JP2006194711A - Performance evaluation method and performance evaluation device of fluorescence-emission pigment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a device for evaluating performance of a fluorescence-emission pigment by staining a cell with the fluorescence-emission pigment and by measuring a fluorescence-emission brightness value and pigment permeability of the fluorescence-emission pigment. <P>SOLUTION: The cell is stained by the fluorescence-emission pigment, and excitation light is irradiated to generate fluorescence emission, and the cell is imaged by a microbe-measuring device having a light-receiving wavelength filter for blocking wavelengths other than an objective wavelength. The brightness of a fluorescence-emission point is measured by measuring the brightness of an imaged image, and binarization is performed in the bounded state by a reference brightness value, to thereby change each emission point having the brightness value over a reference value into a bright point, and it is determined whether the emission is a cell or not by recognizing the shape or the area thereof, and then each bright point is measured. Then, each bright point is flattened 13 on the acquired image by a brightness value analysis means 12 shown in Figure 3, and binarization 14 is performed again, to thereby extract 15 bright points inconfirmable by a naked eye. The number of brightness values in one picture is expressed by a frequency in a histogram 16 relative to the extracted 15 image, and the maximum peak of the brightness values is clarified. Hereby, the fluorescence-emission brightness value of the fluorescence-emission pigment to the cell can be measured 17. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method and an apparatus for evaluating the performance of a fluorescent dye that recognizes the site and number of cells by staining cells with a fluorescent dye and irradiating the stained cells with excitation light to emit fluorescence. .

Conventionally, a fluorescence spectrophotometer is mainly used as an apparatus used in this kind of evaluation method, and a cell as a test material is stained with a fluorescent luminescent dye, and the test material is used as a light source using, for example, a xenon lamp. , And the fluorescence spectrum and fluorescence intensity emitted by cells labeled with a fluorescent dye can be measured, or the intracellular pH and calcium concentration can be measured (see, for example, Patent Document 1).
JP-A-9-159609

  In the measurement of fluorescent luminescent dyes using such a conventional fluorescent spectrophotometer, although the fluorescence emission intensity of the fluorescent luminescent dye becomes clear, there is a problem that the membrane permeability of the fluorescent luminescent dye to cells cannot be measured, and the fluorescence emission There is a demand for a fluorescent light emitting dye performance evaluation method and a fluorescent light emission measuring apparatus capable of measuring the fluorescent light emission intensity and membrane permeability of the dye.

  In addition, when measuring cells, which are test materials stained with a fluorescence spectrophotometer, there is a problem that the same test material cannot be measured with good reproducibility because the cells are not fixed in the cell. Thus, there is a demand for a fluorescence emission measurement method that can be measured in the same manner.

  The light source used in the fluorescence spectrophotometer is mainly a xenon lamp or halogen lamp, but it is a very expensive light source and requires a cooling device, which makes it difficult to reduce the size of the device. Has been.

  The present invention solves such a conventional problem, and it is possible to measure the membrane permeability in addition to the fluorescence emission intensity by evaluating and counting a plurality of fluorescent luminescent dyes with the same type of cells. In addition, the same specimen can be measured with good reproducibility of cells as test materials. Another object of the present invention is to provide a fluorescent light emitting dye evaluation method and apparatus that can be easily reduced in size because the use of an LED as the light source makes the light source very inexpensive and does not require a cooling device.

  In order to achieve the above object, the fluorescent dye evaluation method and apparatus of the present invention stain cells with a fluorescent light-emitting dye, and irradiate the stained cells with excitation light to cause fluorescent light emission, whereby the fluorescence emission luminance value and the dye The penetrability is quantified by an image analysis means, and the performance of the fluorescent luminescent dye with respect to the cells is evaluated.

  This provides a method for evaluating a fluorescent luminescent dye that measures the fluorescence emission luminance value and the dye dye permeability of the fluorescent luminescent dye to cells.

  In addition, the present invention stains cells with a fluorescent luminescent dye, irradiates the stained cells with excitation light and fluoresces, thereby quantifying the fluorescence emission luminance value and the dye permeability with an image analysis means, and fluorescing the cells. The performance of the dye is evaluated, and an excitation light and a light receiving wavelength filter that match the characteristics of the absorption wavelength and the fluorescence emission wavelength of the fluorescence emitting dye are provided.

  Thereby, optimal fluorescence conditions can be given to various fluorescent light-emitting dyes.

  In the present invention, cells are filtered through a microporous membrane support, irradiated with excitation light and imaged, and then the shape, area and fluorescence emission luminance are recognized by the image analysis means.

  This makes it possible to immobilize cells stained with fluorescent dyes, enable highly reproducible measurements, and to recognize the shape, area, and fluorescence emission brightness, so that they exist in addition to the stained cells. Differentiation from foreign matters becomes possible.

  The present invention also includes excitation light having an excitation wavelength of 350 nm to 400 nm and a light receiving wavelength filter having a wavelength of 430 nm to 580 nm.

  As a result, it is possible to measure a fluorescent luminescent dye having a wavelength belonging to the excitation wavelength or more and the light reception wavelength or less.

  The present invention also includes excitation light having an excitation wavelength of 430 nm to 485 nm and a light receiving wavelength filter having a light receiving wavelength of 520 nm to 565 nm.

  As a result, it is possible to measure a fluorescent luminescent dye having a wavelength belonging to the excitation wavelength or more and the light reception wavelength or less.

  Further, the present invention includes excitation light having an excitation wavelength of 472 nm to 545 nm and a light receiving wavelength filter having a light receiving wavelength of 590 nm to 646 nm.

  As a result, it is possible to measure a fluorescent luminescent dye having a wavelength belonging to the excitation wavelength or more and the light reception wavelength or less.

  Further, the present invention includes excitation light having an excitation wavelength of 550 nm to 570 nm and a light receiving wavelength filter having a light receiving wavelength of 590 nm to 646 nm.

  As a result, it is possible to measure a fluorescent luminescent dye having a wavelength belonging to the excitation wavelength or more and the light reception wavelength or less.

  The present invention also includes excitation light having an excitation wavelength of 630 nm to 670 nm and a light receiving wavelength filter having a light receiving wavelength of 690 nm or more.

  As a result, it is possible to measure a fluorescent luminescent dye having a wavelength belonging to the excitation wavelength or more and the light reception wavelength or less. Moreover, when measuring microorganisms, there is a means for dyeing live and dead bacteria and dead bacteria, but there is a means for dyeing live and dead bacteria and dead bacteria by combining with short wavelength excitation according to the previous excitation wavelength.

  In the present invention, the fluorescence emission luminance value and the dye permeability value obtained with the fluorescent light emitting dye performance evaluation apparatus are represented by a histogram.

  Thereby, it is possible to grasp at a glance at which wavelength the fluorescence emission luminance value has a peak, and smooth measurement is possible.

  Further, the present invention determines that the phenomenon that the half width of the histogram is small is that the fluorescent dye does not affect the cell activity.

  As a result, when screening for fluorescent luminescent dyes, it is possible to make a judgment based on the half-value width if there is no difference between the fluorescent light emission luminance value and the dye permeability.

  Further, in the present invention, in order to screen a fluorescent luminescent dye having good performance, the irradiation time of excitation light is adjusted as a means for adjusting the measurement conditions, and the fluorescence emission luminance value is converted after the measurement.

  As a result, it is possible to compare fluorescent luminescent dyes having a large difference in fluorescence emission luminance values at the same level, and it is also possible to evaluate fluorescent luminescent dyes having performance exceeding the limit of the apparatus.

  In addition, the present invention can evaluate the change in dyeability of fluorescent dyes by applying a load to cells.

  As a result, it is possible to evaluate a fluorescent luminescent dye assuming actual use.

  Moreover, in this invention, the time-dependent change of a cell can be measured.

  Thereby, the performance of the fluorescent luminescent dye with respect to the cells at the time of change can be grasped.

  In addition, in the present invention, the ability of the fluorescent luminescent dye to discolor the cells can be measured.

  Thereby, the fading ability of the fluorescent luminescent dye can be evaluated.

  ADVANTAGE OF THE INVENTION According to this invention, the evaluation method and apparatus of the fluorescent luminescent pigment | dye which can evaluate the fluorescence luminescence luminance value and pigment | dye permeability of a fluorescent luminescent pigment | dye can be provided.

  In addition, it is possible to provide a method and an apparatus for evaluating a fluorescent luminescent dye that can fix cells by filtering the cells as the test material on the microporous membrane support and can perform measurement with good reproducibility.

  Further, by adopting an LED as the light source, it is possible to provide a fluorescent light emitting dye evaluation device that extends the life of the light source, can be used inexpensively, in a small size and can be easily used, and can reduce the total energy used for evaluation.

  According to the first aspect of the present invention, cells are stained with a fluorescent luminescent dye, and the fluorescent emission luminance value and the dye permeability are digitized by an image analysis means by irradiating the stained cells with excitation light to cause fluorescence emission. , Which is intended to evaluate the performance of fluorescent dyes for cells, and has the effect of obtaining a method for evaluating fluorescent dyes by simply measuring the fluorescence emission luminance value and dye permeability of cells with fluorescent dyes. .

  The invention according to claim 2 of the present invention is provided with excitation light and light receiving wavelength filters that match the characteristics of the absorption wavelength and fluorescence emission wavelength of the fluorescent light-emitting dye, and is optimal for various fluorescent light-emitting dyes. By giving the fluorescence condition, it has an action that can be distinguished from the autofluorescent substance by making use of the characteristics of the fluorescent dye.

  Further, according to the third aspect of the present invention, after filtering a cell on a microporous membrane support, irradiating with excitation light and taking an image, the shape, area and fluorescence emission luminance are recognized by the image analysis means. It can immobilize cells stained with fluorescent dyes, enables reproducible measurement, and recognizes the shape, area, and fluorescence emission brightness to recognize stained cells and others. It has the effect of being able to differentiate from existing impurities.

  According to a fourth aspect of the present invention, there is provided an excitation light having an excitation wavelength of 350 nm to 400 nm and a light receiving wavelength filter having a wavelength of 430 nm to 580 nm, and having a wavelength belonging to the excitation wavelength or more and the reception wavelength or less. It has the effect | action which becomes possible to measure a fluorescent luminescent pigment | dye.

  The invention according to claim 5 of the present invention is provided with excitation light having an excitation wavelength of 430 nm to 485 nm and a reception wavelength filter having a reception wavelength of 520 nm to 565 nm, and a wavelength belonging to the excitation wavelength or more and the reception wavelength or less. It has the effect | action which becomes possible to measure the fluorescent luminescent pigment | dye which has.

  According to a sixth aspect of the present invention, the present invention comprises excitation light having an excitation wavelength of 472 nm to 545 nm and a light receiving wavelength filter having a light receiving wavelength of 590 nm to 646 nm. It has the effect | action which becomes possible to measure the fluorescent luminescent pigment | dye which has the wavelength which belongs to.

  According to a seventh aspect of the present invention, there is provided an excitation light having an excitation wavelength of 550 nm to 570 nm and a reception wavelength filter having a reception wavelength of 590 nm to 646 nm, and a wavelength belonging to the excitation wavelength or more and the reception wavelength or less. It has the effect | action which becomes possible to measure the fluorescent luminescent pigment | dye which has.

  The invention according to claim 8 of the present invention is provided with excitation light having an excitation wavelength of 630 nm to 670 nm and a light receiving wavelength filter having a light receiving wavelength of 690 nm or more, and a wavelength belonging to the excitation wavelength or more and the light receiving wavelength or less. Fluorescent dyes can be measured, and when measuring microorganisms, there is a means to distinguish between live and dead bacteria and dead bacteria, but by combining with short wavelength excitation by the previous excitation wavelength, live and dead bacteria and dead bacteria Has the effect of differentiating dyes.

  Further, the invention according to claim 9 of the present invention is a histogram showing the fluorescence emission luminance value and the dye permeability value obtained with the fluorescent light emitting dye performance evaluation apparatus, and at which wavelength the fluorescence emission luminance value is. It can be grasped at a glance that the peak is shown, and has an effect of enabling smooth measurement.

  In the invention according to claim 10 of the present invention, the phenomenon that the half width of the histogram is small is judged that the fluorescent luminescent dye does not affect the cell activity. In addition, when there is no difference in dye permeability, it has an effect that it can be determined by a half width.

  According to the eleventh aspect of the present invention, in order to screen a fluorescent dye having good performance, the irradiation time of the excitation light is adjusted as a means for adjusting the measurement conditions, and the fluorescence emission luminance value is converted after the measurement. The fluorescent light emitting dyes having a large difference in fluorescence emission luminance values can be compared at the same level.

  Further, the invention according to claim 12 of the present invention is capable of evaluating the change in staining property of the fluorescent luminescent dye by applying a load to the cells, and the effect of enabling evaluation of the fluorescent luminescent dye assuming actual use. Have

  The invention according to claim 13 of the present invention is capable of measuring changes with time of cells, and has an effect of grasping the performance of the fluorescent luminescent dye with respect to the cells at the time of change.

  In addition, the invention according to claim 14 of the present invention can measure the fading ability of a fluorescent luminescent dye to cells, and has an effect of evaluating the fading ability of the fluorescent luminescent dye.

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

(Embodiment)
Cells are fluorescent dyes (for example, SYTO11 to SYTO16, SYTO20 to SYTO25, SYTO40 to SYTO45, SYTO17, SYTO59 to SYTO64, SYTO80 to SYTO85, DAPI, Prodium Iodide, SYTOXGreen, SYBRGreen33, HoB33, HoB33, HoB 1) YO-PRO-1, TOTO-1, POPO-3, TO-PRO3, SYTOXBlue, SYTOXOrange, SYTO9), and the stained cells are composed of the microporous membrane 1 shown in FIG. The test material is filtered on the microporous membrane support 4, and an LED that is excitation light (for example, 350 nm to 400 nm, 430 nm to 485 nm, 472 nm to 472 nm) A light-receiving wavelength filter (for example, 430 nm) that emits fluorescent light by irradiating cells with a wavelength of 545 nm, 550 nm to 570 nm, or 630 nm to 670 nm and is suitable for a fluorescent dye that causes cells to emit fluorescence. The cells are imaged with a microorganism measuring device (not shown) having ˜580 nm, 520 nm to 565 nm, 590 nm to 646 nm, 590 nm to 646 nm, 690 nm or more suitable for the wavelength at which the cells emit fluorescence. Then, as shown in FIG. 2, the image analysis means 5 measures the brightness of the fluorescent emission point of the image taken 6 by the brightness measurement 7, and binarizes 8 with the reference brightness value as a boundary and exceeds the reference value. A luminescent spot having a luminance value is converted into a luminescent spot, the area of the shape converted into a luminescent spot in the shape determination 9 is recognized to determine whether the luminescence is a cell, and the luminescent spot determined to be a cell is counted 10 and measured 11 To do. As a result, the image analysis means 5 is used to prevent erroneous measurement of contaminants in the test material other than the cells. The cells collected in the microporous membrane 1 are weighed, and the fluorescent dye for the cells is dyed. It becomes possible to measure the permeability. Next, the brightness value analyzing means 12 shown in FIG. 3 is used to flatten 13 bright points and binarize 14 again to extract 15 bright points that cannot be confirmed with the naked eye. Using the histogram 16, the extracted image 15 represents the number of luminance values in one screen by frequency, and the maximum peak of the luminance value is clarified. As a result, the measurement 17 of the fluorescence emission luminance value of the fluorescence emitting dye with respect to the cell becomes possible.

Example 1
When screening for fluorescent luminescent dyes, first, the concentration of the fluorescent luminescent dye is adjusted. Since the fluorescent dye is an organic compound, dimethyl sulfoxide was selected as the diluent solvent. Note that not all fluorescent dyes are diluted with dimethyl sulfoxide because they are organic compounds. The manufacturer's recommended concentration was used as the dilution concentration, and the manufacturer's recommended concentration was also used as the dilution concentration. Next, the number of cells was adjusted, and microorganisms were used as test materials. In order to adjust the number of microorganisms, a pH 7.2 phosphate buffer solution was used as a diluting solvent so as not to affect the cell membrane due to the denaturation of the pH of the test material. As a staining method, staining was performed for 10 minutes in a microtube.

  After the dyeing is completed, it is filtered to the microporous membrane support 4, and based on the image analysis means 5 and the luminance value analysis means 12, the fluorescent dyes A, B, C, D, E, F, and G are shown in FIG. The evaluation results are shown. By measuring and comparing the fluorescence emission luminance value and the dye permeability for each fluorescence emitting dye and screening the fluorescence emitting dye, it is possible to grasp the characteristics of each fluorescence emitting dye.

(Example 2)
When confirming the staining property of the fluorescent luminescent dye, in order to grasp the influence of the staining property by applying a load to the cells, for example, when measuring microorganisms, a diluent solvent (preferably physiological saline, phosphoric acid is preferably used in a dedicated container. Take a means to leave the cells whose number of bacteria has been adjusted with a buffer solution or distilled water) for up to 700 hours, and spot-measure microorganisms between 0 hours and 700 hours. As shown in FIG. It was confirmed that the fluorescence emission luminance value and the dye permeability of the fluorescence emission dye with respect to the microorganism having the cell membrane in the state.

(Example 3)
Since the state in which the cell membrane of the microorganism that is the test material is damaged is defined as the dead cell state, the cell membrane of the microorganism was dissolved with an organic solvent (preferably ethanol). After the dissolution, the image analysis means 5 and the brightness value analysis means 12 can be used to quantitatively measure the fluorescence emission brightness value and dye permeability of the fluorescence emitting dye for dead microorganisms.

Example 4
Further, as shown in FIG. 6, by allowing the cells stained with the fluorescent luminescent dye to stand in the conditions and environment for drying, it is possible to measure the fading ability of the fluorescent luminescent dye to the cells. In addition, as a loading step applied to the fluorescent luminescent dye, it is also possible to measure 17 the fading ability of the fluorescent luminescent dye to the cells by leaving the cells stained with the fluorescent luminescent dye under irradiation with excitation light.

  In experiments using fluorescent staining methods in the pharmaceutical and laboratory fields, cells can be stained with fluorescent dyes and screened by measuring fluorescence emission luminance values and dye permeability using image analysis means and luminance value analysis means. It can be applied to screening fluorescent fluorescent dyes that can stain cells.

Whole sectional view showing a microporous membrane support according to Embodiment 1 of the present invention Schematic diagram from imaging to weighing according to Embodiment 1 of the present invention Schematic diagram from flattening to measurement of Embodiment 1 of the present invention Diagram of fluorescent dye screening results of Example 1 of the present invention Time-dependent evaluation chart of fluorescent luminescent dye for cells of Example 2 of the present invention Discoloration progress diagram of fluorescent luminescent dye on the cells of Example 4 of the present invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microporous film 2 Holding ring 3 Base 4 Microporous film support body 5 Image analysis means 6 Imaging 7 Luminance measurement 8 Binarization 9 Shape judgment 10 Count 11 Measurement 12 Luminance value analysis means 13 Flattening 14 Binarization 15 Extraction 16 Histogram 17 measurement

Claims (14)

The cells are stained with a fluorescent dye, and the fluorescence emission luminance value and the dye permeability are quantified by image analysis means by irradiating the stained cells with excitation light to emit fluorescence, and the performance of the fluorescent dye on the cells is evaluated. A method for evaluating the performance of a fluorescent luminescent dye characterized by the above. The cells are stained with a fluorescent dye, and the fluorescence emission luminance value and the dye permeability are quantified by image analysis means by irradiating the stained cells with excitation light to emit fluorescence, and the performance of the fluorescent dye on the cells is evaluated. An apparatus for evaluating the performance of a fluorescent luminescent dye, comprising: an excitation light and a light receiving wavelength filter that match the characteristics of the absorption wavelength and the fluorescent emission wavelength of the apparatus fluorescent luminescent dye. 2. The fluorescent luminescent dye according to claim 1, wherein the cell is filtered through a microporous membrane support, irradiated with excitation light and imaged, and then the shape, area and fluorescence emission luminance are recognized by the image analysis means. Performance evaluation method. The fluorescent light emitting dye performance evaluation apparatus according to claim 2, comprising excitation light having an excitation wavelength of 350 nm to 400 nm and a light receiving wavelength filter having a light receiving wavelength of 430 nm to 580 nm. 5. The fluorescent light emitting dye performance evaluation apparatus according to claim 2, further comprising excitation light having an excitation wavelength of 430 nm to 485 nm and a light receiving wavelength filter having a light receiving wavelength of 520 nm to 565 nm. 6. The fluorescent light emitting dye performance evaluation apparatus according to claim 2, comprising excitation light having an excitation wavelength of 472 nm to 545 nm and a light receiving wavelength filter having a light receiving wavelength of 590 nm to 646 nm. 7. The fluorescent light emitting dye performance evaluation apparatus according to claim 2, comprising excitation light having an excitation wavelength of 550 nm to 570 nm and a light receiving wavelength filter having a light receiving wavelength of 590 nm to 646 nm. 8. The fluorescent light emitting dye performance evaluation apparatus according to claim 2, comprising excitation light having an excitation wavelength of 630 nm to 670 nm and a light receiving wavelength filter having a light receiving wavelength of 690 nm or more. The fluorescent light emission luminance value and the dye membrane permeability value obtained by the fluorescent light emitting dye performance evaluation apparatus according to claim 2, 4, 5, 6, 7, or 8 are represented by a histogram. Or the method for evaluating the performance of the fluorescent light-emitting dye according to 3. The method for evaluating the performance of a fluorescent luminescent dye according to claim 1, 3 or 9, wherein the phenomenon in which the half width of the histogram is relatively small is determined that the fluorescent luminescent dye does not affect the cell activity. The fluorescent light emission according to claim 1, 3, 9 or 10, wherein the irradiation time of the excitation light is adjusted as a means for adjusting the measurement conditions, and the fluorescent light emission luminance value is converted after the measurement in order to screen the fluorescent light emitting dye having good performance. Method for evaluating dye performance. The method for evaluating the performance of a fluorescent light-emitting dye according to claim 1, 3, 9, 10 or 11, wherein the change in staining property of the fluorescent light-emitting dye can be evaluated by applying a load to the cells. 13. The method for evaluating the performance of a fluorescent luminescent dye according to claim 1, wherein the time-dependent change of cells is measured. The method for evaluating the performance of a fluorescent light-emitting dye according to claim 1, wherein the ability of the fluorescent light-emitting dye to fade is measured.

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