700 nm, it is possible to detect the state of the human cells more accurately.

Furthermore, since the excitation light irradiation means irradiates the human cell with excitation light (G excitation) having a wavelength of 530 nm, it is possible to detect the state of the human cell more accurately.

The cell state detection apparatus of the present invention is particularly useful for detecting the state of human mesenchymal stem cells as claimed in claim 2 .

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a schematic diagram illustrating a configuration of a cell state detection device 10 according to an embodiment of the present invention. The cell state detection device detects excitation light irradiation means 1 (light source) that irradiates a human cell to be detected with excitation light L1 and autofluorescence L2 emitted from the cell by the excitation light L1 from the excitation light irradiation means 1. Auto-fluorescence detecting means 7 and cell state output means 8 for outputting the state of cells based on the detection result of the auto-fluorescence detecting means 7. The cell state detection apparatus 10 according to the present embodiment reflects light from the excitation light irradiation means 1 by the dichroic mirror 2 and irradiates the cell from the lower surface of the culture container 4 in which the cell is stored, so that the cell is self-generated. An epi-illumination type detection device that generates fluorescence. That is, the excitation light L1 from the excitation light irradiation means 1 (light source) is irradiated to the cells from the lower surface of the culture vessel 4 in which the cells are stored via the dichroic mirror 2 and the objective lens 3, and the excitation light L1 is irradiated. The autofluorescence L2 emitted from the cells was sequentially passed through the objective lens 3, the dichroic mirror 2 and the absorption filter 6 installed on the lower side of the culture vessel 4 to remove light of a specific wavelength from the autofluorescence L2. The object is condensed by the eyepiece 9, detected by the autofluorescence detection means 7, and the cell state output means 8 outputs the cell state based on the detection result.

  The excitation light irradiation means 1 irradiates a cell with excitation light L1 having a noninvasive wavelength with respect to a cell to be observed, and includes a lamp (for example, a mercury lamp), an optical filter, and a lens. ing. That is, the light of the lamp includes wavelengths of various components, but by passing through the optical filter, only light other than the specific wavelength is cut by the optical filter, and light of a specific wavelength (a preset wavelength) Only light) passes through the optical filter, and the light passing therethrough is emitted as excitation light L1 from the excitation light irradiation means 1 through the lens.

  The excitation light L1 irradiated to the cell from the excitation light irradiation means 1 is light having a wavelength excluding wavelengths in the ultraviolet region and the infrared region that may damage the cell by irradiation of the excitation light L1. In this way, the excitation light L1 irradiated to the cell from the excitation light irradiation means 1 is the excitation light L1 having a noninvasive wavelength, and in particular, the excitation light L1 excluding the wavelengths in the ultraviolet region and the infrared region. It is possible to avoid inconvenience that the cells are damaged by the irradiation of the light L1. In addition, if the wavelength of the excitation light L1 irradiated to the cell from the excitation light irradiation means 1 is set so that the autofluorescence L2 emitted from the cell becomes visible light, it is output from the cell state output means 8 described later. Since the autofluorescence L2 can be visually observed, a simple device such as a display described later can be applied as the cell state output means 8. In addition, the wavelength of the excitation light L1 that is actually applied to the cells was determined by measuring the fluorescence of the cells to be detected with a fluorescence spectrophotometer. For detection of human mesenchymal stem cells described later, autofluorescence L2 can be effectively generated from the cells by irradiating excitation light L1 having a wavelength of 400 nm or more and 600 nm or less.

  On the other hand, the autofluorescence detection means 7 may be any detection means as long as it can detect autofluorescence emitted from a cell. For example, light such as a photodiode that detects autofluorescence for each point. A detection element or a CCD image sensor that detects autofluorescence two-dimensionally and captures a fluorescence image may be used. The cell state output means 8 is a means for outputting the state of the cell based on the result of the autofluorescence detection means 7. Specifically, based on the output result of the autofluorescence detection means 7, the cell state output means 8 Any one of the intensity of emitted autofluorescence or the spectrum of autofluorescence spectra, or a combination of these, or any information that can be output as a result of processing the output Even so, it is applicable. For example, a personal computer or the like can be used as the cell state output means 8.

  In addition to the personal computer, if the excitation light irradiation means 1 as described above irradiates the cells with excitation light L1 in which the autofluorescence emitted from the cells becomes visible light, the autofluorescence detection means 7 is a CCD image element. In addition, the cell state output means 8 may be a display that displays an image captured by the CCD video device or a printer that prints the captured image. Thus, if the autofluorescence L2 emitted from the cell is set to be visible light, the excitation light L1 irradiated to the cell from the excitation light irradiation means 1 is a simple device such as a display as the cell state output means 8. Therefore, it is possible to reduce costs and simplify the entire apparatus.

  Further, when the autofluorescence L2 emitted from the cell becomes visible light and a personal computer is used as the cell state output means 8, it is of course possible to simply display the image captured by the CCD image device as described above on the display as it is. Other than that, for example, some processing (amplification, etc.) may be added to the image and output to the display, or cell discrimination (life / death discrimination, type discrimination, etc.) based on the image data Various outputs are possible, such as displaying the results on a display or printer.

  On the other hand, when light having a wavelength other than the visible region is emitted as the autofluorescence L2, it is impossible to visually observe the autofluorescence L2. Therefore, the cell state output means 8 is the result of the autofluorescence detection means 7. Based on the above, the autofluorescence intensity and autofluorescence spectrum pattern emitted from the cells are processed by a data processing device (such as a personal computer), converted into numerical values and visible light, and output.

  The dichroic mirror 2 described above is a so-called band-pass filter that transmits only light of a specific wavelength and reflects light of other wavelengths, and the absorption filter 6 removes light of a specific wavelength. It is a filter to do.

  The cells described above in the present invention are human (human) cells, and human mesenchymal stem cells are used as cells in this example.

  Next, a cell state detection method using the cell state detection apparatus 10 of the present embodiment having the above configuration will be described. In this example, human mesenchymal stem cells are cultured and detached by trypsin treatment, and then the collected cells are used as target 1, and the same human mesenchymal stem cells as subject 1 are cultured in the same manner and detached by trypsin treatment. The collected cells were heat-treated at 70 ° C. for 20 minutes, and the state of each cell was detected for the target 2. That is, the cells used for subject 1 and subject 2 are the same, but subject 1 is a living cell, and subject 2 is different only in that subject 2 is a dead cell obtained by subjecting subject 1 to heat treatment.

  First, the case where a fluorescence spectrophotometer is used as the autofluorescence detecting means 7 will be described. In this case, detection was performed by irradiating the cells with excitation light L1 having two wavelengths of 470 nm and 530 nm. After culturing human mesenchymal stem cells as described above and detaching with trypsin treatment, culture vessel 4 (target 1) in which the collected cells are enclosed, or culturing human mesenchymal stem cells and detaching with trypsin treatment Then, the culture vessel 4 (target 2) in which the collected cells are heat-treated at 70 ° C. for 20 minutes is mounted at a predetermined position of the cell state detection device 10. And the wavelength of the excitation light L1 emitted from the excitation light irradiation means 1 is set to either 470 nm or 530 nm. Note that the dichroic mirror 2 has a characteristic of reflecting at least light having a wavelength equal to or shorter than the excitation light L1 and transmitting light having a longer wavelength. The dichroic mirror 2 is preliminarily adjusted in angle so as to reflect the excitation light L1 from the excitation light irradiation means 1 and irradiate it into the culture vessel 4.

  When the cell state detection device 10 is turned on, excitation light L1 having a wavelength (470 nm or 530 nm) set from the excitation light irradiation means 1 is emitted. The excitation light L1 is reflected by the dichroic mirror 2 and irradiated into the culture vessel 4 through the objective lens 3. Thereby, the excitation light L1 is irradiated to the cell in the culture container 4, and the autofluorescence L2 is emitted from the cell. At this time, the autofluorescence L2 emitted from the cell becomes light having a longer wavelength than the excitation light L1 irradiated to the cell.

  Then, the autofluorescence L2 from the cells is collected by the objective lens 3 and passes through the dichroic mirror 2. Further, the excitation light L <b> 1 irradiated and reflected on the cell also reaches the dichroic mirror 2 through the objective lens 3. At this time, since the dichroic mirror 2 reflects the wavelength below the excitation light L1 and transmits light having a longer wavelength as described above, the excitation light L1 is reflected and excited without passing through the dichroic mirror 2. It will return to the light irradiation means 1.

  On the other hand, since the autofluorescence L2 emitted from the cell is light having a wavelength longer than that of the excitation light L1 as described above, it passes through the dichroic mirror 2 and passes through the absorption filter 6. In this process, after light of a specific wavelength is removed, it enters the autofluorescence detection means 7 (fluorescence spectrophotometer) through the eyepiece 9. The autofluorescence detecting means 7 detects the state of the incident autofluorescence L2. In this case, since the fluorescence spectrophotometer is used as the autofluorescence detection means 7, the state of the autofluorescence L2 is detected by the light detection element of the fluorescence spectrophotometer, and the detection information is the cell state output means 8 (PC) Etc.).

  The cell state output means 8 outputs an autofluorescence spectrum pattern emitted from the cell based on the input signal. FIG. 2 shows an output result when the cell is irradiated with excitation light having a wavelength of 470 nm (during B excitation), and FIG. 3 shows an output result when the cell is irradiated with excitation light having a wavelength of 530 nm (during G excitation). is there. 2 and 3, a solid line A is a spectrum obtained from the target 1 (live cells), and a solid line B is a spectrum obtained from the target 2 (target 2).

  When each object (object 1 and object 2) is irradiated with excitation light having a wavelength of 470 nm (at the time of B excitation), both objects show fluorescence peaks at wavelengths near 470 nm and 700 nm as shown in FIG. A slow increase in fluorescence is observed at wavelengths near 500 nm to 570 nm. Since the peak near 470 nm is the excitation light L1 irradiated to the cell, the wavelength of the autofluorescence L2 emitted from the cell is considered to be around 500 nm to 570 nm. In addition, it can be seen from FIG. 2 that the dead cells of the subject 2 have higher fluorescence than the living cells of the subject 1.

  Moreover, when each object (object 1 and object 2) is irradiated with excitation light having a wavelength of 530 nm, both objects have a fluorescence peak at a wavelength near 530 nm as shown in FIG. An increase in fluorescence is observed at the wavelength. Since the peak near 530 nm is the excitation light L1 irradiated to the cell, the wavelength of the autofluorescence L2 emitted from the cell is considered to be around 600 nm to 700 nm. Moreover, it became clear that the fluorescence intensity of the dead cell of the subject 2 is remarkably higher than that of the living cell of the subject 1 as in the result of FIG.

  Thus, even in the same cell, the autofluorescence spectrum pattern emitted from the cell differs depending on the state of the cell (in this example, the life and death of the cell), so the cell state detection device 10 of this example is used. By detecting the autofluorescence of the cell and outputting the autofluorescence spectrum pattern, it is possible to clearly determine the state of the cell without causing a reagent such as a fluorescent dye to act on the cell.

  Next, a case where a CCD camera (CCD imaging device) is used as the autofluorescence detecting means 7 will be described. In this case, the fluorescence of the autofluorescence emitted from both objects is higher when the excitation light with the wavelength of 530 nm in FIG. 3 is irradiated with the excitation light with the wavelength of 470 nm in FIG. Since it is clear that irradiation with 530 nm excitation light L1 can generate autofluorescence L2 from cells more effectively than irradiation with 470 nm excitation light L1, it is emitted from excitation light irradiation means 1 to cells. Autofluorescence L2 was detected by setting the wavelength of the excitation light L1 to 530 nm.

  First, human mesenchymal stem cells are cultured as described above and detached by trypsin treatment, and then the culture vessel 4 (target 1) in which the collected cells are enclosed or human mesenchymal stem cells are cultured and trypsinized. After peeling, the culture vessel 4 (target 2) in which the collected cells are heat-treated at 70 ° C. for 20 minutes is attached to a predetermined position of the cell state detection device 10. And the wavelength of the excitation light L1 emitted from the excitation light irradiation means 1 is set to 530 nm. Note that the dichroic mirror 2 has a characteristic of reflecting at least light having a wavelength of 530 nm or less, which is the excitation light L1, and transmitting light having a longer wavelength. The dichroic mirror 2 is preliminarily adjusted in angle so as to reflect the excitation light L1 from the excitation light irradiation means 1 and irradiate it into the culture vessel 4.

  When the cell state detection device 10 is turned on, excitation light L1 having a wavelength (530 nm) set from the excitation light irradiation means 1 is emitted. The excitation light L1 is reflected by the dichroic mirror 2 and irradiated into the culture vessel 4 through the objective lens 3. Thereby, the excitation light L1 is irradiated to the cell in the culture container 4, and the autofluorescence L2 is emitted from the cell. At this time, the autofluorescence L2 emitted from the cell becomes light having a longer wavelength than the excitation light L1 irradiated to the cell, as is apparent from FIG.

  Then, the autofluorescence L2 from the cells is collected by the objective lens 3 and passes through the dichroic mirror 2. Further, the excitation light L <b> 1 irradiated and reflected on the cell also reaches the dichroic mirror 2 through the objective lens 3. At this time, since the dichroic mirror 2 reflects the wavelength below the excitation light L1 and transmits light having a longer wavelength as described above, the excitation light L1 is reflected and excited without passing through the dichroic mirror 2. It will return to the light irradiation means 1.

  On the other hand, since the autofluorescence L2 emitted from the cell is light having a wavelength longer than that of the excitation light L1 as described above, it passes through the dichroic mirror 2 and passes through the absorption filter 6. In this process, after light of a specific wavelength is removed, the light enters the autofluorescence detection means 7 (CCD camera) through the eyepiece 9. The autofluorescence detecting means 7 detects the state of the incident autofluorescence L2. In this case, since a CCD camera is used as the autofluorescence detection means 7, the state of the autofluorescence L2 is imaged by the CCD image element of the CCD camera and input to the cell state output means 8 as an image signal.

  The cell state output means 8 outputs the cell state based on the input image signal. In this embodiment, since the display is used as the cell state output means 8 for outputting the autofluorescence intensity emitted from the cells, the image captured by the CCD camera is displayed on the display. Here, FIG. 4 is a diagram schematically showing a photograph showing the autofluorescence intensity of the object 1 (live cells) displayed on the display, and FIG. 5 shows the autofluorescence intensity of the object 2 (dead cells). It is the figure which showed the photograph shown typically. The autofluorescence L2 obtained from each cell when irradiated with the excitation light L1 of 530 nm was red (background is black). In FIGS. 4 and 5, the background color is reversed to white, and the cell The autofluorescence L2 emitted from is shown in black. Therefore, the more black the cells are, the stronger the fluorescence intensity is, and the smaller the color difference from the background (the cells are closer to white), the weaker the fluorescence intensity. That is, it can be seen that the live cell of the target 1 in FIG. 4 has a low fluorescence intensity and the dead cell of the target 2 in FIG. 5 having a large color difference from the background has a high fluorescence intensity. As a result, it was found that the fluorescence intensity was significantly different between the living cells and the dead cells, and there was a clear intensity difference. Further, the result is consistent with the result of the autofluorescence spectrum pattern shown in FIG.

  Thus, even in the same cell, the fluorescence intensity of the autofluorescence emitted from the cell differs depending on the state of the cell (in this embodiment, the life and death of the cell). By detecting the autofluorescence of the cells and outputting the fluorescence intensity, it is possible to clearly determine the state of the cells without causing a reagent such as a fluorescent dye to act on the cells.

  In the present embodiment, the cell state output means 8 displays the autofluorescence intensity emitted from the cell on the display based on the image of the autofluorescence L2 imaged by the CCD camera. May be displayed on the display, or may be subjected to processing such as amplification on the image and output to the display. In addition, when performing cell discrimination based on image data, for example, cell viability as in this embodiment, dead cells have higher fluorescence intensity than live cells, so a predetermined threshold is set. When the cell state output means 8 has a fluorescence intensity higher than a predetermined threshold, it is determined to be a dead cell, and when the fluorescence intensity is lower than the predetermined threshold, it is determined to be a living cell, and the result is displayed on a display or printer. It is also possible.

  Further, by detecting the autofluorescence L2 emitted from the cell, it is possible to determine the type of the cell. That is, since the autofluorescence L2 emitted from each cell differs depending on the type of cell, the autofluorescence L2 of various cells is measured in advance, and the measurement results are input to the cell state output means 8, and the cell state output means In 8, based on the input information, the fluorescence detection means 7 described above outputs the result to a display or a printer so that the cell type can also be determined. Further, it can be applied to detection of the differentiation state of cells. In particular, the use of the cell state detection device 10 makes it possible to easily determine the state, type, etc. while the cells are non-invasive, so that the state of the cells returned to the human body after culture such as regenerative medicine can be evaluated. become.

  The cell state detection device 10 of the above embodiment reflects the light from the excitation light irradiation means 1 by the dichroic mirror 2 and irradiates the cells from the lower surface of the culture container 4 in which the cells are stored, so that the cells are self-generated. Although the epi-illumination type detection device that generates fluorescence is used, the cell state detection device of the present invention is not limited to this. For example, a transmission detecting device as shown in FIG. 6 may be used. In FIG. 6, the same reference numerals as those in FIG. 1 have the same or similar effects, and thus description thereof is omitted.

  In this case, the excitation light L1 from the excitation light irradiation means 1 (light source) is emitted from the upper side of the culture vessel 4 to the cells stored in the culture vessel 4 and emitted from the cells by the irradiation of the excitation light L1. The autofluorescence L2 is condensed by the objective lens 3 installed on the lower side. Then, the autofluorescence L2 from which light of a specific wavelength is removed by passing through the absorption filter 6 is detected by the autofluorescence detection means 7 via the eyepiece 9, and the cell state output means 8 is based on the detected result. Will output the cell status.

  Since the autofluorescence detection means 7 and the cell state output means 8 used in this embodiment are the same as those in the above embodiment, description thereof will be omitted.

  As described above, even if the cell state detection device 20 of the present embodiment is used, it is possible to determine the state and type of the cell without causing a reagent such as a fluorescent dye to act on the cell.

  In addition to the above embodiments, autofluorescence may be observed and detected using the evanescent light shown in FIG. 7 or FIG. First, the cell state detection device 30 shown in FIG. 7 will be described. In this case, when the culture vessel 4 is irradiated with the excitation light L1 at a predetermined angle at which the light from the excitation light irradiation means 1 is totally reflected, total reflection occurs on the lower surface of the culture vessel 4, and all the excitation light L1 is directed to the L3 side. Although it seems to be removed, in reality, evanescent light is generated from the lower surface of the culture vessel 4 to the upper side. Since the evanescent light is a very thin layer, it does not propagate far away and cannot be confirmed visually.

  However, when the evanescent light is irradiated to the cells (cells stored in the culture vessel 4) in the region where the evanescent light reaches (in the thin layer), the cells once absorb the evanescent light, It emits autofluorescence. At this time, the autofluorescence emitted from the cells is normal propagation light. The autofluorescence L2 emitted from the cells is collected by the objective lens 3 and passes through the absorption filter 6. In this process, after light of a specific wavelength is removed, the light enters the autofluorescence detection means 7 through the eyepiece 9. Then, as described in detail in each of the embodiments, the autofluorescence detection means 7 detects the autofluorescence, and based on the result, the cell state output means 8 outputs the cell state. Note that the autofluorescence detection means 7 and the cell state output means 8 used in this embodiment are the same as those in the above-described embodiment, and thus description thereof is omitted.

  On the other hand, the cell state detection device 40 shown in FIG. 8 irradiates the excitation light L1 from the objective lens 3 to generate vanescent light from the lower surface to the upper side of the culture vessel 4, and emits autofluorescence from the cells by irradiation with the evanescent light. It is an example of the apparatus to generate. In this case, the autofluorescence L2 emitted from the cells is collected by the objective lens 3 and passes through the dichroic mirror 2. Further, the excitation light L <b> 1 irradiated and reflected on the cell also reaches the dichroic mirror 2 through the objective lens 3. At this time, since the dichroic mirror 2 reflects the wavelength below the excitation light L1 and transmits light having a longer wavelength as described above, the excitation light L1 is reflected and excited without passing through the dichroic mirror 2. It will return to the light irradiation means 1.

  The autofluorescence L2 that has passed through the dichroic mirror 2 then passes through the absorption filter 6, and light of a specific wavelength is removed in this process. Thereafter, the autofluorescence L2 that has passed through the absorption filter 6 enters the autofluorescence detection means 7 via the eyepiece 9. Then, as described in detail in each of the embodiments, the autofluorescence detection means 7 detects the autofluorescence, and based on the result, the cell state output means 8 outputs the cell state.

  Thus, even if the autofluorescence L2 of the cell is detected using evanescent light and the detection result is output, the cells are not affected by a reagent such as a fluorescent dye as in the above embodiments. It becomes possible to determine the state and type. In particular, by using evanescent light, it becomes possible to detect only a very thin layer of cells housed in the culture vessel 4, so that the autofluorescence of a single cell present in the focused portion is observed. It is also possible to monitor the cells continuously. As a result, the state of the cell can be detected more clearly, and the detection accuracy can be greatly improved.

It is a schematic diagram which shows the structure of the cell state detection apparatus of one Example of this invention. It is a figure which shows the autofluorescence spectrum pattern at the time of irradiating the excitation light of a wavelength of 470 nm. It is a figure which shows the autofluorescence spectrum pattern at the time of irradiating the excitation light of a wavelength of 530 nm. It is the figure which showed typically the photograph of the object 1 at the time of irradiating the excitation light of a wavelength of 530 nm (live cell). It is the figure which showed typically the photograph of the object 2 at the time of irradiating the excitation light of a wavelength of 530 nm (dead cell). It is a schematic diagram which shows the structure of the cell state detection apparatus of the other Example of this invention. It is a schematic diagram which shows the structure of the cell state detection apparatus of another another Example of this invention. It is a schematic diagram which shows the structure of the cell state detection apparatus of another another Example of this invention.

1 Excitation light irradiation means (light source)
2 Dichroic mirror 3 Objective lens 4 Culture vessel 6 Absorption filter 7 Autofluorescence detection means 8 Cell state output means 9 Eyepiece 10, 20, 30, 40 Cell state detection device

Claims (2)

Excitation light irradiation means for irradiating human cells with excitation light (G excitation) having a wavelength of 530 nm ;
Autofluorescence detecting means for detecting autofluorescence having a wavelength of 600 nm to 700 nm emitted from the human cell by excitation light from the excitation light irradiation means;
Cell state output means for outputting a state relating to the life or death of the human cell based on the detection result of the autofluorescence detection means,
The cell state output means outputs any one of autofluorescence intensity or autofluorescence spectrum pattern emitted from the human cells, or a combination thereof, or outputs information obtained by processing the output. A cell state detection device characterized by: The cell state detection device according to claim 1, wherein the human cell is a human mesenchymal stem cell .

Images