CN106645713A

CN106645713A - Cell information obtaining method and cell information obtaining apparatus

Info

Publication number: CN106645713A
Application number: CN201610958244.XA
Authority: CN
Inventors: 吉川景子
Original assignee: Sysmex Corp
Current assignee: Sysmex Corp
Priority date: 2015-10-30
Filing date: 2016-10-28
Publication date: 2017-05-10
Anticipated expiration: 2036-10-28
Also published as: JP6824654B2; CN106645713B; JP2017083426A

Abstract

The invention discloses a cell information obtaining method and a cell information obtaining apparatus. The cell information obtaining method comprises causing a plurality of fluorescent substances having different fluorescence wavelengths from each other to be bound to a test substance contained in a cell, applying light to the cell to cause fluorescences having different wavelengths and intensities to be generated from the plurality of fluorescent substances, and obtaining fluorescence information on the basis of the generated fluorescences. For example, the fluorescences generated from the plurality of fluorescent substances are separated into a first fluorescence and a second fluorescence with intensity lower than that of the first fluorescence by a filter member. The distribution of the substance detected in the cell is determined according to the fluorescence information.

Description

Cellular informatics acquisition methods and cellular informatics acquisition device

Technical field

The present invention relates to cellular informatics acquisition methods and cellular informatics acquisition device.

Background technology

In various biological phenomenas headed by the propagation of cell, differentiation, the various molecules such as protein, mRNA, microRNA Locally lie in cell.When locally lying in of various molecules in parsing cell, expects function parsing, the protein of molecule The solution of the substantial amounts of biological phenomenas such as the parsing of interphase interaction, the parsing of signaling path is bright.

In International Publication No. 2005/098430, disclose by fluorescence microscope and imaging flow cytometer come The method for locally lying in of the intracellular molecule of parsing.

Even origin identical cell of the same race, each cell is also diversified, so, for example, both just like figure The cell that specific molecule shown in 23 (a) locally lies at specific position, also just like specific molecule shown in Figure 23 (b) at it The cell that its position locally lies in.In addition, for example, also there is the amount of the molecule as shown in Figure 23 (c) due to various factors thin Uneven situation between born of the same parents.In addition, inventor is found that distribution and diversified point of amount if it is intended to from cell Sub- acquisition information, then can produce deviation in result is obtained.Therefore, it is desirable to precision parse distribution in this like cell well with And the gimmick of the diversified molecule of amount.

The content of the invention

The scope of the present invention is limited only by the accompanying claims, not by any impact of this content of the invention explanation.

1st scheme of the present invention is related to cellular informatics acquisition methods.In the cellular informatics acquisition methods of this programme, make glimmering The mutually different multiple fluorescent materials of optical wavelength are combined with the tested substance included in cell, to cell irradiation light cause from Multiple fluorescent materials produce the different fluorescence of wavelength and intensity, and according to each fluorescence for producing multiple fluorescence informations are obtained.

In the cellular informatics acquisition methods of this programme, " the mutually different multiple fluorescent materials of wavelength of fluorescence " are referred to, Multiple fluorescent materials send respectively the fluorescence of mutually different wavelength during illuminated light.In order that producing from multiple fluorescent materials The different fluorescence of raw wavelength and intensity, such as in multiple fluorescent materials, the mutually different situation of the wavelength of the light of excitation Under, for the different multiple light of cell irradiation wavelength and intensity.Image of the fluorescence information e.g. based on fluorescence.Additionally, " making Multiple fluorescent materials are combined with tested substance " refer to, it is also possible to it is not necessarily the tested substance of the same race included in cell Each molecule is all combined with multiple fluorescent materials, multiple fluorescent materials especially with least one of tested substance of the same race Molecule combine.

In the case that the distribution of the tested substance in cell and amount are varied, exist from fluorescent material and produce Fluorescence too strong situation, the excessively weak situation of intensity, so may not suitably differentiate detected using 1 fluorescence information Distribution situation of material etc..However, according to the cellular informatics acquisition methods of this programme so that many from what is combined with tested substance Individual fluorescent material produces the different fluorescence of intensity, can obtain multiple fluorescence informations according to the different fluorescence of intensity.Thus, even if In the case of cannot suitably differentiating distribution situation of tested substance etc. because fluorescence is too strong during using a fluorescence information, If using other fluorescence informations, it becomes possible to suitably differentiate distribution situation of tested substance etc..Therefore, as long as using a certain Individual fluorescence information just can suitably differentiate distribution situation of tested substance etc., even if so tested substance in cell Distribution and amount are varied, it is also possible to which precision parses well tested substance.

2nd scheme of the present invention is related to cellular informatics acquisition methods.In the cellular informatics acquisition methods of this programme, base is made Matter contacts to cause to produce the mutually different multiple fluorescent materials of wavelength of fluorescence with the tested substance included in cell, to cell Irradiation light and cause to produce wavelength and the different fluorescence of intensity from multiple fluorescent materials, obtain multiple according to each fluorescence for producing Fluorescence information.

In the cellular informatics acquisition methods of this programme, matrix is set to contact to cause with the tested substance included in cell The mutually different multiple fluorescent materials of wavelength of fluorescence are produced, thus tested substance is identified with multiple fluorescent materials.In this programme Cellular informatics acquisition methods in, also in the same manner as the 1st scheme, as long as just can suitably be differentiated using some fluorescence information Distribution situation of tested substance etc., it is possible to precision parses well tested substance.

3rd scheme of the present invention is related to cellular informatics acquisition methods.In the cellular informatics acquisition methods of this programme, make glimmering Stimulative substance is combined with the tested substance included in cell, cell irradiation light is caused to produce fluorescence from fluorescent material, from product Raw fluorescence obtains the different multiple fluorescence of wavelength and intensity, and according to each fluorescence for obtaining multiple fluorescence informations are obtained, according to Multiple fluorescence informations, differentiate the distribution situation of the tested substance in cell.

In the cellular informatics acquisition methods of this programme, a kind of fluorescent material is combined with tested substance.In order to from generation Fluorescence obtains the different multiple fluorescence of wavelength and intensity, for example, makes the fluorescence produced from fluorescent material pass through transmission peak wavelength section Different multiple filter members and separate.Additionally, in the cellular informatics acquisition methods of this programme, fluorescence information is also for example Image based on fluorescence.Alternatively, it is also possible to be not necessarily the tested substance of the same race included in cell each molecule all with it is glimmering Stimulative substance is combined, and fluorescent material is especially combined with the molecule of at least one of tested substance of the same race.In we In the cellular informatics acquisition methods of case, also in the same manner as the 1st scheme, as long as just can suitably be sentenced using some fluorescence information Distribution situation of other tested substance etc., it is possible to precision parses well tested substance.

4th scheme of the present invention is related to cellular informatics acquisition methods.In the cellular informatics acquisition methods of this programme, base is made Matter is combined to cause to produce fluorescent material with the tested substance included in cell, and cell irradiation light is caused from fluorescent material Fluorescence is produced, from the fluorescence for producing the different multiple fluorescence of wavelength and intensity are obtained, obtain multiple according to each fluorescence for obtaining Fluorescence information, according to multiple fluorescence informations, differentiates the distribution situation of the tested substance in cell.

In the cellular informatics acquisition methods of this programme, by matrix is contacted with the tested substance included in cell come So that producing fluorescent material, with fluorescent material tested substance is identified.In the cellular informatics acquisition methods of this programme, also with 3 schemes similarly, as long as just can suitably differentiate distribution situation of tested substance etc. using some fluorescence information, so Can precision parse tested substance well.

5th scheme of the present invention is related to cellular informatics acquisition device.The cellular informatics acquisition device of this programme possesses：Illumination Portion is penetrated, to the cell irradiation light comprising the tested substance for combining the mutually different multiple fluorescent materials of wavelength of fluorescence so that The different fluorescence of wavelength and intensity is produced from multiple fluorescent materials；Light accepting part, receives from each glimmering of multiple fluorescent materials generation Light；And acquisition unit, multiple fluorescence informations are obtained according to the different fluorescence of intensity.

According to the cellular informatics acquisition device of this programme, the effect same with the 1st scheme can be played.

6th scheme of the present invention is related to cellular informatics acquisition device.The cellular informatics acquisition device of this programme possesses：Illumination Portion is penetrated, the cell irradiation light comprising the tested substance for combining fluorescent material is caused to produce fluorescence from fluorescent material；Receive Light portion, receives from the different multiple fluorescence of the wavelength and intensity of fluorescent material generation；Acquisition unit, it is multiple glimmering according to what is received Light, obtains multiple fluorescence informations；And analysis unit, according to multiple fluorescence informations, differentiate the distribution of the tested substance in cell Situation.

According to the cellular informatics acquisition device of this programme, the effect same with the 3rd scheme can be played.

7th scheme of the present invention is related to cellular informatics acquisition device.The cellular informatics acquisition device of this programme possesses：Illumination Portion is penetrated, to comprising the cell irradiation light that the tested substance for producing fluorescent material is caused by contact with matrix and so that from glimmering Stimulative substance produces fluorescence；Light accepting part, receives from the different multiple fluorescence of the wavelength and intensity of fluorescent material generation；Acquisition unit, root According to the multiple fluorescence for being received, multiple fluorescence informations are obtained；And analysis unit, according to multiple fluorescence informations, in differentiating cell Tested substance distribution situation.

According to the cellular informatics acquisition device of this programme, the effect same with the 4th scheme can be played.

In accordance with the invention it is possible to precision parses well the distribution in cell and measures diversified molecule.

Description of the drawings

Fig. 1 is the flow chart of the cellular informatics acquisition methods for illustrating embodiment 1.

Fig. 2 is the figure for illustrating the summary that the fluorescence of embodiment 1 is obtained.

Fig. 3 is the concept for illustrating the image obtained according to the fluorescence and low intensive fluorescence of the high intensity of embodiment 1 Figure.

Fig. 4 (a) is the figure for illustrating the image obtained in the checking of embodiment 1.Fig. 4 (b) is illustrated in embodiment 1 Checking in obtain numerical value figure.

Fig. 5 is the block diagram of the structure of the device for illustrating embodiment 1.

Fig. 6 is the figure of the structure in the optical detection portion for illustrating embodiment 1.

Fig. 7 is the figure of the structure in the optical detection portion of the modification for illustrating embodiment 1.

Fig. 8 is the flow chart for illustrating the process that the utilization cellular informatics acquisition device of embodiment 1 is carried out.

Fig. 9 is the figure for illustrating the picture shown in the display part of embodiment 1.

Figure 10 is the figure that the fluorescence of the high intensity for illustrating the modification according to embodiment 1 and low intensive fluorescence are obtained The concept map of table.

Figure 11 is the figure for illustrating the summary that the fluorescence of embodiment 2 is obtained.

Figure 12 is the figure for illustrating the summary that the fluorescence of embodiment 3 is obtained.

Figure 13 is the wavelength period for illustrating the fluorescence of the transmission peak wavelength section and acquisition of the filter member of embodiment 3 Figure.

Figure 14 is the figure for illustrating the image obtained in the checking of embodiment 3.

Figure 15 is the figure of the structure in the optical detection portion for illustrating embodiment 3.

Figure 16 is the figure for illustrating the summary that the fluorescence of embodiment 4 is obtained.

Figure 17 is the figure of the structure in the optical detection portion for illustrating embodiment 4.

Figure 18 is the figure for illustrating the summary that the fluorescence of embodiment 5 is obtained.

Figure 19 is the figure of the structure in the optical detection portion for illustrating embodiment 5.

Figure 20 is the figure for illustrating the summary that the fluorescence of embodiment 6 is obtained.

Figure 21 is the figure for illustrating the image obtained in the checking of embodiment 6.

Figure 22 is the figure for illustrating the summary that the fluorescence of embodiment 7 is obtained.

Figure 23 is the schematic diagram of the problem solved for illustrating invention to want.

Specific embodiment

Below with reference to Description of Drawings the preferred embodiments of the present invention.

In embodiment 1, what is included in the multiple fluorescent materials for making wavelength of fluorescence mutually different with cell is detected Material combines and differentiates the cell for locally lying in situation of tested substance according to the multiple fluorescence produced from fluorescent material In information getting method, the present invention is applied.In embodiment 1, tested substance is NF- κ B.As the NF- of transcription factor κ B are present in cytoplasm in the state of complex is defined with I κ B, it is believed that by stimulating dividing for the I κ B for causing due to various Solve and be transferred to the inside of core.In embodiment 1, parsed as follows：Using NF- κ B as tested substance, fluorescence is used Matter is especially identified, and according to the fluorescence from fluorescent material, which in cytoplasm and core judgement NF- κ B be present in.Additionally, Tested substance can also be protein, the molecule beyond NF- κ B.For example, tested substance can also be turning beyond NF- κ B The record factor, for example, it is also possible to be STAT (Signal Transducer and Activator of Transcription, letter Number transduction and activating transcription factor), NFAT (nuclear factor of activated T cells, the core of activating T cell The factor), HIF (hypoxia-inducible factor, hypoxia inducible factor).In addition, tested substance can also be mRNA, microRNA.In addition, " multiple fluorescent materials is combined with tested substance " refers to, it is not necessarily the quilt of the same race included in cell Each molecule of detection material is all combined with multiple fluorescent materials, and multiple fluorescent materials are especially of the same race with least one of The molecule of tested substance is combined.In addition, the differentiation for locally lying in situation is not limited to tested substance locally lying in core In still locally lie in the differentiation in cytoplasm.For example, in the case where there is shape for lugs as nerve cell, also may be used To differentiate whether tested substance is locally lain in the front end of projection.

As shown in figure 1, the step of cellular informatics acquisition methods include step S1～S4.Hereinafter, illustrate that operator uses energy Enough flow cytometers imaged to fluoroscopic image and the processing meanss that can be parsed to the image that images are performing Fig. 1 Cellular informatics acquisition methods situation.Each step of Fig. 1 can also be performed by the process in cellular informatics acquisition device. Subsequently, with reference to after Fig. 5, illustrate that cellular informatics acquisition device carries out the structure in the case of each step of Fig. 1 and process.

In step sl, operator identifies what is extracted from examinee with the mutually different fluorescent material 11,12 of wavelength of fluorescence The NF- κ B included in cell.For example, as shown in Fig. 2 via an antibody and secondary antibodies, in fluorescent material 11,12 and cell Comprising NF- κ B combine.Fluorescent material 11,12 both can be combined by multiple antibody with NF- κ B, it is also possible to by anti- A part for body or the whole of antibody and combined with NF- κ B.In addition, in step sl, operator's wavelength of fluorescence and fluorescence The different fluorescent material 13 of material 11,12, the core included in marked cells.

Fluorescent material 11,12,13 is fluorchrome.Fluorescent material 11,12,13 is respectively structured as when illuminated wavelength X 1, λ 2nd, the fluorescence of mutually different wavelength period is encouraged during the light of λ 3.That is, for from the ripple of the light of the excitation fluorescence of fluorescent material 11～13 Length is set to mutually different.So, sample is modulated by step S1.Additionally, being mRNA, microRNA in tested substance In the case of, fluorescent material is combined via nucleic acid probe with tested substance.

In step s 2, operator drives flow cytometer, makes the examination comprising the cell with the mark of fluorescent material 11～13 Sample is flowed into flow cell, the light of the 1～λ of cell irradiation wavelength X 3 to flowing in flow cell so that from fluorescent material 11～13 Produce fluorescence.

As shown in Fig. 2 when the laser to 1～λ of cell irradiation wavelength X 3, from fluorescent material 11～13 difference is produced respectively Wavelength period fluorescence.Filter member 21 passes through the fluorescence of wavelength period B1 produced from fluorescent material 11, interdicts wavelength period Light beyond B1.By filter member 21, the fluorescence of wavelength period B1 produced from fluorescent material 11 is separated.Filter member 22 pass through the fluorescence of wavelength period B2 produced from fluorescent material 12, interdict the light beyond wavelength period B2.By filter member 22, the fluorescence of wavelength period B2 produced from fluorescent material 12 is separated.Filter member 23 makes the ripple produced from fluorescent material 13 The fluorescence of long section B3 passes through, and interdicts the light beyond wavelength period B3.By filter member 23, from the wavelength that fluorescent material 13 is produced The fluorescence of section B3 is separated.

Here, the laser with high power to cell irradiation wavelength X 1, with laser of the low-power to cell irradiation wavelength X 2.It is logical The laser to cell irradiation wavelength X 1 with high power is crossed, having passed through the fluorescence of wavelength period B1 of filter member 21 becomes high-strength Degree.By the laser with low-power to cell irradiation wavelength X 2, having passed through the fluorescence of wavelength period B2 of filter member 22 becomes Low-intensity.

In step s3, processing meanss obtain 3 according to the fluorescence produced from fluorescent material 11～13 for each cell Fluorescence information.Flow cytometer possesses for distinguishing the fluorescence by detached wavelength period B1 of filter member 21～23～B3 It is imaged onto the structure that the image based on each fluorescence is obtained by the imaging apparatus light accepting part for constituting.Processing meanss are according to fluidic cell The image pickup signal of the light accepting part output of instrument, acquisition is based on the image of the fluorescence of the high intensity of wavelength period B1, based on wavelength period B2 The image of low intensive fluorescence and based on wavelength period B3 fluorescence image as fluorescence information.

With regard to the fluorescence of wavelength period B1～B3, both can individually be imaged respectively by 3 light accepting parts, it is also possible to logical Cross 1 light accepting part to be imaged.Optical system is configured to, and the fluorescence of wavelength period B1～B3 is being taken the photograph by 1 light accepting part The fluorescence of wavelength period B1～B3 is imaged on the sensitive surface of light accepting part in different regions as in the case of.

As shown in figure 3, in the case of ought being the cell that NF- κ B are locally lain in core, in step s3, such as figure is obtained As 31,32.Image 31 is the image of the fluorescence based on the high intensity of wavelength period B1, and image 32 is based on the low-intensity of wavelength period B2 Fluorescence image.In image 31,32, the region 33 that core is present is set.Region 33 is from based on simultaneously raw with image 31,32 Into wavelength period B3 fluorescence i.e. from core produce fluorescence image obtain.

Be in the case that NF- κ B locally lie in the cell in cytoplasm, in step s3, for example obtain image 41, 42.Image 41 is the image of the fluorescence based on the high intensity of wavelength period B1, and image 42 is based on the low intensive glimmering of wavelength period B2 The image of light.The region 43 of core presence is all set in image 41,42.Region 43 be from based on and image 41,42 generate simultaneously Wavelength period B3 fluorescence image obtain.

In the case of the few cell of the discovery amount for being NF- κ B, according to image 31, the intensity of fluorescence is appropriate, and in core There is difference in fluorescence intensity between cytoplasm, it is possible to differentiating that NF- κ B are locally lain in the region 33 of core.The opposing party Face, according to image 32, the intensity of fluorescence is too low, so cannot differentiate that NF- κ B are locally lain in the region 33 of core.On the other hand, Be NF- κ B discovery amount more than cell in the case of, according to image 41, the intensity of fluorescence is too high, thus become core with it is thin Do not have in fluorescence intensity discrepant state between kytoplasm.Therefore, it is impossible to differentiate that NF- κ B are locally lain in the region 43 of core and ratio Which region in the wide cytoplasmic region in the region 43 of core.On the other hand, according to image 42, the intensity of fluorescence is appropriate, And difference is produced in intensity between core and cytoplasm, it is possible to differentiating that NF- κ B are locally lain in the region 43 than core Wide cytoplasmic region.

In the case where NF- κ B are locally lain in cytoplasm, NF- κ B to surround core in the way of be distributed in the cell.That is, When observing cell on direction is imaged, NF- κ B are also distributed with the front of core.Therefore, in the figure of the fluorescence based on high intensity In 41, strong fluorescence is also produced in the region of core by being distributed in the NF- κ B of the front of core.Therefore, in image 41, deposit Cannot suitably differentiate that NF- κ B locally lie in the trend in cytoplasm that still locally lies in core.In contrast, in base In the image 42 of low intensive fluorescence, although fluorescence is also produced in the region of core by being distributed in the NF- κ B of the front of core, But the low intensity of the fluorescence.Therefore, in image 41, even if in the case that NF- κ B are locally lain in cytoplasm, it is also possible to suitable Locality differentiation locally lies in situation.

In addition, in the case where NF- κ B are locally lain in core, a part of NF- κ B is distributed in cytoplasm, but most It is distributed in core.Therefore, in the image 31 based on the fluorescence of high intensity, strong fluorescence is produced from the NF- κ B being distributed in core, Can suitably differentiate that NF- κ B are locally lain in core.On the other hand, in the image 32 based on low intensive fluorescence, from The fluorescence of the NF- κ B being distributed in core is excessively weak, so exist cannot suitably differentiate that NF- κ B are locally lain in core or local The trend being present in cytoplasm.

So, the amount according to the NF- κ B as intracellular tested substance and distribution, for differentiating the local of NF- κ B The appropriate intensity for existing is different.

Therefore, be set to can be appropriate in the cell that NF- κ B are locally lain in core for the power of the laser of wavelength X 1 Ground differentiates that the NF- κ B as shown in image 31 are locally lain in core.The power of the laser of wavelength X 2 is set to be deposited in NF- κ B local It is can suitably to differentiate that NF- κ B are locally lain in cytoplasm as shown in image 42 in the cell in cytoplasm.Thus, exist Become differentiate object cell in, though NF- κ B locally lie in which in core and cytoplasm in, can use 2 At least one party in image is differentiating locally lying in for NF- κ B.

Fig. 1 is returned to, in step s 4, operator's reference is based on the image of the fluorescence of the high intensity of wavelength period B1 and is based on The image of the low intensive fluorescence of wavelength period B2, differentiates the distribution situation of the NF- κ B as tested substance.Specifically, grasp Author is selected in image and the image based on the low intensive fluorescence of wavelength period B2 based on the fluorescence of the high intensity of wavelength period B1 , NF- κ B can the be differentiated image for locally lying in position, according to the image, differentiate in the cell NF- κ B locally lie in In which in core and cytoplasm, locally lie in situation.Can also replace locally lying in situation, differentiate that NF- κ B are distributed in Which position, for example intracellular distribution etc..

As described above, in embodiment 1,2 produced from the fluorescent material 11,12 for identifying NF- κ B by adjustment Fluorescence, becoming the one party in the image based on the fluorescence of high intensity and the image based on low intensive fluorescence can suitably sentence Determine the state for locally lying in of NF- κ B.Therefore, operator can well parse the distribution in cell according to 2 images, precision Diversified NF- κ B.Specifically, can precision differentiate that NF- κ B's in cell locally lies in situation, i.e. NF- κ well Which in core and cytoplasm B locally lie in.

In addition, in embodiment 1, it is also contemplated that following situation：In the case that even if NF- κ B are locally lain in core, by Many in the amount of NF- κ B, so the too high state of the intensity for becoming the fluorescence of image 31, the intensity for becoming the fluorescence of image 32 is appropriate State.In this case, can differentiate that NF- κ B are locally lain in core also by the appropriate image 32 of the intensity using fluorescence In.In addition, it is also contemplated that following situation：Even if in the case where NF- κ B are locally lain in cytoplasm, due to the amount of NF- κ B it is few, So the appropriate state of the intensity for becoming the fluorescence of image 41, becomes the too low state of the intensity of the fluorescence of image 42.In the feelings Under condition, can differentiate that NF- κ B are locally lain in cytoplasm also by the appropriate image 41 of the intensity using fluorescence.So, According to embodiment 1, according to 2 images, no matter the amount of NF- κ B is how many, can precision differentiate NF- κ B in cell well Locally lie in situation.

Here, vascular endothelial cell is peeled off from the inwall of blood vessel and is flowed into blood.The stripping of vascular endothelial cell, removes Beyond due to producing the stimulation because of inflammation, due also to because the change of the pressure caused by compressing etc. is also produced.Due to In the vascular endothelial cell peeled off because of the stimulation caused by inflammation, NF- κ B tend to locally lie in core, due to because of inflammation Stimulation beyond caused stimulation and in the vascular endothelial cell peeled off, NF- κ B tend to not locally lie in core.According to reality Apply mode 1, as mentioned above can precision differentiate locally lying in for NF- κ B well, it is possible to according to as signaling molecule Whether NF- κ B locally lie in core the stripping to judge to be produced due to the stimulation of the inflammation in these stripping reasons From can determine that vascular endothelial cell whether there is activation.Thus, for example, the stripping that can interpolate that vascular endothelial cell be due to Compressing during blood sampling and produce or produced as main cause with disease etc., have meaning clinically.

Can also be by the laser of the middle power of the wavelength different from wavelength X 1, λ 2, the image of the fluorescence of intensity in acquisition. I.e., it is also possible to the mutually different 3 fluorescent materials mark NF- κ B of wavelength of fluorescence, 3 laser of cell irradiation are caused from 3 Individual fluorescent material produces image of the different fluorescence of intensity to obtain based on each fluorescence.Thus, it is different by the intensity from fluorescence 3 images used in optimal image, can precision differentiate locally lying in for NF- κ B better.From tested substance The intensity of the fluorescence of generation can also be more than 4 grades, it is also possible to obtain based on the intensity of more than 4 for 1 cell The image of more than 4 of fluorescence.

Further, in step s 4, operator locally lies in situation according to each cell for differentiating as described above, obtains examination In the cell included in sample, NF- κ B locally lie in the ratio of the cell in privileged site.Specifically, if will differentiate The quantity for locally lying in the cell in core for NF- κ B is set to N1, will be determined as NF- κ B and locally lies in the cell in cytoplasm Quantity be set to N2, then operator is by following formula, obtains that core locally lies in rate and cytoplasm locally lies in rate.Additionally, In step S4, operator can also replace core to locally lie in rate and cytoplasm locally lies in rate and obtains core and locally lie in number and thin Kytoplasm locally lies in number.

Core locally lies in rate={ N1/ (N1+N2) } × 100

Cytoplasm locally lies in rate={ N2/ (N1+N2) } × 100

In step s 2, flow cytometer used as discussed above also may be used obtaining the image of each fluorescence, but not limited to this To use microscope, the image of each fluorescence is obtained as fluorescence information.I.e., it is also possible to by microscope, obtain from fluorescent material The fluoroscopic image of 11 high intensity for producing, the low intensive fluoroscopic image produced from fluorescent material 12 and produce from fluorescent material 13 Raw image corresponding with core.

Next, illustrating the checking of the embodiment 1 that inventor is carried out.

1. prepare

As cell, prepared human heart tiny blood vessels endothelial cell (HMVEC-C) (Lonza CatNo.CC-7030, Lot No.0000296500(P4)).As an antibody, NF- κ B p65 (D14E12) XP Rabbit mAb (Cell are prepared Signaling Technologies #8242S).As secondary antibodies, Goat anti-Rabbit IgG (H+L) is prepared The conjugate of Secondary Antibody, Alexa Fluor 647 (Life technologies A-21245), Goat Conjugate (the Life of anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 technologies A-11008).To secondary antibodies, as fluorchrome, Alexa Fluor 647, Alexa are combined Fluor 488.As nuclear staining pigment, the solution (DOjinDO of Cellstain Hoechst 33342 are prepared H342).In addition, having prepared EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit (Lonza Cat No.CC-3202)、PBS pH7.4(GIBCO Cat No.10010-023)、BSA(LAMPIRE Cat No.7500805)、PFA(WAKO Cat No.160-16061)、TritonX100(Nacalai Tesque CatNo.35501- 15)。

2. reagent modulation

To the reagent beyond the FBS of the EGM-2MV Medium addition EGM-2MV SingleQuots Kit of 500mL, will 100mL moves on to aseptic bottle, has made serum free medium.Surplus (400mL) to free serum culture matrix manufacturing, adds 20mL Single Quots Kit FBS, produce culture medium.In the PBS using pH12 paraformaldehyde is dissolved with its ultimate density After for 8%w/v, pH7.4 is adjusted to.The BSA of 1.5g is added to PBS and is dissolved and is complemented into 50mL, modulated 3%BSA/ PBS.The BSA of 0.5g is added to PBS and is dissolved and is complemented into 50mL, modulated 1%BSA/PBS.Modulated using PBS TritonX100 is with its ultimate density as 0.1%w/v.

3. step

HMVEC-C is to recommend agreement in accordance with manufacturer, is cultivated using EGM-2MV culture mediums.Used in this checking Cell within subculture number is 6 times after buy.With regard to culture medium, during the useful life behind Kaifeng is set into 3 weeks. Stimulate with regard to TNF-α and cultivate, remove the culture supernatant of the HMVEC-C cells at about 70% interflow, add to become ultimate density The mode of 25ng/mL with the addition of the EGM-2MV culture mediums of Recombinant Human TNF-alpha, and in 37 DEG C of CO₂It is permanent 1 hour has been stood in incubator.Retain 3mL or so and remove culture medium with electric pipettor, and cell has been peeled off with scraper.Add With the 8%PFA/PBS of the suspension equivalent for reclaiming, 15 minutes have been reacted at room temperature.At room temperature, 3 have been carried out with 1000rpm The centrifugation of minute.Cell granulations 2 times have been rinsed into the PBS of 1mL.Supernatant is removed, the 0.1%Triton X- of 1mL are added 100/PBS, has reacted at room temperature 15 minutes.At room temperature, the centrifugation of 3 minutes has been carried out with 1000rpm.With 1mL's 1%BSA/PBS has rinsed 2 times.Supernatant is removed, the 3%BSA/PBS of 1mL is added, 30 minutes have been stood at room temperature.3% An antibody of 1/1,600 400 μ L is with the addition of in BSA/PBS.1 hour has been reacted at room temperature.At room temperature, with 1000rpm The centrifugation of 3 minutes is carried out.Rinsed with the 1%BSA/PBS of 1mL.1/1000 is with the addition of in 3%BSA/PBS 400 μ L secondary antibodies.30 minutes have been reacted at room temperature.2 times have been rinsed with the 1%BSA/PBS of 1mL.Supernatant is removed, is added The 1%BSA/PBS of 50 μ L is added.

4. detected using flow cytometer

As the flow cytometer that can obtain fluoroscopic image, ImageStreamX Mark II Imaging have been used Flow Cytometer(Merck Millipore).Make the circulation that the flow cytometer is flowed into according to the sample of above-mentioned 3 modulation Chi Zhong, to the sample flowed in flow cell, has irradiated the laser of wavelength 488nm, 647nm, 405nm.Wavelength 488nm, The laser of 647nm, 405nm is corresponding with the laser of above-mentioned wavelength X 1, λ 2, λ 3.The laser of wavelength 488nm, 647nm, 405nm is penetrated Go out power and be respectively 55mW, 10mW, 120mW.The laser of wavelength 488nm, 647nm is irradiated to 2 kinds of iridescent of mark NF- κ B Element, thus generates respectively the fluorescence and low intensive fluorescence of high intensity.The laser of wavelength 405nm be irradiated to nuclear staining pigment and Generate fluorescence.

In above-mentioned flow cytometer, via the filter member of transmission peak wavelength section 505nm～560nm, to by wavelength The fluorescence that the laser of 488nm is produced is imaged, and obtains the fluoroscopic image of high intensity.Via transmission peak wavelength section 642nm～ The filter member of 740nm, images to the fluorescence that the laser by wavelength 647nm is produced, and obtains low intensive fluorescence Image.Via the filter member of transmission peak wavelength section 430nm～505nm, the fluorescence that the laser by wavelength 405nm is produced is entered Row shooting, obtains fluoroscopic image corresponding with core.In addition, to the sample flowed in flow cell, having irradiated wavelength and being set For the laser between 430nm～480nm.Via the filter member of transmission peak wavelength section 430nm～480nm, to the laser transmission Light after cell is imaged, and obtains bright field image.Additionally, in above-mentioned flow cytometer, by filter member Light accepting part is suitably incided etc. come the light that removes the light of unwanted wavelength period to become the wavelength period of object.Additionally, Bright field image, but not limited to this are obtained in this checking, it is also possible to obtain scotopia field picture.

With reference to Fig. 4 (a), the image obtained by above-mentioned detection is illustrated.

" bright field " represents the bright field image of cell." fluorescence of high intensity " and " low intensive fluorescence " is based on respectively From the image of the fluorescence of the high intensity of the fluorchrome generation for identifying NF- κ B and based on from the fluorchrome for identifying NF- κ B The image of the low intensive fluorescence for producing." from the fluorescence of core " is to be based on to be produced with pigment from the nuclear staining for dyeing core The image of raw fluorescence." synthesis " be synthesis on the left of 4 images obtained from image.5 transversely arranged images are from 1 The image that cell is obtained.The figure that " fluorescence of high intensity ", " from the fluorescence of core ", " low intensive fluorescence " and " synthesis " is represented Seem for convenience image obtained from GTG to be carried out to acquired coloured image." fluorescence of high intensity ", " from In the image that the fluorescence of core " and " low intensive fluorescence " are represented, white part represents that the intensity of fluorescence is strong.

In the case of the cell shown in epimere, the intensity based on the image of low intensive fluorescence is too low, so being difficult to sentence Other NF- κ B's locally lies in.On the other hand, the intensity based on the image of the fluorescence of high intensity is appropriate, it is possible to being determined as NF- κ B are locally lain in core.In the case of the cell shown in hypomere, the intensity based on the image of the fluorescence of high intensity is too high, institute To be difficult to differentiate locally lying in for NF- κ B.On the other hand, the intensity based on the image of low intensive fluorescence is appropriate, it is possible to It is determined as NF- κ B to locally lie in cytoplasm.

5. core locally lies in the calculating of rate

By carrying out visually, for each cell locally lying in for NF- κ B having been differentiated to acquired image.With above-mentioned step Rapid S4 has similarly carried out the differentiation.That is, the region of core is set according to the fluoroscopic image from core, by the area beyond the region of core Domain is used as cytoplasmic region.In addition, in the situation of more than about 2 times that the fluorescence intensity for thinking core is cytoplasmic fluorescence intensity Under, it is determined as the NF- κ B in the cell and locally lies in core, it is less than cytoplasmic fluorescence intensity in the fluorescence intensity for thinking core About 2 times in the case of, be determined as the NF- κ B in the cell and locally lie in cytoplasm.

With reference to Fig. 4 (b), illustrate that 131 cells for being recognized with above-mentioned flow cytometer have differentiated that the local of NF- κ B is deposited Result.

The quantity for being determined as locally lying in the cell in core is 44, the cell for being determined as locally lying in cytoplasm Quantity is 87.The quantity that the cell for locally lying in cannot be differentiated is 0.It is 44/131=34% that core now locally lies in rate.

Here, explanation has differentiated the comparative example 1 for locally lying in and according only to low-intensity according only to the fluoroscopic image of high intensity Fluoroscopic image differentiated the comparative example 2 for locally lying in.In the case of comparative example 1, it is determined as locally lying in thin in core The quantity of born of the same parents is 42, and the quantity for being determined as locally lying in the cell in cytoplasm is 10.The intensity of fluorescence is too high, so cannot The quantity of the cell that differentiation locally lies in is 79.It is 81% that the core of comparative example 1 locally lies in rate.In the case of comparative example 2, sentence The quantity that the cell in core Wei not locally lain in is 16, and the quantity for being determined as locally lying in the cell in cytoplasm is 84. Fluorescence intensity is too low, so the quantity that cannot differentiate the cell for locally lying in is 31.The core of comparative example 2 locally lies in rate 16%.

As described above, understood according to this checking, as Embodiment 1 according to 2 fluoroscopic images that intensity is different In the case that differentiation locally lies in, with regard to the cell that can not be differentiated in the case of comparative example 1,2, it is also possible to differentiate NF- κ B's Locally lie in.In addition we know, in the case where as Embodiment 1 differentiation locally lies in, the cell that can not be differentiated is few, so Can precision differentiate locally lying in for NF- κ B in cell well.Therefore, according to embodiment 1, can not will can differentiate The quantity of cell suppresses relatively low, and precision differentiates well locally lying in for NF- κ B.Thus, for example, even if extracting from examinee Cell it is few in the case of, it is also possible to while the cell for differentiating is guaranteed, precision differentiates that well the local of NF- κ B is deposited .

In embodiment 1, as the differentiation of the distribution situation of tested substance, show that the local for differentiating NF- κ B is deposited In the example of situation, but in the case where the amount of the molecule of tested substance changes, it is also possible to differentiate the amount of the molecule. In this case, operator identifies molecule with fluorescent material 11,12, and processing meanss are according to the intensity produced from fluorescent material 11,12 2 different fluorescence, obtain image.Operator is differentiated in the case where the amount of molecule is more using low intensive fluoroscopic image Amount, in the case where the amount of molecule is few, using the fluoroscopic image of high intensity come the amount of differentiation.Differentiate well thereby, it is possible to precision The amount of molecule.

Illustrate for the cellular informatics acquisition methods according to embodiment 1 cell image to be imaged and differentiated in cell Tested substance the cellular informatics acquisition device for locally lying in structure.

As shown in figure 5, cellular informatics acquisition device 100 possesses processing unit 110, sample modulation portion 120, optical detection portion 130th, drive division 140, display part 150, input unit 160 and storage part 170.

Processing unit 110 is made up of microcomputer and CPU etc..Storage part 170 is made up of RAM, ROM, hard disk etc..Storage Portion 170 stores the various data such as the processing routine, the image that are performed by processing unit 110.Processing unit 110 is obtaining dress with cellular informatics The transmission that signal is carried out between each portion for putting 100 is received, and controls each portion.In processing unit 110, by being stored in storage part 170 In program, give the function of acquisition unit 111 and analysis unit 112.

Thus sample modulation portion 120 modulates sample according to S1 the step of Fig. 1, cell mixing and reagent.Can also be by operating Person carries out the modulation of sample.In this case, sample modulation portion 120 is omitted from cellular informatics acquisition device 100.Optical detection portion 130 is flow cytometer.Optical detection portion 130 images to the cell irradiation light included in sample to the fluorescence of generation.With Afterwards, the structure in optical detection portion 130 is illustrated with reference to Fig. 6.Drive division 140 drive the light source 301 in aftermentioned optical detection portion 130～ 304。

Display part 150 is made up of display.Display part 150 shows the image, thin for each obtained for each cell The locally lying in of NF- κ B that born of the same parents differentiate, the cell number that NF- κ B are locally lain in core, NF- κ B are locally lain in cytoplasm Cell number, core locally lie in rate and cytoplasm locally lies in rate etc..Input unit 160 is made up of mouse and keyboard.Operator Indicate for the input of cellular informatics acquisition device 100 via input unit 160.

As shown in fig. 6, optical detection portion 130 possesses flow cell 200, illumination part 300, light collecting part 400 and light accepting part 501～504.Stream 210 is defined in flow cell 200, in stream 210, the examination modulated using sample modulation portion 120 is flow through Sample.In figure 6, for convenience, it is illustrated that mutually orthogonal XYZ axles.

The cell irradiation light included in the sample that illumination part 300 pairs circulates in flow cell 200, from glimmering shown in Fig. 2 Stimulative substance 11,12 produces the different fluorescence of intensity.In addition, illumination part 300 is caused from glimmering shown in Fig. 2 to cell irradiation light Stimulative substance 13 produces fluorescence, and then, the light to cell irradiation bright field.Illumination part 300 possesses light source 301～304, optically focused Lens 311～314 and dichroscope 321,322.

Light source 301～304 is made up of semiconductor laser light source.The light projected from light source 301～304 is respectively 1～λ of wavelength X 4 Laser.1～λ of wavelength X 4 is respectively such as 488nm, 647nm, 405nm, 785nm.1～λ of wavelength X 3 is to be used for as shown in Figure 2 The light of fluorescence is encouraged from fluorescent material 11～13.Light of the collector lens 311～314 respectively to projecting from light source 301～304 is carried out Optically focused.Dichroscope 321 makes the light transmission of wavelength X 1, and the light for making wavelength X 2 reflects.Dichroscope 322 makes wavelength X 1, the light of λ 2 Transmission, the light for making wavelength X 3 reflects.

So, the cell included in the sample that illumination part 300 pairs flows in stream 210, with overlapped state Irradiate the light of the 1～λ of wavelength X 3 projected from light source 301～303.In addition, stream of the illumination part 300 to illuminated 1～λ of wavelength X 3 The position on road 210, the light of illumination wavelength lambda 4.If the light of the 1～λ of sample illumination wavelength lambda 3 to flowing in flow cell 200, Fluorescence as produced different wavelength periods from fluorescent material 11～13 with reference to Fig. 2 explanations.If to flowing in flow cell 200 The light of dynamic sample illumination wavelength lambda 4, then the light transmission cell.The light for having transmitted the wavelength X 4 of cell is used for bright field image Acquisition.

Here, light source 301 projects the light of wavelength X 1 with high power, light source 302 projects the light of wavelength X 2 with low-power.Pass through Drive division 140 shown in Fig. 5, controls the injection power of light source 301,302.Thus, from fluorescent material as illustrating with reference to Fig. 2 11 fluorescence for producing become high intensity, and the fluorescence produced from fluorescent material 12 becomes low-intensity.Additionally, preferably 1 with And in aftermentioned embodiment 2,3, it is also possible to do not adjust the injection power of light source 301 and 302.By selecting even same penetrating Go out power but the discrepant fluorescence labelling of resulting fluorescence intensity, the fluorescence produced from fluorescent material 11 becomes high intensity, from The fluorescence that fluorescent material 12 is produced becomes low-intensity.

Light collecting part 400 makes the fluorescence optically focused that the irradiation of the light by 1～λ of wavelength X 3 is produced from flow cell 200.Light collecting part 400 make the fluorescence produced from fluorescent material 11～13 be condensed to light accepting part 501～503 respectively.In addition, light collecting part 400 is made from stream The light of the wavelength X 4 that logical pond 200 produces is condensed to light accepting part 504.Light collecting part 400 possesses collector lens 401, filter member 411 ～413,421～424 and collector lens 431～434.

Collector lens 401 makes the fluorescence produced from the sample flowed in flow cell 200 and has transmitted in flow cell 200 The light optically focused of the wavelength X 4 of the sample of flowing.Filter member 411～413 is made up of dichroscope.

Filter member 411 reflects the light of in the light by the optically focused of collector lens 401, wavelength period B1, makes wavelength period Light transmission beyond B1.Filter member 421 only makes the light of in the light reflected by filter member 411, wavelength period B1 saturating Penetrate, interdict the light beyond wavelength period B1.So, filter member 411,421 is configured to be merely able to separate produce from flow cell 200 Light in, the fluorescence of wavelength period B1.Similarly, filter member 412,422 is configured to be merely able to separate produce from flow cell 200 The fluorescence of in raw light, wavelength period B2, filter member 413,423 is configured to be merely able to separate what is produced from flow cell 200 In light, wavelength period B3 fluorescence.Filter member 424 makes in the light for having transmitted filter member 411～413, wavelength X 4 Light transmission, interdict wavelength X 4 beyond light.

Light accepting part 501 receives the light of wavelength period B1 by the optically focused of collector lens 431, by the image based on the light for receiving Information is exported as image pickup signal.Light accepting part 502 receives the light by wavelength period B2 of the optically focused of collector lens 432, will be based on connecing The image information of the light being subject to is exported as image pickup signal.Light accepting part 503 receives wavelength period B3 by the optically focused of collector lens 433 Light, the image information of light for receiving will be based on and exported as image pickup signal.Light accepting part 504 receives to pass through collector lens 434 The light of the wavelength X 4 of optically focused, exports the image information based on the light for receiving as image pickup signal.Light accepting part 501～504 by The imaging apparatus such as such as colored CCD are constituted.

Light collecting part 400 makes the light of the light of wavelength period B1～B3 and wavelength X 4 be condensed to light accepting part 501～504 respectively, but Can be imaged in 1 light accepting part.In this case, optical detection portion 130 is configured to：Make the light and wavelength of wavelength period B1～B3 The light of λ 4 is imaged on the sensitive surface of light accepting part in different regions.

In the structure shown in Fig. 6, in order to separate the light of wavelength period B1 multiple filter members are used, but it is also possible to As shown in fig. 7, separating the light produced from flow cell 200 by 1 filter member.As shown in fig. 7, light collecting part 400 possesses poly- Optical lens 441～444, filter member 451～454 and collector lens 461～464.The light of wavelength period B1～B3 leads to respectively Cross filter member 451～453 to be separated, the light of wavelength X 4 is separated by filter member 454.

Next, with reference to the flow chart of Fig. 8, illustrating the place of the step of carrying out Fig. 1 by cellular informatics acquisition device 100 S4 The situation of reason.

As shown in figure 8, in step s 11, processing unit 110 drives sample modulation portion 120, the step of Fig. 1 in the same manner as S1, With the NF- κ B included in fluorescent material 11,12 marked cells, with the core included in the marked cells of fluorescent material 13 examination is modulated Sample.In step s 12, processing unit 110 makes sample flow into flow cell 200, and by drive division 140 light source 301～304 is driven, right The cell irradiation light flowed in flow cell 200.In step s 13, processing unit 110 passes through 501～503 pairs of wavelength periods of light accepting part The fluorescence of B1～B3 is imaged, and the light of wavelength X 4 is imaged by light accepting part 504.Then, the acquisition unit of processing unit 110 111 image pickup signals exported according to light accepting part 501～504, obtain image.

In step S14, the analysis unit 112 of processing unit 110 from high intensity and low intensive fluoroscopic image, be selected into The image of the differentiation that row locally lies in.Specifically, analysis unit 112 selects the fluoroscopic image and low intensive fluorogram of high intensity The brightness of the intensity of fluorescence as in, obtaining from image such as image entirety image within a predetermined range.Thus, it is glimmering Cannot in the same manner as the image 32 of the terrifically big image of the intensity of light, the terrifically little image of intensity of fluorescence and Fig. 3, image 41 Locally lying in for NF- κ B is differentiated, so removing from the image used in differentiation.

In addition, analysis unit 112 selects the analysis object position of in high intensity and low intensive fluoroscopic image, cell The difference of the fluorescence intensity in cell beyond the fluorescence intensity and analysis object position image bigger than predetermined threshold value.In embodiment party In formula 1, analysis object position is core.That is, the difference ratio for selecting the fluorescence intensity of the fluorescence intensity in core and the cell outside core makes a reservation for The big image of threshold value.Thus, the little image of the difference of the fluorescence intensity in core and outside core is same with the image 32 of Fig. 3, image 41 Ground cannot differentiate locally lying in for NF- κ B, so removing from the image used in differentiation.

Next, in step S15, analysis unit 112 is sentenced using the image selected in step S14 for each cell NF- κ B's in other cell locally lies in.That is, deposit the local of the NF- κ B at the analysis object position of the calculating of analysis unit 112 cell The ratio of the amount of locally lying in of the NF- κ B in amount versus cell entirety.For example, the figure that analysis unit 112 is selected in step S14 As in, by the intensity of the fluorescence in the region of core divided by the fluorescence intensity in the overall region of cell.Analysis unit 112 is tied in division In the case that fruit is more than 2, it is determined as NF- κ B and locally lies in core, in the case where result of division is less than 2, is determined as NF- κ B are locally lain in cytoplasm.The value for judging result of division is not limited to 2, or other values.

Additionally, in the case of 2 images are have selected in step S14, in step S15, for the image of two sides, as above It is described acquirement result of division and differentiate locally lie in.In addition, in step S14 in the case of non-selected image, in step S15 In, locally lying in for the cell is considered as " can not differentiate ".

In step s 16, analysis unit 112 calculates above-mentioned core office according to the differentiation result of all cells for being processed Portion exist number, cytoplasm locally lie in number, core locally lies in rate and cytoplasm locally lies in rate.In step S17, processing unit 110 is aobvious by differentiation result of the numerical value for calculating in step s 16, the image obtained for each cell and each cell etc. It is shown in display part 150.Specifically, processing unit 110 will be shown in display part 150 including the picture 161 of the above.

As shown in figure 9, picture 161 possesses region 161a, 161b.Region 161a shows that core locally lies in number, cytoplasm office Portion has that number, core locally lie in rate and cytoplasm locally lies in rate.Region 161b display images and NF- κ B's locally lies in Differentiate result.In the 161b of region, the image of the differentiation for being used to locally lie in is enclosed in step S15 with solid line, to know The image has been locally lain in used in differentiation.

The fluorescence information for obtaining in step s 13 can also be the waveform signal for representing time dependent fluorescence intensity. In this case, in optical detection portion 130, as light accepting part 501～503, the photodetectors such as photomultiplier are respectively configured. 3 photodetectors receive the glimmering of the fluorescence of high intensity, the low intensive fluorescence of wavelength period B2 and wavelength period B3 of wavelength period B1 Light, respectively output represents the waveform signal of the intensity of each fluorescence.

As shown in Figure 10, the waveform signal that analysis unit 112 is exported according to photodetector is few in the discovery amount for being NF- κ B In the case of cell, for example obtain chart 51,52, be NF- κ B discovery amount more than cell in the case of, for example obtain chart 61、62.In addition, the waveform signal that analysis unit 112 is exported according to photodetector, obtains chart corresponding with core.Analysis unit 112 According to the chart of the core obtained with chart 51,52 simultaneously, the width W1 of waveform corresponding with core is set in chart 51,52, according to The chart of the core obtained simultaneously with chart 61,62, sets the width W2 of waveform corresponding with core in chart 61,62.

According to chart 51, there is waveform in the peak value of fluorescence between threshold value Sh1, Sh2 in width W1 corresponding with core Peak value, so analysis unit 112 can be determined as NF- κ B and locally lie in core.On the other hand, according to chart 52, fluorescence Peakedness ratio threshold value Sh1 is little, so analysis unit 112 cannot differentiate locally lying in for NF- κ B.According to chart 61, the peakedness ratio of fluorescence Threshold value Sh2 is big, so analysis unit 112 cannot differentiate locally lying in for NF- κ B.On the other hand, according to chart 62, the peak value of fluorescence Between threshold value Sh1, Sh2, there is the depression of waveform in width W2 corresponding with core, so analysis unit 112 can differentiate Locally lie in cytoplasm for NF- κ B.Therefore, in this case, also can in the same manner as the situation of image is used as shown in Figure 3 It is enough to differentiate locally lying in for NF- κ B well come precision by 2 different fluorescence of intensity.

In embodiment 2, and 2 light of non-usage, but mutually different intensity is only obtained using the light of wavelength X 1 Fluorescence.In embodiment 2, compared to embodiment 1, the step of cellular informatics acquisition methods shown in Fig. 1 in, only A part of step in step S1, S2 is different.Hereinafter, the steps different from embodiment 1 are illustrated.

In step sl, as shown in figure 11, with including in the mutually different fluorescent material 14 of wavelength of fluorescence, 15 marked cells NF- κ B.Fluorescent material 14,15 is fluorchrome.If the light of the illuminated wavelength X 1 of fluorescent material 14, encourage and embodiment party The fluorescence of the same wavelength period of the fluorescent material 11 of formula 1.If the light of the illuminated wavelength X 1 of fluorescent material 15, encourage and Fig. 2 The same wavelength period of fluorescent material 12 fluorescence.That is, the wavelength of the light of the excitation of fluorescent material 14,15 is substantially the same.

In step s 2, the sample comprising the cell identified with fluorescent material 14,15,13 is flowed into flow cell, wavelength X 1, The illumination of λ 3 is mapped to the cell flowed in flow cell, and from fluorescent material 14,15,13 fluorescence is produced.Produce from fluorescent material 14,15 Raw fluorescence becomes the fluorescence of wavelength period B1, B2 by filter member 21,22 respectively.Now, fluorescent material 14,15 is constituted For the fluorescence of wavelength period B1 becomes high intensity, and the fluorescence of wavelength period B2 becomes low-intensity.

In the apparatus structure of embodiment 2, compared to embodiment 1, the optical detection portion 130 shown in Fig. 6 is eliminated In, light source 302, collector lens 312 and dichroscope 321.

In embodiment 2, also in the same manner as embodiment 1, the fluorescence and ripple of the high intensity of wavelength period B1 can be produced The low intensive fluorescence of long section B2, obtains the fluoroscopic image and low intensive fluoroscopic image of high intensity.Therefore, with embodiment 1 Similarly, the distribution in cell can well be parsed according to the fluoroscopic image of high intensity and low intensive fluoroscopic image, precision And the diversified NF- κ B of amount.

In embodiment 3, and 2 light of non-usage and 2 fluorescent materials, but the light using 1 wavelength X 1 and 1 it is glimmering Stimulative substance 11, obtains the fluorescence of mutually different intensity.In embodiment 3, compared to embodiment 1, the cell shown in Fig. 1 In the step of information getting method, a part of step difference only having in step S1, S2.Hereinafter, illustrate with embodiment 1 not Same step.

In step sl, as shown in figure 12, only with including in the marked cells of fluorescent material 11 same with embodiment 1 NF-κB.Now, fluorescent material 11 can also be combined via 1 antibody with NF- κ B.In step s 2, comprising using fluorescent material 11st, the sample of the cell of 13 marks is flowed into flow cell, and wavelength X 1, the illumination of λ 3 are mapped to the cell flowed in flow cell, from Fluorescent material 11,13 produces fluorescence.

As shown in figure 12, the fluorescence produced from fluorescent material 11 is divided into 2, makes a side by same with embodiment 1 The filter member 21 of sample, makes the opposing party by the filter member 22 same with embodiment 1.Filter member 21 only makes ripple The light transmission of long section B1, filter member 21 only makes the light transmission of wavelength period B4.As shown in figure 13, wavelength period B1 comprising for example from The intensity of the fluorescence that fluorescent material 11 is produced is the wavelength of peak value.Wavelength period B4 is for example set to the wavelength bigger than wavelength period B1 Section and, be set to not Chong Die with wavelength period B1 wavelength period.Thus, as shown in figure 12, filter member 21 has been passed through The fluorescence of wavelength period B1 becomes high intensity, has passed through the fluorescence of wavelength period B4 of filter member 22 and has become low-intensity.

Additionally, wavelength period B1 can also include the wavelength of the intensity for peak value of the fluorescence produced from fluorescent material 11. Wavelength period B4 can also be set as the wavelength period less than wavelength period B1, it is also possible to a part Chong Die with wavelength period B1.

Next, illustrating the checking of the embodiment 3 that inventor is carried out.

1. prepare

As cell, prepared human heart tiny blood vessels endothelial cell (HMVEC-C) (Lonza CatNo.CC-7030, Lot No.0000296500(P4)).As an antibody, NF- κ B p65 (D14E12) XP Rabbit mAb (Cell are prepared Signaling Technologies #8242S).As secondary antibodies, Goat anti-Rabbit IgG (H+L) is prepared The conjugate of Secondary Antibody, Alexa Fluor 647 (Life technologies A-21245).To two Secondary antibody, as fluorchrome, combines Alexa Fluor 647.In addition, having prepared EGM-2MV Medium (Lonza Cat No.CC-3202)、EGM-2MV SingleQuots Kit(LonzaCat No.CC-3202)、PBS pH7.4(GIBCO Cat No.10010-023)、BSA(LAMPIRE Cat No.7500805)、PFA(WAKO Cat No.160-16061)、 TritonX100(Nacalai Tesque CatNo.35501-15)。

2. reagent modulation

EGM-2MV SingleQuots Kit are added to the EGM-2MV Medium of 500mL, culture medium has been made.In profit After dissolving paraformaldehyde with its ultimate density as 8%w/v with the PBS of pH12, pH7.4 is adjusted to.To PBS plus 1.5g's BSA simultaneously dissolves and is complemented into 50mL, has modulated 3%BSA/PBS.The BSA of 0.5g is added to PBS and is dissolved and is complemented into 50mL, 1%BSA/PBS is modulated.Using PBS TritonX100 is modulated with its ultimate density as 0.1%w/v.

3. step

HMVEC-C is to recommend agreement in accordance with manufacturer, using EGM-2MV medium cultures.Used in this checking Cell within subculture number is 6 times after buy.With regard to culture medium, during the useful life behind Kaifeng is set into 3 weeks.Close Stimulate in TNF-α and cultivate, remove the culture supernatant of the HMVEC-C cells at about 70% interflow, add to become ultimate density 25ng/ The mode of mL with the addition of the EGM-2MV culture mediums of Recombinant Human TNF-alpha, and in 37 DEG C of CO₂Insulating box 1 hour is inside stood.Retain 3mL or so and remove culture medium with electric pipettor, and cell has been peeled off with scraper.Plus with return The 8%PFA/PBS of the suspension equivalent of receipts, has reacted at room temperature 15 minutes.At room temperature, 3 minutes have been carried out with 1000rpm Centrifugation.Cell granulations 2 times have been rinsed into the PBS of 1mL.Supernatant is removed, the 0.1%Triton X-100/ of 1mL are added PBS, has reacted at room temperature 15 minutes.At room temperature, the centrifugation of 3 minutes has been carried out with 1000rpm.With the 1% of 1mL BSA/PBS has rinsed 2 times.Supernatant is removed, the 3%BSA/PBS of 1mL is added, 30 minutes have been stood at room temperature.In 3%BSA/ An antibody of 1/1,600 400 μ L is with the addition of in PBS.1 hour has been reacted at room temperature.At room temperature, carried out with 1000rpm The centrifugation of 3 minutes.Rinsed with the 1%BSA/PBS of 1mL.The 400 of 1/1000 are with the addition of in 3%BSA/PBS The secondary antibodies of μ L.30 minutes have been reacted at room temperature.2 times have been rinsed with the 1%BSA/PBS of 1mL.Supernatant is removed, be with the addition of The 1%BSA/PBS of 50 μ L.

4. detected using flow cytometer

As the flow cytometer that can obtain fluoroscopic image, ImageStreamX Mark II Imaging have been used Flow Cytometer(Merck Millipore).Make the stream that the flow cytometer is flowed into according to above-mentioned 3 samples for modulating In logical pond, to the sample flowed in flow cell, the laser of wavelength 647nm has been irradiated.Shown in the laser and Figure 12 of wavelength 647nm Wavelength X 1 laser correspondence.The injection power of the laser of wavelength 647nm is 10mW.By the fluorchrome to identifying NF- κ B The laser of illumination wavelength 647nm, generates fluorescence.

In above-mentioned flow cytometer, via the filter member of transmission peak wavelength section 642nm～740nm, to by wavelength The fluorescence that the laser of 647nm is produced is imaged, and obtains the fluoroscopic image of high intensity.In addition, via transmission peak wavelength section 740nm The filter member of～800nm, images to the fluorescence that the laser by wavelength 647nm is produced, and obtains low intensive glimmering Light image.Additionally, in above-mentioned flow cytometer, the light of unwanted wavelength period is eliminated by filter member etc., with The light for becoming the wavelength period of object suitably incides light accepting part.

With reference to Figure 14 (a), (b), the image obtained by above-mentioned detection is illustrated.

" fluorescence of high intensity " and " low intensive fluorescence " is respectively based on from the fluorchrome generation for identifying NF- κ B The image of the fluorescence of high intensity and the image based on the low intensive fluorescence produced from the fluorchrome for identifying NF- κ B.In figure In 14 (a), (b), 2 transversely arranged images are the images obtained from 1 cell.Each image is for convenience to acquired Coloured image carry out image obtained from GTG.In each image, white part represents that the intensity of fluorescence is strong.

In the case of 3 cells shown in Figure 14 (a), the intensity based on the image of low intensive fluorescence is too low, so It is difficult to differentiate locally lying in for NF- κ B.On the other hand, the intensity based on the image of the fluorescence of high intensity is appropriate, it is possible to sentencing Not Wei NF- κ B locally lie in cytoplasm.Fluorescence in the case of 3 cells shown in Figure 14 (b), based on high intensity The intensity of image is too high, so being difficult to differentiate locally lying in for NF- κ B.On the other hand, the image based on low intensive fluorescence Intensity is appropriate, it is possible to be determined as NF- κ B locally lying in cytoplasm.

As described above, understood according to this checking, even if as Embodiment 3 according to 2 fluorescence that intensity is different In the case that image discriminating locally lies in, it is also possible to differentiate locally lying in for NF- κ B.So, according to embodiment 3, make from a kind The fluorescence that fluorchrome is produced obtains 2 fluorescence by the filter member 21,22 for passing through the fluorescence of different wavelength periods Image, thus, it is possible to be directed to 1 cell locally lying in for NF- κ B is correctly obtained in measure once.

As shown in figure 15, in the apparatus structure of embodiment 3, compared to embodiment 1, the optics shown in Fig. 6 is omitted In test section 130, light source 302, collector lens 312 and dichroscope 321, replace filter member 412,422 and distinguish Additional filter member 414,425.Filter member 414 makes the light of in the light for having transmitted filter member 411, wavelength period B4 Reflection, makes the light transmission beyond wavelength period B4.Filter member 425 only makes in the light reflected by filter member 414, ripple The light transmission of long section B4, interdicts the light beyond wavelength period B4.So, filter member 414,425 be configured to only to separate from The fluorescence of in the light that flow cell 200 is produced, wavelength period B4.Light accepting part 502 is taken the photograph to the low intensive fluorescence of wavelength period B4 Picture.

In embodiment 3, also in the same manner as embodiment 1 so that produce the fluorescence of high intensity and low intensive respectively Fluorescence, can obtain the fluoroscopic image and low intensive fluoroscopic image of high intensity.Therefore, in the same manner as embodiment 1, Neng Gougen According to the fluoroscopic image and low intensive fluoroscopic image of high intensity, precision parses well the distribution in cell and measures varied NF- κ B.

In embodiment 4, compared to embodiment 1, the step of cellular informatics acquisition methods shown in Fig. 1 in, only There is a part of step in step S1, S2 different.Hereinafter, the steps different from embodiment 1 are illustrated.

In step sl, as shown in figure 16, only with including in the marked cells of fluorescent material 11 same with embodiment 1 NF-κB.In step s 2, the sample comprising the cell identified with fluorescent material 11,13 is flowed into flow cell, wavelength X 1, λ 3 Illumination is mapped to the cell flowed in flow cell, and from fluorescent material 11,13 fluorescence is produced.Now, for cell with high power photograph The light of wavelength X 1 is penetrated, so the fluorescence of wavelength period B1 of transmission filter part 21 becomes high intensity in the same manner as embodiment 1. Further, the cell is made to move in flow cell, the light to mobile cell irradiation wavelength X 1 produces fluorescence from fluorescent material 11. Now, for cell with the light of low-power illumination wavelength lambda 1.Therefore, the fluorescence of wavelength period B1 of transmission filter part 21 becomes Low-intensity.

In the apparatus structure of embodiment 4, compared to embodiment 1, in omitting the optical detection portion 130 shown in Fig. 6 , light source 302, collector lens 312, dichroscope 321, filter member 412,422, collector lens 432 and light accepting part 502.In addition, as shown in figure 17, in the apparatus structure of embodiment 3, compared to embodiment 1, illumination part 300 is added Light source 305 and collector lens 315, collector lens 471, filter member 472 and collector lens 473 are added to light collecting part 400. In addition, in the apparatus structure of embodiment 3, compared to embodiment 1, adding light accepting part 505.

Additionally, Figure 17 is the figure that optical detection portion 130 is observed on the direction parallel with X/Y plane.In fig. 17, it is For the sake of convenient, with regard to illumination part 300, it is illustrated that in the state that Y-axis pros look up, with regard to light collecting part 400 and light accepting part 505, it is illustrated that in the state that X-axis losing side is looked up.

In the stream 210 of flow cell 200, the light of the wavelength X 1 projected from light source 301 is irradiated to position 211.Light source 305 Constitute in the same manner as light source 301, light is projected with the power lower than light source 301.Collector lens 315 makes the light projected from light source 305 The position 212 of the Z axis positive side positioned at position 211 is condensed in stream 210.Collector lens 471 is made from the glimmering of the generation of position 212 Light optically focused.Filter member 472 only makes the light transmission of in the light by the optically focused of collector lens 471, wavelength period B1.Light accepting part 505 Receive by the low intensive light of wavelength period B1 of the optically focused of collector lens 473, using based on the image information of light for receiving as taking the photograph As signal output.

In advance acquirement cell aligns the time T to position 212 from position 211.Thus, if connect by light accepting part 501 After by the light based on certain cell, elapsed time T is then received based on homocellular light by light accepting part 505.Accordingly, it is capable to Enough images obtained by the image obtained according to light accepting part 501 and according to light accepting part 505 and the image pair from the acquisition of same cell Should get up.

In embodiment 4, also in the same manner as embodiment 1 so that produce the fluorescence of high intensity and low intensive respectively Fluorescence, can obtain the fluoroscopic image and low intensive fluoroscopic image of high intensity.Therefore, in the same manner as embodiment 1, Neng Gougen According to the fluoroscopic image and low intensive fluoroscopic image of high intensity, precision parses well the distribution in cell and measures varied NF- κ B.

In embodiment 5, compared to embodiment 1, the step of cellular informatics acquisition methods shown in Fig. 1 in, only There is a part of step in step S2 different.Hereinafter, the steps different from embodiment 1 are illustrated.

In step s 2, as shown in figure 18, the sample comprising the cell identified with fluorescent material 11～13 is flowed into circulation Pond, the illumination of 1～λ of wavelength X 3 is mapped to the cell flowed in flow cell, and from fluorescent material 11～13 fluorescence is produced.From fluorescence The fluorescence that matter 11,12 is produced incides altogether filter member 24.Filter member 24 is made up of prism.From fluorescent material 11st, 12 fluorescence for producing pass through fluorescence and wavelength period B2 that filter member 24 is divided into wavelength period B1 according to the difference of wavelength period Fluorescence.Here, in the same manner as embodiment 1, for cell with the light of high power illumination wavelength X 1, for cell with low-power The light of illumination wavelength lambda.Thus, in the same manner as embodiment 1, the fluorescence of wavelength period B1 of transmission filter part 21 becomes high-strength Degree, the fluorescence of wavelength period B2 of transmission filter part 22 becomes low-intensity.

In addition it is also possible to be, the fluorescence produced from fluorescent material 11～13 incides altogether filter member 24, according to The difference of wavelength period is respectively classified into the fluorescence of wavelength period B1～B3 by filter member 24.Here, showing for embodiment party The example that the structure of formula 1 is used, but it is also possible to the filter member 24 used in the structure of embodiment 2～4.

As shown in figure 19, in the apparatus structure of embodiment 5, compared to embodiment 1, the optics shown in Fig. 6 is omitted In test section 130, filter member 411,412,421,422, to light collecting part 400 filter member 481 is added.Optical filter portion Part 481 is made up of prism.

The fluorescence produced from the sample flowed in flow cell 200 incides filter member 481.According to the ripple of fluorescence It is long, project the fluorescence for inciding filter member 481 at different angles from filter member 481.Collector lens 431 and light Portion 501 is configured at direction corresponding with the fluorescence of wavelength period B1 in the fluorescence projected from filter member 481.Thus, light Portion 501 can image to the fluorescence of the high intensity of wavelength period B1.Collector lens 432 and light accepting part 502 be configured at from filter The corresponding direction of fluorescence of wavelength period B2 in the fluorescence that optical sheet unit 481 is projected.Thus, light accepting part 502 can be to wavelength period The low intensive fluorescence of B2 is imaged.

In embodiment 5, also in the same manner as embodiment 1 so that produce the fluorescence of high intensity and low intensive respectively Fluorescence, can obtain the fluoroscopic image and low intensive fluoroscopic image of high intensity.Therefore, in the same manner as embodiment 1, Neng Gougen According to the fluoroscopic image and low intensive fluoroscopic image of high intensity, precision parses well the distribution in cell and measures varied NF- κ B.

In embodiment 6, make matrix touch the tested substance that includes in cell to cause to produce fluorescent material, it is right The fluorescent material irradiation light of generation, according to the fluorescence produced from fluorescent material by the irradiation of light, differentiates the office of tested substance Portion's existence.In embodiment 6, tested substance is cytoplasm.Matrix includes being cut when tested substance is touched Disconnected place of incision, if place of incision is cut off fluorescent material is produced.More specifically, matrix is used as quilt by touching The cytoplasm of detection material, the digestion being present in cytoplasm is broken.In embodiment 6, according to from marked cells matter The fluorescence of fluorescent material, judges cytoplasmic locally lying in.Additionally, tested substance can also be the albumen in such as cytoplasm The material in addition to the cytoplasm included in cell such as matter, cell organella, cell membrane.

In embodiment 6, compared to embodiment 3, the step of cellular informatics acquisition methods shown in Fig. 1 in, step The step of a part in rapid S1 is different.Hereinafter, the steps different from embodiment 3 are illustrated.

In step sl, as shown in figure 20, cell and matrix 16a mix.Matrix 16a is included in by cytoplasm The material of fluorescent material 16b is produced during esterase hydrolyzable.If cell and matrix 16a mix, the base of cell membrane has been transmitted Matter 16a, by the esterase hydrolyzable included in cytoplasm, produces fluorescent material 16b by contacting with cytoplasm.So, profit With fluorescent material 16b marked cells matter.

Next, in the same manner as embodiment 3, carrying out the later process of step S2.I.e., in step s 2, comprising with glimmering The sample of the cell of stimulative substance 16b marks is flowed into flow cell, and as shown in figure 20, wavelength X 1, the illumination of λ 3 are mapped in flow cell The cell of middle flowing, produces fluorescence from fluorescent material 16b, 13 respectively.Then, as shown in figure 20, produce from fluorescent material 16b Fluorescence is divided into 2, makes a side by filter member 21, makes the opposing party pass through filter member 22.Thus, with embodiment 3 similarly, has transmitted the fluorescence of wavelength period B1 of filter member 21 and has become high intensity, has transmitted the wavelength of filter member 22 The fluorescence of section B4 becomes low-intensity.Additionally, the device based on embodiment 6 is constituted in the same manner as embodiment 3.

In embodiment 6, also in the same manner as embodiment 3 so that produce 2 different fluorescence of intensity, can obtain The fluoroscopic image of high intensity and low intensive fluoroscopic image.Therefore, it is possible to according to the fluoroscopic image of high intensity and low intensive glimmering Light image, precision differentiates well cytoplasmic locally lying in.So, if it is possible to which precision differentiates well cytoplasmic local Exist, then can precision be well defined cytoplasmic scope.Thus, for example, can be by cytoplasmic scope and other parsings Combine and parsed in more detail.In addition, for example, cytoplasmic scope can be made to play a role in the research of cell etc..

Next, illustrating the checking of the embodiment 6 that inventor is carried out.

1. prepare

As cell, prepared human heart tiny blood vessels endothelial cell (HMVEC-C) (Lonza CatNo.CC-7030, Lot No.0000296500(P4)).As cell qualitative character reagent, Cell Explorer Fixable Live are prepared Cell Tracking Kit*Green Fluorescence*(Cosmobio 22621).Cell qualitative character reagent is included and figure The corresponding material of matrix 16a shown in 20.Cell qualitative character reagent has hydrophobicity.Cell qualitative character reagent passes through cell membrane, Due to producing fluorescent material by intracellular esterase hydrolyzable.The fluorescent material that here is produced and the fluorescence shown in Figure 20 Matter 16b correspondence.As nuclear staining pigment, the solution (DOjinDO of Cellstain Hoechst 33342 are prepared H342).In addition, having prepared EGM-2MV Medium (Lonza Cat No.CC-3202), EGM-2MV SingleQuots Kit (LonzaCat No.CC-3202)、PBS pH7.4(GIBCO Cat No.10010-023)、BSA(LAMPIRE Cat No.7500805)、PFA(WAKO Cat No.160-16061)、TritonX100(Nacalai Tesque CatNo.35501- 15)。

2. reagent modulation

EGM-2MV SingleQuots Kit are added to the EGM-2MV Medium of 500mL, culture medium has been made.In profit After dissolving paraformaldehyde with its ultimate density as 8%w/v with the PBS of pH12, pH7.4 is adjusted to.To PBS plus 1.5g's BSA simultaneously dissolves and is complemented into 50mL, has modulated 3%BSA/PBS.The BSA of 0.5g is added to PBS and is dissolved and is complemented into 50mL, 1%BSA/PBS is modulated.Using PBS TritonX100 is modulated with its ultimate density as 0.1%w/v.In Track kit Add the DMSO of 100 μ L in the bottle of Green, make 1000 × Track kit Green stock solution, it is attached to Kit Assay buffer add 1/1000 amount, so as to have adjusted Track kit working solution.

3. step

HMVEC-C is to recommend agreement in accordance with manufacturer, using EGM-2MV medium cultures.In this checking, use Cell within subculture number is 6 times after buy.With regard to culture medium, during the useful life behind Kaifeng is set into 3 weeks. After cell is cultivated according to 70% interflow, the culture medium for retaining 3mL or so simultaneously removes culture medium with electric pipettor, uses scraper Cell is peeled off.The suspension of the 3mL to reclaiming adds the Track kit working solution of 3 μ L, in 37 DEG C of CO₂ 30 minutes have been stood in insulating box.30 minutes are being stood afterwards, at room temperature, with the centrifugation point that 1000rpm has carried out 3 minutes From.Cell granulations is rinsed 3 times with the PBS of 5mL.Supernatant is removed, the 1%BSA/PBS of 50 μ L is with the addition of.

4. detected using flow cytometer

As the flow cytometer that can obtain fluoroscopic image, ImageStreamX Mark II Imaging have been used Flow Cytometer(Merck Millipore).Make the circulation that the flow cytometer is flowed into according to the sample of above-mentioned 3 modulation Pond, the sample to flowing in flow cell has irradiated the laser of wavelength 488nm, 405nm.The laser of wavelength 488nm, 405nm with The laser correspondence of wavelength X 1, λ 3 shown in Figure 20.The injection power of the laser of wavelength 488nm, 405nm is respectively 50mW, 20mW. Fluorescence is generated because the laser of wavelength 488nm is irradiated to the fluorchrome of marked cells matter.Due to the laser of wavelength 405nm It is irradiated to nuclear staining pigment and generates fluorescence.

In above-mentioned flow cytometer, via the filter member of transmission peak wavelength section 480nm～560nm, to by wavelength The fluorescence that the laser of 488nm is produced is imaged, and obtains the fluoroscopic image of high intensity.In addition, via transmission peak wavelength section 560nm The filter member of～595nm, images to the fluorescence that the laser by wavelength 488nm is produced, and obtains low intensive glimmering Light image.Via the filter member of transmission peak wavelength section 420nm～505nm, the fluorescence produced to the laser by wavelength 405nm Imaged, obtained fluoroscopic image corresponding with core.In addition, the sample to flowing in flow cell has irradiated wavelength and has been set as Laser between 420nm～480nm.Via the filter member of transmission peak wavelength section 420nm～480nm, the laser is transmitted Light after cell is imaged, and obtains bright field image.Additionally, in above-mentioned flow cytometer, by filter member etc. To eliminate the light of unwanted wavelength period, to become the light of wavelength period of object light accepting part is suitably incided.

With reference to Figure 21, the image obtained by above-mentioned detection is illustrated.

" bright field " represents the bright field image of cell." fluorescence of high intensity " and " low intensive fluorescence " is based on respectively From the image of the fluorescence for identifying the high intensity that cytoplasmic fluorchrome is produced and based on from identifying cytoplasmic iridescent The image of the low intensive fluorescence that element is produced." from the fluorescence of core " is based on from the nuclear staining pigment dyeed to core The image of the fluorescence of generation.4 transversely arranged images are the images obtained from 1 cell.For convenience, bright field image Image in addition is to carry out image obtained from GTG to acquired coloured image.Image beyond bright field image In, white part represents that the intensity of fluorescence is strong.

In the case of the cell shown in lower 1 section of the cell shown in uppermost and uppermost, based on low intensive glimmering The intensity of the image of light is too low, thus it is cytoplasmic locally lie in it is unclear.On the other hand, based on high intensity fluorescence image Intensity it is appropriate, it is possible to precision differentiates well cytoplasmic locally lying in.Under in the cell shown in lowermost and most In the case of cell shown in upper 1 section of section, the intensity based on the image of the fluorescence of high intensity is too high, so being difficult to differentiate cell Matter locally lies in.On the other hand, the intensity based on the image of low intensive fluorescence is appropriate, it is possible to precision differentiates well It is cytoplasmic to locally lie in.

As described above, understood according to this checking, if as Embodiment 6 based on 2 fluorograms that intensity is different Picture, then can differentiate cytoplasmic locally lying in.So, it can be seen from embodiment 6, a kind of fluorescence from marked cells matter is made The fluorescence that pigment is produced obtains 2 fluoroscopic images by the filter member 21,22 for passing through the fluorescence of different wavelength periods, Cytoplasmic locally lying in correctly is obtained in measure once thus, it is possible to be directed to 1 cell.

In embodiment 7, make 2 kinds of matrix touch the tested substance that includes in cell to cause to produce 2 kinds of fluorescence Material, to the 2 kinds of fluorescent material irradiation lights for producing, according to the fluorescence produced from 2 kinds of fluorescent materials by the irradiation of light, differentiates quilt That detects material locally lies in situation.That is, in embodiment 7,1 light is not used as Embodiment 6, but use The light of mutually different wavelength is obtaining the fluorescence of mutually different intensity.In addition it is also possible to it is detected to touch a kind of matrix Material come cause produce 2 kinds of fluorescent materials.

In embodiment 7, compared to embodiment 6, the step of cellular informatics acquisition methods shown in Fig. 1 in, only There is a part of step in step S1, S2 different.Hereinafter, the steps different from embodiment 6 are illustrated.

In step sl, as shown in figure 22, cell and matrix 17a, 18a mix.Matrix 17a, 18a is when by cytoplasm In include esterase hydrolyzable when produce the material of fluorescent material 17b, 18b respectively.Fluorescent material 17b, 18b are respectively structured as The fluorescence of mutually different wavelength period is encouraged when illuminated wavelength X 1, the light of λ 2.If cell and matrix 17a, 18a mix, Matrix 17a, 18a for then having transmitted cell membrane is contacted with cytoplasm, so as to pass through cytoplasm in the esterase hydrolyzable that includes, produce Raw fluorescent material 17b, 18b.So, using fluorescent material 17b, 18b marked cells matter.

In step s 2, the sample comprising the cell identified with fluorescent material 17b, 18b is flowed into flow cell, such as Figure 22 institutes Show, wavelength X 1, λ 2, the illumination of λ 3 are mapped to the cell flowed in flow cell, produce from fluorescent material 17b, 18b, 13 respectively glimmering Light.Now, the laser with high power to cell irradiation wavelength X 1, with laser of the low-power to cell irradiation wavelength X 2.By making The fluorescence produced from fluorescent material 17b becomes the fluorescence of wavelength period B1 by filter member 21.By making from fluorescent material The fluorescence that 18b is produced becomes the fluorescence of wavelength period B2 by filter member 22.So, the ripple of filter member 21 has been passed through The fluorescence of long section B1 becomes high intensity, has passed through the fluorescence of wavelength period B2 of filter member 22 and has become low-intensity.Additionally, being based on The device of embodiment 7 is constituted in the same manner as embodiment 1.

In embodiment 7, also in the same manner as embodiment 6,2 different fluorescence of intensity can be produced, be obtained high-strength The fluoroscopic image of degree and low intensive fluoroscopic image.Therefore, in the same manner as embodiment 6, can be according to the fluorogram of high intensity Picture and low intensive fluoroscopic image, precision differentiates well cytoplasmic locally lying in.

Claims

1. a kind of cellular informatics acquisition methods, it is characterised in that

The multiple fluorescent materials for making wavelength of fluorescence mutually different are combined with the tested substance included in cell,

The cell irradiation light is caused to produce the different fluorescence of wavelength and intensity from the plurality of fluorescent material,

According to each described fluorescence for producing, multiple fluorescence informations are obtained.

2. cellular informatics acquisition methods according to claim 1, it is characterised in that

According to the plurality of fluorescence information, the distribution situation of the tested substance in the cell is differentiated.

3. cellular informatics acquisition methods according to claim 1, it is characterised in that

According to the plurality of fluorescence information, differentiate the tested substance in the cell locally lies in situation.

4. cellular informatics acquisition methods according to claim 1, it is characterised in that

According to the plurality of fluorescence information, differentiate that the tested substance locally lies in and still locally lie in cytoplasm in core In.

5. cellular informatics acquisition methods according to claim 1, it is characterised in that

The wavelength of the light of the excitation of the plurality of fluorescent material is mutually different.

6. cellular informatics acquisition methods according to claim 1, it is characterised in that

To the cell, the 2nd weak light of the 1st light and the 1st light described in strength ratio is irradiated.

7. cellular informatics acquisition methods according to claim 1, it is characterised in that

The wavelength and intensity of the fluorescence that the plurality of fluorescent material is produced by the light of illuminated Same Wavelength is mutually different.

8. cellular informatics acquisition methods according to claim 1, it is characterised in that

The each fluorescence produced from the plurality of fluorescent material is separated into weak the 2nd glimmering of the 1st fluorescence described in the 1st fluorescence and strength ratio Light.

9. cellular informatics acquisition methods according to claim 1, it is characterised in that

The 1st fluoroscopic image of the 1st wavelength of fluorescence produced from the cell of illuminated 1st light is obtained,

Obtain the 2nd wavelength of fluorescence produced from the cell of illuminated the 2nd weak light of 1st light described in strength ratio the 2nd is glimmering Light image.

10. cellular informatics acquisition methods according to claim 1, it is characterised in that

The sample comprising the cell is set to be flowed into flow cell,

The cell irradiation light flowed in the flow cell is caused to produce the fluorescence.

11. cellular informatics acquisition methods according to claim 3, it is characterised in that

Locally lying in the differentiation of situation described in the tested substance, including the analysis object position of the cell The ratio of the amount of locally lying in of the tested substance in the relatively described cell entirety of the amount of locally lying in of the tested substance The calculating of example.

12. cellular informatics acquisition methods according to claim 3, it is characterised in that

It is in the fluorescence information obtained from the different multiple described fluorescence of intensity, be contained in from the intensity it is predetermined The fluorescence information that the fluorescence of scope is obtained, carries out the differentiation for locally lying in situation of the tested substance.

13. cellular informatics acquisition methods according to claim 3, it is characterised in that

The analysis object position of in the fluorescence information obtained from the different multiple described fluorescence of the intensity, cell The difference of the fluorescence intensity in the fluorescence intensity at place and the cellular portions in addition to the analysis object position is than predetermined The big fluorescence information of threshold value, carries out the differentiation for locally lying in situation of the tested substance.

14. cellular informatics acquisition methods according to claim 1, it is characterised in that

The tested substance is protein, mRNA, microRNA, cytoplasm, cell organella or cell membrane.

15. cellular informatics acquisition methods according to claim 1, it is characterised in that

According to the plurality of fluorescence information, calculate in the cell included in the sample, tested substance and locally lie in The ratio or quantity of the cell in specific position.

A kind of 16. cellular informatics acquisition methods, it is characterised in that

Matrix is contacted with the tested substance included in cell and cause to produce the mutually different multiple fluorescences of wavelength of fluorescence Matter,

A kind of 17. cellular informatics acquisition methods, it is characterised in that

Fluorescent material is set to be combined with the tested substance included in cell,

The cell irradiation light is caused to produce fluorescence from the fluorescent material,

From the fluorescence for producing, the different multiple fluorescence of wavelength and intensity are obtained,

According to each described fluorescence for obtaining, multiple fluorescence informations are obtained,

18. cellular informatics acquisition methods according to claim 17, it is characterised in that

19. cellular informatics acquisition methods according to claim 17, it is characterised in that

The fluorescence produced from the fluorescent material is separated into weak the 2nd glimmering of the 1st fluorescence described in the 1st fluorescence and strength ratio Light.

A kind of 20. cellular informatics acquisition methods, it is characterised in that

Matrix is contacted with the tested substance included in cell and cause to produce fluorescent material,

21. a kind of cellular informatics acquisition device, it is characterised in that possess：

Illumination part, to the cell irradiation comprising the tested substance for combining the mutually different multiple fluorescent materials of wavelength of fluorescence Light so that produce the different fluorescence of wavelength and intensity from the plurality of fluorescent material；

Light accepting part, receives each described fluorescence produced from the plurality of fluorescent material；And

Acquisition unit, according to the different fluorescence of intensity, obtains multiple fluorescence informations.

22. cellular informatics acquisition device according to claim 21, it is characterised in that

The illumination part possesses：

1st light source, irradiates the 1st light；And

2nd light source, illumination wavelength and the 2nd light that the 1st light is different and the 1st light described in strength ratio is weak.

23. cellular informatics acquisition device according to claim 21, it is characterised in that

Possesses the detached filter member of each fluorescence for making to be produced from the plurality of fluorescent material.

24. cellular informatics acquisition device according to claim 21, it is characterised in that possess：

1st light accepting part, receives the 1st fluorescence；And

2nd light accepting part, receives the 2nd weak fluorescence of the 1st fluorescence described in strength ratio.

25. cellular informatics acquisition device according to claim 21, it is characterised in that

The acquisition unit obtain the 1st fluoroscopic image of the 1st wavelength of fluorescence produced from the cell of illuminated 1st light and from 2nd fluoroscopic image of the 2nd wavelength of fluorescence that the cell of illuminated the 2nd weak light of 1st light described in strength ratio is produced.

26. a kind of cellular informatics acquisition device, it is characterised in that possess：

Illumination part, to the cell irradiation light comprising the tested substance for combining fluorescent material so that from the fluorescent material Produce fluorescence；

Light accepting part, receives from the different multiple fluorescence of the wavelength and intensity of fluorescent material generation；

Acquisition unit, according to the plurality of fluorescence for being received, obtains multiple fluorescence informations；And

Analysis unit, according to the plurality of fluorescence information, differentiates the distribution situation of the tested substance in the cell.

27. cellular informatics acquisition device according to claim 26, it is characterised in that

Possess weak for the fluorescence produced from the fluorescent material to be separated into the 1st fluorescence described in the 1st fluorescence and strength ratio The 2nd fluorescence filter member.

28. a kind of cellular informatics acquisition device, it is characterised in that possess：

Illumination part, to the cell irradiation light comprising the tested substance for causing to produce fluorescent material by contacting with matrix, So that producing fluorescence from the fluorescent material；