JP6977287B2 - Labeling method using multiple fluorescent substances - Google Patents

Description

The present invention relates to a method for detecting target particles contained in a sample by using a plurality of fluorescent substances having different maximum fluorescence wavelengths from each other.

When trying to estimate the characteristics and cell types of cells contained in samples such as blood samples, tissue suspensions, and culture mediums, the cells are labeled by staining with a substance that specifically binds to the organelles of the cells. It is usual to estimate the cells based on the labeled site and its intensity (labeling degree). For example, since tetramethylrhodamine methyl ester (TMRM) specifically binds to mitochondria, it is possible to know whether or not midchondria is present in the cell. Moreover, since mitochondria are organelles characteristically found in eukaryotic cells, it can be presumed that the cells stained (labeled) by TMRM are eukaryotic cells. Furthermore, when trying to estimate the cells contained in the sample from a plurality of characteristics, the cells are stained (labeled) with a plurality of substances corresponding to the plurality of characteristics, and the localized position and intensity of the labeled substance are determined. Based on this, the cells are estimated by detecting the characteristics.

When estimating the cells contained in the sample based on the degree of labeling, the degree of labeling is determined by labeling with a fluorescent substance that can specifically bind to the cells and measuring the fluorescence intensity derived from the labeled fluorescent substance. Fluorescence observation methods for judgment are often used. Since the fluorescence observation method can suppress the influence of ambient light, more accurate measurement is possible. When trying to estimate the cells contained in the sample by the fluorescence observation method based on a plurality of features, the cells are labeled with a plurality of fluorescent substances corresponding to the plurality of features, and the fluorescence intensity derived from the fluorescent substance is measured. In many cases, the optical system corresponding to the fluorescence spectrum of the fluorescent substance is sequentially switched one by one. While this method can measure the labeling condition of each labeled fluorescent substance with high accuracy, it requires measurement with multiple optical systems or multiple measurements with a single optical system for the same cell, and the measurement time. There was a problem that it became long.

As an example of a method that can shorten the measurement time in the fluorescence observation method, the fluorescence intensity derived from a plurality of fluorescent substances labeled on cells is digitally applied to a multi-bandpass filter (an optical filter having a plurality of transmission and shading ranges). A method of measuring using a color camera can be mentioned. Since this method can measure the dyeing condition of a plurality of fluorescent substances at the same time, the measurement time can be shortened. However, at the time of fluorescence detection, a part of the fluorescence derived from the specific labeled fluorescent substance is detected as the fluorescence derived from another labeled fluorescent substance (fluorescence leakage), and the accuracy of the fluorescence measurement is lowered. There was a problem. Normally, digital color cameras are designed to have a sensitivity in the range of 400 nm to 700 nm, which is recognizable by the human eye, while the fluorescence spectrum of the fluorescent substance used for labeling is wide, so multi-band pass filters and digital color cameras are used. Even if an attempt is made to correspond to the fluorescence spectra of a plurality of (particularly 3 or more) fluorescent substances, the above-mentioned fluorescence leakage may occur.

An object of the present invention is to provide a method for more accurately and quickly detecting fluorescence derived from each fluorescent substance labeled with a target particle.

The present inventor has found that leakage to other transmission bands can be reduced by using a fluorescent substance having a maximum fluorescence wavelength shorter than the lower limit of the transmission band on the shortest wavelength side of the multiband pass filter. It came to be completed.

That is, the present invention comprises a step of labeling the target particles with three or more fluorescent substances having different maximum fluorescence wavelengths from each other.
The step of irradiating the target particles with excitation light and
A step of measuring the fluorescence derived from the fluorescent substance labeled on the target particles with a digital color camera through a multi-bandpass filter, and
A method for measuring the target particles containing
The measurement method is characterized in that the maximum fluorescence wavelength of the fluorescent substance having the shortest maximum fluorescence wavelength is shorter than the lower limit of the transmission band on the shortest wavelength side of the multi-bandpass filter.

Hereinafter, the present invention will be described in detail.

In the present invention, the target particles include biomaterials that can be labeled with fluorescent substances such as cells, viruses, organelles, and vesicles. When the target particle is a particle made of the above-mentioned biological material, as an example of the sample containing the target particle, whole blood, diluted blood, serum, plasma, umbilical cord blood, component blood sampling, spinal fluid, urine, saliva, semen, etc. Biological samples such as feces, sputum, sheep's water, ascites, and peritoneal lavage fluid, tissue suspensions in which a piece of tissue such as liver, lung, spleen, kidney, skin, tumor, and lymph node is suspended, or the biological sample or Examples thereof include a fraction containing the biological sample or cells derived from the tissue obtained by separating from the tissue suspension, a culture solution of cells isolated in advance, and the like. Among these, as an example of a fraction containing cells derived from a biological sample or tissue, there is a fraction obtained by layering a biological sample or tissue suspension on a medium for forming a density gradient and then centrifuging the density gradient.

When the solution containing the target particles is a blood sample such as whole blood, diluted blood, serum, plasma, umbilical cord blood, or component blood sample, an example of the target particles is a tumor cell such as a blood circulation tumor cell (CTC). Various stem cells such as circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), circulating fetal cells (CFC), and antigen-specific T cells can be mentioned. The blood sample in the present specification is not limited to blood samples such as whole blood, serum, plasma, umbilical cord blood, and component blood sample, but is separated from a sample obtained by diluting the blood sample with physiological saline or the like or the blood sample. The fraction containing the cells derived from the blood sample obtained is also included in the blood sample.

In the present invention, the fluorescent substance used for labeling the target particles is not particularly limited as long as it is a substance having a maximum fluorescence wavelength different from each other. As an example of the embodiment of labeling a fluorescent substance on a target particle in the present invention, a single target particle contained in a sample is subjected to a fluorescent substance having different maximum fluorescence wavelengths (for example, a fluorescent substance having a maximum fluorescence wavelength A, a maximum fluorescence wavelength). A mode of labeling with a fluorescent substance of B and a fluorescent substance having a maximum fluorescence wavelength of C) and a plurality of types of target particles contained in the sample (for example, three types of target particle A, target particle B and target particle C). Each target particle is labeled with a fluorescent substance having a different maximum fluorescence wavelength from each other (for example, a fluorescent substance having a maximum fluorescence wavelength A for the target particle A, a fluorescent substance having a maximum fluorescence wavelength B for the target particle B, and a target). An embodiment of labeling the particles C with a fluorescent substance having a maximum fluorescence wavelength C) can be mentioned. A plurality of types of the fluorescent substances having the maximum fluorescence wavelength A (or B, C) may be used in combination as long as the maximum fluorescence wavelengths are the same or substantially the same (for example, the difference between the maximum fluorescence wavelengths is within 5 nm).

In the present invention, "labeling the target particles with a fluorescent substance" means physically and chemically binding the target particles and the fluorescent substance. The labeling method is not particularly limited, and for example, the target particle may be labeled by directly dyeing it with a fluorescent dye, or by binding the target particle and a fluorescent substance-labeled antibody against the target-specific substance by an antigen-antibody reaction. It may be labeled, and after binding the target particle and an antibody against the specific substance (primary antibody) by an antigen-antibody reaction, the fluorescent substance-labeled antibody (secondary antibody) against the primary antibody is bound by an antigen-antibody reaction. It may be labeled by letting it.

In the present invention, the fluorescence derived from the fluorescent substance labeled on the target particles is measured by a digital color camera through a multi-bandpass filter. The multi-bandpass filter is a bandpass filter having a plurality of transmission bands, and any filter having a transmission band corresponding to the fluorescence spectrum of each fluorescent substance may be appropriately selected from commercially available products. In the present specification, the "transmission band corresponding to the fluorescence spectrum" does not necessarily mean that the maximum fluorescence wavelength of the fluorescent substance is included in the transmission band of the multiband pass filter, and the fluorescence intensity at the maximum fluorescence wavelength of the fluorescent substance is used. It suffices if the wavelength exhibiting the fluorescence intensity of 80% or more (preferably 90% or more) of the above is included in the transmission band of the multi-band pass filter. As a specific example, in the case of a multi-bandpass filter having a transmission band of 425 nm to 465 nm, the maximum fluorescence wavelength of 421 nm of Alexa Fluor 405 is not included in the transmission band. However, since the transmission band is included in the wavelength (405 nm to 435 nm, see FIGS. 1 and 5) showing a fluorescence intensity of 80% or more of the fluorescence intensity at the maximum fluorescence wavelength of the Alexa Fluor 405, the multi-bandpass filter is used. It can be said that the filter has a transmission band corresponding to the fluorescence spectrum of Alexa Fluor 405.

In addition, a digital color camera is a device that has a sensor that converts the intensity of light into an electric signal and can obtain an image for each wavelength band. Typical sensor elements include a charge-coupled device (CCD) and a complementary metal oxide semiconductor (CMOS), but what kind of element is used in the present invention? You may. Many digital color cameras are made to correspond to the three primary colors of light, blue, green, and red, but the digital color camera in the present invention is not limited to this, and obtains images in two or four or more wavelength bands. It may be a thing. There are single-plate type and three-plate type as typical methods for dividing by wavelength band. The single plate type is a method in which a wavelength band shared by each light receiving element is assigned on one sensor. The three-plate type is a method in which an image for each wavelength band is obtained by each sensor after being optically decomposed by a dichroic mirror or the like. The present invention may be either a single plate type or a three-plate type, and is not limited to these, and may be any type as long as it receives light separately for each wavelength band.

The present invention is characterized in that the maximum fluorescence wavelength of the fluorescent substance having the shortest maximum fluorescence wavelength is shorter than the lower limit of the transmission band on the shortest wavelength side of the multi-bandpass filter. In other words, among the transmission bands of the multi-bandpass filter, the transmission band corresponding to the fluorescence spectrum of the fluorescent substance having the shortest maximum fluorescence wavelength is characterized by being on the longer wavelength side than the maximum fluorescence wavelength of the fluorescent substance. There is. The transmission band other than the transmission band may be included in the maximum fluorescence wavelength of the fluorescent substance having the fluorescence spectrum corresponding to the transmission band.

When the target particle is a cell, DAPI (4', 6-DiAmidino-2-PhenylIndole), which is a blue fluorescent substance, is often used as a fluorescent substance that labels the nucleus. However, the maximum fluorescence wavelength of this substance is 461 nm, which is relatively long on the long wavelength side for a blue fluorescent substance. Therefore, cells labeled at least with DAPI and a green fluorescent substance (eg, SYSTEM Green (manufactured by Thermo Fisher Scientific) having a maximum fluorescence wavelength of 523 nm) are passed through a multi-band pass filter having a transmission band corresponding to each fluorescence spectrum. When attempting to detect, a large amount of fluorescence derived from DAPI leaks into the fluorescence that has passed through the transmission band corresponding to the fluorescence spectrum of the green fluorescent substance. For example, when the blue fluorescent substance is DAPI and the transmission band of the multi-bandpass filter has a wavelength of 425 nm to 465 nm (hereinafter referred to as channel A) and a wavelength of 500 nm to 565 nm (hereinafter referred to as channel B), from channel B. DAPI-derived fluorescence corresponding to 50% of the amount of fluorescence from channel A leaks into the fluorescence of. Therefore, by changing the blue fluorescent substance to Alexa Fluor 405 (maximum fluorescence wavelength: 421 nm) having the maximum fluorescence wavelength on the shorter wavelength side than the channel A, the amount of leakage to the channel B can be reduced to about the amount of fluorescence from the channel A. It can be reduced to 5% amount.

In addition, the target particle is a cell, the label on the cell contains at least the label on the cell nucleus, and the label on the cell is at least a blue fluorescent substance (for example, a fluorescent substance having a maximum fluorescent wavelength of 400 nm to 500 nm) and a green fluorescent substance (for example,). , A transmission band and a transmission band comprising a red fluorescent material (eg, a fluorescent material having a maximum fluorescent wavelength of 600 nm to 650 nm) or a multiband pass filter corresponding to at least the fluorescence spectrum of the blue fluorescent material. When the transmission bands corresponding to the fluorescence spectra of the green fluorescent substance or the red fluorescent substance do not overlap each other, the labeling substance for the cell nucleus is preferably a green fluorescent substance or a red fluorescent substance. As described above, the maximum fluorescence wavelength of the blue fluorescent substance known as a labeling substance for cell nuclei is on the long wavelength side (specifically, 450 nm or more), and the transmission of a multi-band pass filter is applied in order to apply the method of the present invention. This is because if the band is set to a longer wavelength side than the maximum fluorescence wavelength, the detection region on the long wavelength side (green / red side) becomes narrow and multicolor detection becomes difficult. As an example of a green fluorescent substance or a red fluorescent substance that can be labeled on the cell nucleus, SYSTEM X Green (manufactured by Thermo Fisher Scientific) having a maximum fluorescence wavelength of 523 nm can be mentioned. Note that SYSTEMG Green has a narrow full width at half maximum, which is a preferable embodiment as a cell nucleus labeling substance used in the method of the present invention.

INDUSTRIAL APPLICABILITY According to the present invention, leakage of fluorescence into a transmission band adjacent to a long wavelength side can be reduced, and rapid and highly sensitive measurement of target particles labeled with a plurality of fluorescent substances having different maximum fluorescence wavelengths becomes possible.

Fluorescence spectrum of a fluorescent substance (Alexa Fluor 405, Alexa Fluor 350). Spectral characteristics of the multi-bandpass filter used in the examples. The spectral sensitivity curve of the color camera (CMOS) used in the examples. Fluorescence images obtained by labeling with Alexa Fluor 405 or Alexa Fluor 350 (blue channel (wavelength: 425 nm to 465 nm), green channel (wavelength: 500 nm to 565 nm) and green channel / blue channel). The area surrounded by the white square in the figure is the area used for calculating the fluorescence intensity ratio (green channel / blue channel). Fluorescence spectrum of a fluorescent substance (SYSTOX Green, Alexa Fluor 633, Alexa Fluor 405). Fluorescent image obtained in the example (originally an image consisting of three colors of blue, green, and red, which is an image combined into one color). It is an image which color-separated (blue channel) the image data of FIG. It is an image which color-separated (green channel) the image data of FIG. It is an image which color-separated (red channel) the image data of FIG.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

(Effect of fluorescence leakage due to difference in fluorescent material)
(1-1) Cancer cell line PC-9 is cultured in a culture dish, and the cultured cancer cells are fixed with a 4% paraformaldehyde solution and membrane permeabilized with ethanol, and then Anti-Pan as a primary antibody. Using CK (cytokeratin) antibody (derived from mouse, homemade) and Alexa Fluor 405-labeled anti-mouse IgG antibody (manufactured by Abcam) or Alexa Fluor 350-labeled anti-mouse IgG antibody (manufactured by Thermo Fisher Scientific) as secondary antibodies, respectively. The CK in the cytoplasm was labeled with a fluorescent substance. The maximum fluorescence wavelength of the Alexa Fluor 405 is 421 nm, and the maximum fluorescence wavelength of the Alexa Fluor 350 is 442 nm (the fluorescence spectrum of the fluorescent substance used in this example is shown in FIG. 1).

(1-2) Cancer cells labeled with a fluorescent substance are irradiated with excitation light (light containing a wavelength of 401 nm for cells labeled with Alexa Fluor 405, light containing a wavelength of 346 nm for cells labeled with Alexa Fluor 350). The obtained fluorescence was detected with a fluorescence microscope (IX83 manufactured by Olympus), and from a multi-band pass filter (transmission band: 425 nm to 465 nm (hereinafter, also referred to as blue channel), 500 nm to 565 nm (hereinafter, also referred to as green channel) and 610 nm. The spectral sensitivity curve of a color camera (DFK23UX236 manufactured by Imaging Source, CMOS (IMX236 manufactured by Sony) provided in this camera) through 685 nm (hereinafter, also referred to as red channel), manufactured by Semirock, and spectral characteristics are shown in FIG. 2). I took an image with.

(1-3) Fluorescence brightness in the green channel with respect to the fluorescence brightness of the blue channel in a specific region (the region surrounded by the square in FIG. 4) of the cytoplasmic portion in the fluorescence image of the blue channel and the fluorescence image of the green channel (FIG. 4). The ratio (fluorescence brightness in the green channel / fluorescence brightness in the blue channel) was calculated, and the leakage of fluorescence into the green channel was evaluated.

As a result of calculating the fluorescence intensity ratio, it is 0.358 ± 0.0141 when Alexa Fluor 405 is used as the fluorescent substance, and 0.384 ± 0.0973 (range is standard deviation) when Alexa Fluor 350 is used. It can be seen that by using Alexa Fluor 405 as the fluorescent substance, the leakage of fluorescence into the green channel is suppressed as compared with the case of using Alexa Fluor 350.

From the above results, Alexa Fluor 405 (maximum fluorescence wavelength: 421 nm) whose maximum fluorescence wavelength is on the lower wavelength side than the transmission band of the channel is used as the fluorescent dye used for fluorescence detection in the blue channel (wavelength: 425 nm to 465 nm). It turns out that it is preferable to use it.

(Fluorescence detection of cancer cells using 3 types of fluorescent substances)
(2-1) Cancer cell line PC-9 is cultured in a culture dish, and the cultured cancer cells are fixed with a 4% paraformaldehyde solution and membrane permeabilized with ethanol, and then the cell nucleus, nuclear envelope, and cytoplasm. The fluorescent substances were labeled on the CKs in the cells by the methods shown below. The fluorescence spectrum of the fluorescent substance used in this example is shown in FIG.
Cell nucleus: Stained with SYSTEMX Green (maximum fluorescence wavelength: 523 nm, manufactured by Thermo Fisher Scientific) Nucleus membrane: Primary antibody Anti-SUN2 antibody (derived from rabbit, manufactured by Sigma Life Science), and then secondary antibody Alexa Fluor 633 (maximum fluorescence) : 633 nm) Labeled cytoplasm (CK) by adding a labeled anti-rabbit antibody (manufactured by Thermo Fisher Scientific): Primary antibody Anti-Pan CK (cytokeratin) antibody (derived from mouse, homemade), and then secondary antibody Alexa Fluor. A fluorescence image was obtained by adding a 405-labeled anti-mouse IgG antibody (manufactured by Abcam) using a fluorescence microscope, a multi-band pass filter and a color camera similar to those labeled (2-2) and (1-2).

The acquired fluorescence image is shown in FIG. Further, from the image of FIG. 6, the images color-separated for each transmission band (each channel) of the multi-band pass filter are shown in FIG. 7 (blue channel, corresponding to the fluorescence image by Alexa Fluor 405) and FIG. 8 (green channel, SYSTEMG Green). (Corresponding to the fluorescence image by) and FIG. 9 (red channel, corresponding to the fluorescence image by Alexa Blue 633). From the results shown in FIG. 6, it was confirmed that the cell nuclei, nuclear envelope and cytoplasm (CK) of the cancer cells were stained with the respective fluorescent substances. In addition, the dyeing condition of each channel (each fluorescent dye) could be confirmed from the color-separated images (FIGS. 7 to 9).

From this result, the maximum fluorescence wavelength of the fluorescent dye for fluorescence detection in the green channel (wavelength: 500 nm to 565 nm) and the red channel (wavelength: 610 nm to 685 nm) is within the channel (transmission band). It can be seen that there is no problem (maximum fluorescence wavelength of SYSTEMG Green: 523 nm, maximum fluorescence wavelength of Alexa Fluor 633: 633 nm).

Claims (2)

A step of labeling the target particles with three or more fluorescent substances having different maximum fluorescence wavelengths from each other.
The step of irradiating the target particles with excitation light and
A step of measuring the fluorescence derived from the fluorescent substance labeled on the target particles with a digital color camera through a multi-bandpass filter, and
It is a method of quantitatively measuring the fluorescence brightness of the target particles containing
A measuring method characterized in that the maximum fluorescence wavelength of a fluorescent substance having the shortest maximum fluorescence wavelength is shorter than the lower limit of the transmission band on the shortest wavelength side of the multi-bandpass filter. The method according to claim 1, wherein the target particle is a cell, and a fluorescent substance having a maximum fluorescence wavelength of the fluorescence spectrum second or later counted from the short wavelength side is used for staining the cell nucleus. ..

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