CN106645713B

CN106645713B - Cellular informatics acquisition methods and cellular informatics acquisition device

Info

Publication number: CN106645713B
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: 2018-09-18
Anticipated expiration: 2036-10-28
Also published as: JP6824654B2; CN106645713A; JP2017083426A

Abstract

The present invention discloses a kind of cellular informatics acquisition methods and cellular informatics acquisition device.In cellular informatics acquisition methods, the multiple fluorescent materials for keeping wavelength of fluorescence mutually different are combined with the tested substance for including in cell, to cell irradiation light so that from the different fluorescence of multiple fluorescent material generation wavelengths and intensity, multiple fluorescence informations are obtained according to each fluorescence of generation.For example, by filter member, each fluorescence generated from multiple fluorescent materials is separated into the 1st fluorescence and intensity 2nd fluorescence weaker than the 1st fluorescence.According to multiple fluorescence informations, the distribution situation of the tested substance in cell is differentiated.

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 proliferation of cell, differentiation, the various molecules such as protein, mRNA, microRNA It is locally lain in cell.When locally lying in of various molecules in parsing cell expects function parsing, the protein of molecule Between interact parsing, signaling path a large amount of biological phenomena such as parsing solution it is bright.

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

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

Invention content

The scope of the present invention is limited only by the accompanying claims, not by any influence of the invention description of contents.

The 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 for including in cell, to cell irradiation light so that from Multiple fluorescent material generation wavelengths and the different fluorescence of intensity obtain multiple fluorescence informations according to each fluorescence of generation.

In the cellular informatics acquisition methods of this programme, " the mutually different multiple fluorescent materials of wavelength of fluorescence " refer to, Multiple fluorescent materials send out the fluorescence of mutually different wavelength respectively when illuminated light.In order to enable being produced from multiple fluorescent materials Raw wavelength and the different fluorescence of 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.Fluorescence information is, for example, fluorescence-based image.In addition, " making Multiple fluorescent materials are combined with tested substance " refer to can also be not necessarily the tested substance of the same race for including in cell Each molecule is all combined with multiple fluorescent materials, multiple fluorescent materials particularly at least part of tested substance of the same race Molecule combine.

In the case that the distribution of tested substance in cell and amount are varied, exist and generated from fluorescent material Fluorescence the too strong situation of intensity, excessively weak situation, so can may not suitably be differentiated using 1 fluorescence information detected The distribution situation etc. of substance.However, according to the cellular informatics acquisition methods of this programme so that more from what is combined with tested substance A fluorescent material generates the different fluorescence of intensity, can obtain multiple fluorescence informations according to the different fluorescence of intensity.Even if as a result, In the case of can not suitably differentiating the distribution situation of tested substance since fluorescence is too strong when using a fluorescence information etc., If using other fluorescence informations, it will be able to suitably differentiate the distribution situation etc. of tested substance.Therefore, as long as using a certain A fluorescence information can suitably differentiate the distribution situation etc. of tested substance, so even if tested substance in cell Distribution and amount are varied, also can precisely parse tested substance.

The 2nd scheme of the present invention is related to cellular informatics acquisition methods.In the cellular informatics acquisition methods of this programme, make base Matter contacts to generate the mutually different multiple fluorescent materials of wavelength of fluorescence with the tested substance for including in cell, to cell Irradiation light and from the different fluorescence of multiple fluorescent material generation wavelengths and intensity, to be obtained according to each fluorescence of generation multiple Fluorescence information.

In the cellular informatics acquisition methods of this programme, matrix is made to be contacted with the tested substance for including in cell to make The mutually different multiple fluorescent materials of wavelength of fluorescence are generated, thus identify tested substance with multiple fluorescent materials.In this programme Cellular informatics acquisition methods in, also in the same manner as the 1st scheme, can suitably differentiate as long as using some fluorescence information The distribution situation etc. of tested substance, so tested substance can be parsed precisely.

The 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 for including in cell, to cell irradiation light so that fluorescence is generated from fluorescent material, from production Raw fluorescence obtains wavelength and the different multiple fluorescence of intensity, and multiple fluorescence informations are obtained according to each fluorescence of acquisition, 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 wavelength and the different multiple fluorescence of intensity, for example, the fluorescence generated from fluorescent material is made to pass through transmission peak wavelength section Different multiple filter members and detach.In addition, in the cellular informatics acquisition methods of this programme, fluorescence information is also for example Fluorescence-based image.Alternatively, it is also possible to be not necessarily the tested substance of the same race for including in cell each molecule all with it is glimmering Stimulative substance combines, and fluorescent material is particularly combined with the molecule of at least part 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, can suitably it sentence as long as using some fluorescence information The distribution situation etc. of other tested substance, so tested substance can be parsed precisely.

The 4th scheme of the present invention is related to cellular informatics acquisition methods.In the cellular informatics acquisition methods of this programme, make base Matter is combined to generate fluorescent material with the tested substance for including in cell, to cell irradiation light so that from fluorescent material Fluorescence is generated, the multiple fluorescence different from the fluorescence of generation acquisition wavelength and intensity obtain multiple according to each fluorescence of acquisition Fluorescence information differentiates the distribution situation of the tested substance in cell according to multiple fluorescence informations.

In the cellular informatics acquisition methods of this programme, by so that matrix is contacted with the tested substance for including in cell come So that generating fluorescent material, tested substance is identified with fluorescent material.In the cellular informatics acquisition methods of this programme, also with 3 schemes similarly, can suitably differentiate the distribution situation etc. of tested substance as long as using some fluorescence information, so Tested substance can precisely be parsed.

The 5th scheme of the present invention is related to cellular informatics acquisition device.The cellular informatics acquisition device of this programme has：Illumination Portion is penetrated, to including the cell irradiation light for the tested substance for combining the mutually different multiple fluorescent materials of wavelength of fluorescence so that The fluorescence different from multiple fluorescent material generation wavelengths and intensity；Acceptance part, receiving generate each glimmering from multiple fluorescent materials 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, effect same as the 1st scheme can be played.

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

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

The 7th scheme of the present invention is related to cellular informatics acquisition device.The cellular informatics acquisition device of this programme has：Illumination Portion is penetrated, to making from glimmering comprising the cell irradiation light for generating the tested substance of fluorescent material is made by being contacted with matrix Stimulative substance generates fluorescence；Acceptance part receives the wavelength that is generated from fluorescent material and the different multiple fluorescence of intensity；Acquisition unit, root According to the multiple fluorescence received, multiple fluorescence informations are obtained；And analysis unit differentiates according to multiple fluorescence informations in cell Tested substance distribution situation.

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

In accordance with the invention it is possible to precisely parse the distribution in cell and the diversified molecule of amount.

Description of the drawings

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

Fig. 2 is the figure for the summary for showing that the fluorescence of embodiment 1 obtains.

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

Fig. 4 (a) is the figure for the image for showing to obtain in the verification of embodiment 1.Fig. 4 (b) is shown in embodiment 1 Verification in the figure of numerical value that obtains.

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

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

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

Fig. 8 is the flow chart for the processing carried out using cellular informatics acquisition device for showing embodiment 1.

Fig. 9 is the figure for the picture for showing to show in the display unit of embodiment 1.

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

Figure 11 is the figure for the summary for showing that the fluorescence of embodiment 2 obtains.

Figure 12 is the figure for the summary for showing that the fluorescence of embodiment 3 obtains.

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

Figure 14 is the figure for the image for showing to obtain in the verification of embodiment 3.

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

Figure 16 is the figure for the summary for showing that the fluorescence of embodiment 4 obtains.

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

Figure 18 is the figure for the summary for showing that the fluorescence of embodiment 5 obtains.

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

Figure 20 is the figure for the summary for showing that the fluorescence of embodiment 6 obtains.

Figure 21 is the figure for the image for showing to obtain in the verification of embodiment 6.

Figure 22 is the figure for the summary for showing that the fluorescence of embodiment 7 obtains.

Figure 23 is the schematic diagram for illustrating to invent the project for wanting solution.

Specific implementation mode

The preferred embodiment of the present invention is described below with reference to attached drawing.

In the embodiment 1, include in the multiple fluorescent materials for keeping wavelength of fluorescence mutually different and cell is detected Substance is combined and differentiates the cell for locally lying in situation of tested substance according to the multiple fluorescence generated from fluorescent material In information acquisition method, the present invention is applied.In the embodiment 1, tested substance is NF- κ B.NF- as transcription factor κ B are present in the state of foring complex with I κ B in cytoplasm, it is believed that pass through point of the I κ B caused by various stimulations Solve and be transferred to the inside of core.In the embodiment 1, it is parsed as follows：Using NF- κ B as tested substance, fluorescence is used Matter particularly identifies, and according to the fluorescence from fluorescent material, judgement NF- κ B are present in which of cytoplasm and core.In addition, Tested substance can also be protein, the molecule other than NF- κ B.For example, tested substance can also be to turn other than NF- κ B The factor is recorded, for example, it can be STAT (Signal Transducer and Activator of Transcription, letters 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 made to be combined with tested substance " refers to being not necessarily the quilt of the same race for including in cell Detection substance each molecule all combined with multiple fluorescent materials, multiple fluorescent materials particularly with it is at least part of of the same race The molecule of tested substance combines.It is locally lain in core in addition, the differentiation for locally lying in situation is not limited to tested substance In still locally lie in the differentiation in cytoplasm.For example, in the case where having shape for lugs as nerve cell, also may be used To differentiate whether tested substance locally lies in the front end of protrusion.

As shown in Figure 1, cellular informatics acquisition methods include the steps that step S1~S4.Hereinafter, illustrating that operator uses energy The flow cytometer imaged to fluorescent image is reached with the processing unit that can be parsed to the image of camera shooting to execute Fig. 1 Cellular informatics acquisition methods the case where.Each step of Fig. 1 can also be executed by the processing in cellular informatics acquisition device. Then, with reference to after Fig. 5, illustrate the structure in the case that cellular informatics acquisition device carries out each step of Fig. 1 and processing.

In step sl, operator is extracted with the mutually different mark of fluorescent material 11,12 of wavelength of fluorescence from examinee The NF- κ B for including in cell.For example, as shown in Fig. 2, via an antibody and secondary antibodies, in fluorescent material 11,12 and cell Including NF- κ B combine.Fluorescent material 11,12 can both be combined by multiple antibody with NF- κ B, can also be 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 substance 11,12, the core for including in marked cells.

Fluorescent material 11,12,13 is fluorchrome.Fluorescent material 11,12,13 is respectively structured as illuminated wavelength X 1, λ 2, the fluorescence of mutually different wavelength period is encouraged when the light of λ 3.That is, the wave of the light for encouraging fluorescence from fluorescent material 11~13 Length is set to mutually different.In this way, modulating sample by step S1.In addition, 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 for including the cell identified with fluorescent material 11~13 Sample is flowed into flow cell, to the light of the 1~λ of cell irradiation wavelength X 3 flowed in flow cell so that from fluorescent material 11~13 Generate fluorescence.

As shown in Fig. 2, when to the laser of 1~λ of cell irradiation wavelength X 3, difference is generated respectively from fluorescent material 11~13 Wavelength period fluorescence.Filter member 21 makes the fluorescence of the wavelength period B1 generated from fluorescent material 11 pass through, and interdicts wavelength period Light other than B1.By filter member 21, the fluorescence of the wavelength period B1 generated from fluorescent material 11 is detached.Filter member 22 make the fluorescence of the wavelength period B2 generated from fluorescent material 12 pass through, the light other than blocking wavelength period B2.Pass through filter member 22, the fluorescence of the wavelength period B2 generated from fluorescent material 12 is detached.Filter member 23 makes the wave generated from fluorescent material 13 The fluorescence of long section B3 passes through, the light other than blocking wavelength period B3.By filter member 23, from the wavelength of the generation of fluorescent material 13 The fluorescence of section B3 is detached.

Here, with high power to the laser of cell irradiation wavelength X 1, with low-power to the laser of 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 the 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 the wavelength period B2 of filter member 22 becomes Low-intensity.

In step s3, processing unit obtains 3 according to the fluorescence generated from fluorescent material 11~13 for each cell Fluorescence information.Flow cytometer has the fluorescence difference of wavelength period B1~B3 for making to detach by filter member 21~23 The structure for being imaged onto the acceptance part being made of photographing element and obtaining the image based on each fluorescence.Processing unit is according to fluidic cell The image pickup signal of the acceptance part output of instrument, obtains the image of the fluorescence of the high intensity based on wavelength period B1, based on wavelength period B2's The image of the image of low intensive fluorescence and fluorescence based on wavelength period B3 is as fluorescence information.

About the fluorescence of wavelength period B1~B3, it both can individually be imaged, can also be led to respectively by 3 acceptance parts 1 acceptance part is crossed to be imaged.Optical system is configured to, and is taken the photograph to the fluorescence of wavelength period B1~B3 by 1 acceptance part The fluorescence of wavelength period B1~B3 is imaged on the light-receiving surface of acceptance 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 of the high intensity based on wavelength period B1, and image 32 is the low-intensity based on wavelength period B2 Fluorescence image.In image 31,32, region 33 existing for core is set.Region 33 is from based on being given birth to simultaneously with image 31,32 At wavelength period B3 fluorescence be is generated from core fluorescence image acquisition.

In the case where being that NF- κ B locally lie in the cell in cytoplasm, in step s3, such as acquisition image 41, 42.Image 41 is the image of the fluorescence of the high intensity based on wavelength period B1, and image 42 is based on the low intensive glimmering of wavelength period B2 The image of light.Region 43 existing for core are all set in image 41,42.Region 43 is from based on being generated simultaneously with image 41,42 Wavelength period B3 fluorescence image obtain.

In the case where being the few cell of discovery amount of NF- κ B, according to image 31, the intensity of fluorescence is appropriate, and in core Occurs difference in fluorescence intensity between cytoplasm, so can differentiate that NF- κ B are locally lain in the region of core 33.Another party Face, according to image 32, the intensity of fluorescence is too low, so can not differentiate that NF- κ B are locally lain in the region of core 33.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 excessively high, thus as core with it is thin Do not have discrepant state between cytoplasm in fluorescence intensity.Therefore, it is impossible to differentiate that NF- κ B are locally lain in the region of core 43 and ratio Which of the wide cytoplasmic region in the region 43 of core region.On the other hand, according to image 42, the intensity of fluorescence is appropriate, And difference is generated in intensity between core and cytoplasm, so can differentiate 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 are distributed in the cell in a manner of surrounding core.That is, When observing cell on imaging direction, NF- κ B are also distributed in the front of core.Therefore, in the figure of the fluorescence based on high intensity In 41, strong fluorescence is also generated in the region of core by being distributed in the NF- κ B of front of core.Therefore, it in image 41, deposits The trend in cytoplasm is still locally lain in core can not suitably differentiate that NF- κ B are locally lain in.In contrast, in base In the image 42 of low intensive fluorescence, although the NF- κ B of the front by being distributed in core also generate fluorescence in the region of core, But the intensity of the fluorescence is low.Therefore, even if can be fitted if in the case that NF- κ B are locally lain in cytoplasm in image 41 Locality, which differentiates, 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 more than half It is distributed in core.Therefore, in the image 31 of the fluorescence based on high intensity, strong fluorescence is generated from the NF- κ B being distributed in core, It can suitably differentiate that NF- κ B are locally lain in core.On the other hand, it in the image 32 based on low intensive fluorescence, comes from The fluorescence for being distributed in the NF- κ B in core is excessively weak, so in the presence of can not suitably differentiate that it is in core or local that NF- κ B are locally lain in The trend being present in cytoplasm.

In this way, according to the amount of the NF- κ B as intracellular tested substance and distribution, the part for differentiating NF- κ B Existing appropriate intensity is different.

Therefore, be set to can be appropriate in the cell that NF- κ B locally lie 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 locally deposit in NF- κ B It is in the cell in cytoplasm suitably differentiate that the NF- κ B as shown in image 42 are locally lain in cytoplasm.Exist as a result, As in the cell for differentiating object, no matter NF- κ B are locally lain in which of core and cytoplasm, 2 can be used At least one party in image differentiates locally lying in for NF- κ B.

Back to Fig. 1, in step s 4, operator with reference to the high intensity based on wavelength period B1 fluorescence image and be 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, behaviour Author selects in the image of the image of the fluorescence of the high intensity based on wavelength period B1 and the low intensive fluorescence based on wavelength period B2 , the image for locally lying in position that can differentiate NF- κ B, according to the image, differentiate in the cell NF- κ B locally lie in In which of core and cytoplasm, locally lie in situation.It can also replace locally lying in situation, differentiate that NF- κ B are distributed in Which position, such as intracellular distribution.

As described above, in the embodiment 1, by adjusting 2 generated from the fluorescent material 11,12 for identifying NF- κ B Fluorescence, the one party become in the image of the fluorescence based on high intensity and the image based on low intensive fluorescence can suitably be sentenced Determine the state of NF- κ B locally lain in.Therefore, operator can precisely parse the distribution in cell according to 2 images Diversified NF- κ B.Specifically, can precisely differentiate that the NF- κ B's in cell locally lies in situation, i.e. NF- κ B is locally lain in which of core and cytoplasm.

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

Here, vascular endothelial cell is removed from the inner wall of blood vessel and is flowed into blood.The stripping of vascular endothelial cell, is removed Other than the stimulation caused by inflammation generates, due also to the variation of the pressure caused by compressing etc. also generates.Due to In the vascular endothelial cell removed by the stimulation caused by inflammation, NF- κ B tend to locally lie in core, due to because of inflammation Stimulation other than caused stimulation and in the vascular endothelial cell removed, NF- κ B tend to not locally lie in core.According to reality Mode 1 is applied, can precisely differentiate locally lying in for NF- κ B as described above, so can be according to as signaling molecule Whether NF- κ B locally lie in judge to remove at these in core due in inflammation caused by stimulation and the stripping that generates From can determine vascular endothelial cell, whether there is or not activations.As a result, for example, can judge the stripping of vascular endothelial cell be due to Compressing when blood sampling and generate or generated for 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. That is, 3 fluorescent materials that wavelength of fluorescence can also be used mutually different identify NF- κ B, to 3 laser of cell irradiation so that from 3 A fluorescent material generates the different fluorescence of intensity to obtain the image based on each fluorescence.It is different by the intensity from fluorescence as a result, 3 images in use 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 4 grades or more, can also be directed to 1 cell and obtain based on 4 or more intensity 4 or more images of fluorescence.

In turn, in step s 4, operator obtains examination according to the situation that locally lies in of each cell differentiated as described above In the cell for including in sample, NF- κ B locally lie in the ratio of the cell in privileged site.Specifically, if will differentiate The quantity that the cell in core is locally lain in for NF- κ B is set as N1, will be determined as NF- κ B and locally lies in the cell in cytoplasm Quantity be set as N2, then operator is by formula below, obtains that core locally lies in rate and cytoplasm locally lies in rate.In addition, In step S4, operator can also locally lie in rate instead of core and cytoplasm locally lies in rate and obtain core and locally lie in number and thin Cytoplasm 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, the image of each fluorescence is obtained using flow cytometer as described above, but not limited to this, also may be used To use microscope, the image of each fluorescence is obtained as fluorescence information.That is, can also be obtained from fluorescent material by microscope The fluorescent image of 11 high intensity generated is produced from the low intensive fluorescent image of the generation of fluorescent material 12 and from fluorescent material 13 Raw image corresponding with core.

Next, illustrating the verification for the embodiment 1 that inventor carries out.

1. preparing

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) are prepared 647 conjugate of Secondary Antibody, Alexa Fluor (Life technologies A-21245), Goat 488 conjugate (Life of anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor technologies A-11008).Alexa Fluor 647, Alexa are combined as fluorchrome to secondary antibodies Fluor 488.As nuclear staining pigment, 33342 solution (DOjinDO of Cellstain Hoechst 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 is modulated

It, will to the reagent other than the FBS of the EGM-2MV Medium addition EGM-2MV SingleQuots Kit of 500mL 100mL moves on to aseptic bottle, has made serum free medium.To the surplus (400mL) of free serum culture matrix manufacturing, 20mL is added Single Quots Kit FBS, produce culture medium.Paraformaldehyde is dissolved with its ultimate density in the PBS using pH12 After 8%w/v, it is adjusted to pH7.4.PBS plus the BSA of 1.5g and is dissolved and is complemented into 50mL, 3%BSA/ has been modulated PBS.PBS plus the BSA of 0.5g and is dissolved and is complemented into 50mL, 1%BSA/PBS has been modulated.It is modulated using PBS TritonX100 is 0.1%w/v with its ultimate density.

3. step

HMVEC-C is to recommend agreement in accordance with manufacturer, is cultivated using EGM-2MV culture mediums.It is used in this verification Subculture number is the cell within 6 times after buy.About culture medium, during the service life behind Kaifeng is set as 3 weeks. It stimulates and cultivates about TNF-α, the culture supernatant of the HMVEC-C cells at about 70% interflow of removal, in addition to become ultimate density The mode of 25ng/mL is added to 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, scraper is used in combination to remove cell.In addition With the 8%PFA/PBS of the suspension equivalent of recycling, react at room temperature 15 minutes.At room temperature, 3 have been carried out with 1000rpm The centrifugation of minute.Cell granulations have been rinsed 2 times with the PBS of 1mL.Supernatant is removed, in addition the 0.1%Triton X- of 1mL 100/PBS has reacted 15 minutes at room temperature.At room temperature, centrifugation in 3 minutes has been carried out with 1000rpm.With 1mL's 1%BSA/PBS has been rinsed 2 times.Supernatant is removed, in addition the 3%BSA/PBS of 1mL, has stood 30 minutes at room temperature.3% An antibody of 1/1,600 400 μ L is added in BSA/PBS.It has reacted at room temperature 1 hour.At room temperature, with 1000rpm Centrifugation in 3 minutes is carried out.It is rinsed with the 1%BSA/PBS of 1mL.1/1000 is added in 3%BSA/PBS 400 μ L secondary antibodies.It has reacted at room temperature 30 minutes.It has been rinsed 2 times with the 1%BSA/PBS of 1mL.Supernatant is removed, is added The 1%BSA/PBS of 50 μ L is added.

4. being detected using flow cytometer

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

In above-mentioned flow cytometer, via the filter member of transmission peak wavelength section 505nm~560nm, to passing through wavelength The fluorescence that the laser of 488nm generates is imaged, and the fluorescent image of high intensity is obtained.Via transmission peak wavelength section 642nm~ The filter member of 740nm, the fluorescence generated to the laser by wavelength 647nm image, and obtain low intensive fluorescence Image.Via the filter member of transmission peak wavelength section 430nm~505nm, fluorescence that the laser by wavelength 405nm is generated into Row camera shooting, obtains fluorescent image corresponding with core.In addition, to the sample flowed in flow cell, wavelength is irradiated and has been set Laser between 430nm~480nm.Via the filter member of transmission peak wavelength section 430nm~480nm, which is transmitted Light after cell is imaged, and obtains bright field image.In addition, in above-mentioned flow cytometer, pass through filter member Acceptance part is suitably incident on the light for becoming the wavelength period of object etc. to remove the light of unwanted wavelength period.In addition, Bright field image is obtained in this verification, but not limited to this, scotopia field picture can also be obtained.

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

" bright field " indicates the bright field image of cell." fluorescence of high intensity " and " low intensive fluorescence " is based on respectively The image of the fluorescence of the high intensity generated from the fluorchrome for identifying NF- κ B and based on from the fluorchrome for identifying NF- κ B The image of the low intensive fluorescence generated." fluorescence from core " is based on being produced from the nuclear staining dyed to core with pigment The image of raw fluorescence." synthesis " is image obtained from 4 images in synthesis left side.5 transversely arranged images are from 1 The image that cell obtains.The figure that " fluorescence of high intensity ", " fluorescence from core ", " low intensive fluorescence " and " synthesis " indicates Seem that image obtained from grayscale is carried out to acquired coloured image for convenience.At " fluorescence of high intensity ", " come from In the image that the fluorescence of core " and " low intensive fluorescence " indicate, white part indicates that the intensity of fluorescence is strong.

Shown in epimere in the case of cell, the intensity of the image based on 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 of the image of the fluorescence based on high intensity is appropriate, so NF- can be determined as κ B are locally lain in core.Shown in hypomere in the case of cell, the intensity of the image of the fluorescence based on high intensity is excessively high, institute To be difficult to differentiate locally lying in for NF- κ B.On the other hand, the intensity of the image based on low intensive fluorescence is appropriate, so can NF- κ B are determined as to locally lie in cytoplasm.

5. core locally lies in the calculating of rate

It is visual by being carried out to acquired image, differentiate locally lying in for NF- κ B for each cell.With above-mentioned step Rapid S4 has been carried out similarly the differentiation.That is, the region of core is set according to the fluorescent image from core, by the area other than the region of core Domain is as cytoplasmic region.In addition, the case where the fluorescence intensity for thinking core is the about 2 times or more of cytoplasmic fluorescence intensity Under, it is determined as the NF- κ B in the cell and locally lies in core, be 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 to be directed to and with 131 cells that above-mentioned flow cytometer identifies differentiated that the part of NF- κ B is deposited Result.

The quantity for being determined as locally lying in the cell in core is 44, is determined as locally lying in the cell in cytoplasm Quantity is 87.It can not differentiate that the quantity of the cell locally lain in is 0.It is 44/131=34% that core at this time, which locally lies in rate,.

Here, explanation has differentiated the comparative example 1 locally lain in and according only to low-intensity according only to the fluorescent image of high intensity Fluorescent image differentiated the comparative example 2 locally lain 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 excessively high, so can not Differentiate that the quantity of the cell locally lain in is 79.It is 81% that the core of comparative example 1, which 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 can not differentiate that the quantity of the cell locally lain in is 31.The core of comparative example 2 locally lies in rate 16%.

As described above, according to this verification it is found that as Embodiment 1 according to 2 different fluorescent images of intensity In the case that differentiation is locally lain in, about the cell that can not be differentiated in the case of Comparative Examples 1 and 2, it can also differentiate NF- κ B's It locally lies in.In addition we know, in the case where differentiation as Embodiment 1 is locally lain in, the cell that can not be differentiated is few, so It can precisely differentiate locally lying in for the NF- κ B in cell.Therefore, according to embodiment 1, can will can not differentiate The quantity of cell inhibits relatively low, precisely differentiates locally lying in for NF- κ B.As a result, for example, even if being extracted from examinee Cell it is few in the case of, also can precisely differentiate that the part of NF- κ B is deposited while the cell for guaranteeing to differentiate .

In the embodiment 1, the differentiation as the distribution situation of tested substance shows and differentiates that the part of 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 can also differentiate the amount of the molecule. In this case, operator identifies molecule with fluorescent material 11,12, processing unit is according to the intensity generated from fluorescent material 11,12 2 different fluorescence obtain image.In the case that operator is more than the amount of molecule, differentiated using low intensive fluorescent image Amount, in the case where the amount of molecule is few, using the fluorescent image of high intensity come the amount of differentiation.Thereby, it is possible to precisely differentiate The amount of molecule.

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

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

Processing unit 110 is made of microcomputer and CPU etc..Storage part 170 is made of RAM, ROM, hard disk etc..Storage Portion 170 stores the various data such as processing routine, the image executed by processing unit 110.Processing unit 110 is obtaining dress with cellular informatics The transmitting and receiving for carrying out signal between 100 each portion is set, each portion is controlled.In processing unit 110, by being stored in storage part 170 In program, assign acquisition unit 111 and analysis unit 112 function.

For sample modulation portion 120 according to the step S1 of Fig. 1, thus cell mixing and reagent modulate sample.It 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 be flow cytometer.Optical detection portion 130 images the fluorescence of generation the cell irradiation light for including in sample.With Afterwards, the structure in optical detection portion 130 is illustrated with reference to Fig. 6.Driving portion 140 drive the light source 301 in aftermentioned optical detection portion 130~ 304。

Display unit 150 is made of display.The display of display unit 150 is directed to the image of each cell acquisition, for each thin The locally lying in of NF- κ B that born of the same parents differentiate, NF- κ B locally lie in cell number 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 of mouse and keyboard.Operator Via input unit 160 for the input instruction of cellular informatics acquisition device 100.

As shown in fig. 6, optical detection portion 130 has flow cell 200, illumination part 300, light collecting part 400 and acceptance part 501~504.Flow path 210 is formd in flow cell 200, in flow path 210, flows through the examination modulated using sample modulation portion 120 Sample.In figure 6, for convenience, it is illustrated that mutually orthogonal XYZ axis.

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

Light source 301~304 is made of semiconductor laser light source.The light projected from light source 301~304 is 1~λ of wavelength X 4 respectively Laser.1~λ of wavelength X 4 is such as 488nm, 647nm, 405nm, 785nm respectively.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.Collector lens 311~314 respectively carries out the light projected from light source 301~304 Optically focused.Dichroscope 321 makes the light transmission of wavelength X 1, makes the light reflection of wavelength X 2.Dichroscope 322 make wavelength X 1, λ 2 light Transmission, makes the light reflection of wavelength X 3.

In this way, illumination part 300 is to the cell that includes in the sample that is flowed in flow path 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 to the light of the 1~λ of sample illumination wavelength lambda 3 flowed in flow cell 200, The fluorescence of different wavelength periods is generated from fluorescent material 11~13 as illustrating with reference to Fig. 2.If to being flowed 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 Driving portion 140 shown in fig. 5 controls the injection power of light source 301,302.As a result, from fluorescent material as illustrating with reference to Fig. 2 11 fluorescence generated become high intensity, and the fluorescence generated from fluorescent material 12 becomes low-intensity.In addition, preferably 1 with And in aftermentioned embodiment 2,3, the injection power of light source 301 and 302 can not also be adjusted.By selecting even same penetrate Going out power but the discrepant fluorescence labelling of obtained fluorescence intensity, the fluorescence generated from fluorescent material 11 becomes high intensity, from The fluorescence that fluorescent material 12 generates becomes low-intensity.

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

Collector lens 401 makes the fluorescence that the sample flowed from flow cell 200 generates 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 of dichroscope.

Filter member 411 makes in the light by 401 optically focused of collector lens, wavelength period B1 light reflection, makes wavelength period Light transmission other than B1.Filter member 421 only makes in the light reflected by filter member 411, wavelength period B1 light saturating It penetrates, the light other than blocking wavelength period B1.In this way, filter member 411,421 is configured to be merely able to separation from the generation of flow cell 200 Light in, the fluorescence of wavelength period B1.Similarly, filter member 412,422 is configured to be merely able to separation from the production of flow cell 200 In raw light, wavelength period B2 fluorescence, filter member 413,423 is configured to be merely able to detach to be generated from flow cell 200 In light, wavelength period B3 fluorescence.Filter member 424 makes to have transmitted in the light of filter member 411~413, wavelength X 4 Light transmission, interdict wavelength X 4 other than light.

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

Light collecting part 400 makes the light of wavelength period B1~B3 and the light of wavelength X 4 be condensed to acceptance part 501~504 respectively, but It can be imaged in 1 acceptance 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 light-receiving surface of acceptance part in different regions.

In structure shown in Fig. 6, multiple filter members are used in order to detach the light of wavelength period B1, but can also As shown in fig. 7, detaching the light generated from flow cell 200 by 1 filter member.As shown in fig. 7, light collecting part 400 have it is poly- Optical lens 441~444, filter member 451~454 and collector lens 461~464.The light of wavelength period B1~B3 leads to respectively It crosses filter member 451~453 to be detached, the light of wavelength X 4 is detached by filter member 454.

Next, with reference to the flow chart of Fig. 8, illustrate the place that the step S4 of Fig. 1 is carried out by cellular informatics acquisition device 100 The case where reason.

As shown in figure 8, in step s 11, processing unit 110 drives sample modulation portion 120, in the same manner as the step S1 of Fig. 1, It is tried with the core for including in 13 marked cells of fluorescent material to modulate with the NF- κ B for including in fluorescent material 11,12 marked cells Sample.In step s 12, processing unit 110 makes sample flow into flow cell 200, and light source 301~304 is driven by driving portion 140, 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 acceptance part The fluorescence of B1~B3 is imaged, and is imaged to the light of wavelength X 4 by acceptance part 504.Then, the acquisition unit of processing unit 110 111 image pickup signals exported according to acceptance part 501~504, obtain image.

In step S14, from high intensity and low intensive fluorescent image, selection can be into for the analysis unit 112 of processing unit 110 The image for the differentiation that row is locally lain in.Specifically, analysis unit 112 selects the fluorescent image of high intensity and low intensive fluorogram The image of the brightness of the intensity such as image entirety of fluorescence as in, being obtained from image within a predetermined range.It is glimmering as a result, The intensity of light terrifically big image, fluorescence intensity terrifically can not in the same manner as small image and the image 32 of Fig. 3, image 41 Locally lying in for NF- κ B is differentiated, so the image used from differentiation removes.

In addition, analysis unit 112 selects in high intensity and low intensive fluorescent image, the analysis object position of cell The difference of the fluorescence intensity in cell other than the fluorescence intensity and analysis object position image bigger than scheduled threshold value.In embodiment party In formula 1, analysis object position is core.That is, the difference of the fluorescence intensity in selection core and the fluorescence intensity of the cell outside core is than predetermined The big image of threshold value.The image 32 of the difference of the fluorescence intensity in core and outside core small image and Fig. 3, image 41 are same as a result, Ground can not differentiate locally lying in for NF- κ B, so the image used from differentiation removes.

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, the part that analysis unit 112 calculates the NF- κ B at the analysis object position of cell is deposited The ratio of the amount of locally lying in of NF- κ B in measuring versus cell entirety.For example, the figure that analysis unit 112 selects in step S14 As in, by the fluorescence intensity in the region of the intensity of the fluorescence in the region of core divided by cell entirety.Analysis unit 112 is in division knot In the case that fruit is 2 or more, it is determined as NF- κ B and locally lies in core, in the case where result of division is smaller than 2, be determined as NF- κ B are locally lain in cytoplasm.The value of judgement result of division is not limited to 2, can also be other values.

In addition, in the case of having selected 2 images in step S14, in step S15, for the image of two sides, as above The 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 according to the differentiation of all cells handled as a result, calculating above-mentioned core office Portion there are 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 calculated numerical value in step s 16, the differentiation result of image and each cell obtained for each cell etc. It is shown in display unit 150.Specifically, the picture 161 including the above is shown in display unit 150 by processing unit 110.

As shown in figure 9, picture 161 has region 161a, 161b.Region 161a shows that core locally lies in number, cytoplasm office Portion locally lies in rate there are number, core and cytoplasm locally lies in rate.Region 161b shows locally lying in for image and NF- κ B Differentiate result.In the 161b of region, the image of the differentiation in step S15 for locally lying in is enclosed in solid line, to know It locally lies in differentiation and has used the image.

The fluorescence information obtained in step s 13 can also be the waveform signal for the fluorescence intensity for indicating to change over time. In this case, in optical detection portion 130, as acceptance part 501~503, the photodetectors such as photomultiplier are respectively configured. 3 photodetectors receive the glimmering of the fluorescence of the high intensity of wavelength period B1, the low intensive fluorescence of wavelength period B2 and wavelength period B3 Light, respectively output indicate 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, be NF- κ B discovery amount it is few In the case of cell, such as obtain chart 51,52, be NF- κ B discovery amount more than cell in the case of, such as 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 simultaneously with chart 51,52, 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, the peak value of fluorescence is between threshold value Sh1, Sh2, and there are waveforms 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 Peak value is smaller than threshold value Sh1, so analysis unit 112 can not differentiate locally lying in for NF- κ B.According to chart 61, the peak value ratio of fluorescence Threshold value Sh2 is big, so analysis unit 112 can not 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 are the recess of waveform in width W2 corresponding with core, so analysis unit 112 can differentiate It is locally lain in cytoplasm for NF- κ B.It therefore, in this case, also can in the same manner as the case where using image as shown in Figure 3 It is enough precisely to differentiate locally lying in for NF- κ B by 2 different fluorescence of intensity.

In embodiment 2,2 light are not used, but obtain mutually different intensity using only 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 Step S1, a part of step in S2 is different.Hereinafter, the step that explanation is different from embodiment 1.

In step sl, as shown in figure 11, include 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 14 illuminated wavelength X 1 of fluorescent material, excitation and embodiment party The fluorescence of the 11 same wavelength period of fluorescent material of formula 1.If the light of 15 illuminated wavelength X 1 of fluorescent material, excitation and Fig. 2 12 same wavelength period of fluorescent material fluorescence.That is, the wavelength of the light of the excitation of fluorescent material 14,15 is substantially the same.

In step s 2, including the sample of 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 fluorescence is generated from fluorescent material 14,15,13.It is produced from fluorescent material 14,15 Raw fluorescence becomes the fluorescence of wavelength period B1, B2 by filter member 21,22 respectively.At this point, 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, optical detection portion 130 shown in fig. 6 is omitted In, light source 302, collector lens 312 and dichroscope 321.

In embodiment 2, also in the same manner as embodiment 1, it is capable of the fluorescence and wave of the high intensity of generation wavelength section B1 The low intensive fluorescence of long section B2 obtains the fluorescent image of high intensity and low intensive fluorescent image.Therefore, with embodiment 1 Similarly, fluorescent image that can be according to high intensity and low intensive fluorescent image precisely parse the distribution in cell And the diversified NF- κ B of amount.

In embodiment 3, not use 2 light and 2 fluorescent materials, but use 1 wavelength X 1 light and 1 it is glimmering Stimulative substance 11 obtains the fluorescence of mutually different intensity.In embodiment 3, compared to embodiment 1, cell shown in FIG. 1 A part of step in the step of information acquisition method, only in step S1, S2 is different.Hereinafter, explanation and embodiment 1 are not Same step.

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

As shown in figure 12, the fluorescence generated from fluorescent material 11 is divided into 2, so that a side is passed through same with embodiment 1 The filter member 21 of sample makes another party pass through filter member same as embodiment 1 22.Filter member 21 only makes wave The light transmission of long section B1, filter member 21 only make the light transmission of wavelength period B4.As shown in figure 13, wavelength period B1 include for example from The intensity for the fluorescence that fluorescent material 11 generates 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 wavelength period not Chong Die with wavelength period B1.As a result, 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 the wavelength period B4 of filter member 22 and has become low-intensity.

In addition, it is the wavelength of peak value that wavelength period B1, which can also may not include the intensity of the fluorescence generated from fluorescent material 11,. Wavelength period B4 can also be set as the wavelength period smaller than wavelength period B1, can also a part Chong Die with wavelength period B1.

Next, illustrating the verification for the embodiment 3 that inventor carries out.

1. preparing

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) are prepared 647 conjugate of Secondary Antibody, Alexa Fluor (Life technologies A-21245).To two Secondary antibody combines Alexa Fluor 647 as fluorchrome.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 is modulated

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

3. step

HMVEC-C is to recommend agreement in accordance with manufacturer, utilizes EGM-2MV medium cultures.It is used in this verification Subculture number is the cell within 6 times after buy.About culture medium, during the service life behind Kaifeng is set as 3 weeks.It closes It stimulates and cultivates in TNF-α, the culture supernatant of the HMVEC-C cells at about 70% interflow of removal, in addition to become ultimate density 25ng/ The mode of mL is added to 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, scraper is used in combination to remove cell.In addition with returning The 8%PFA/PBS of the suspension equivalent of receipts has reacted 15 minutes at room temperature.At room temperature, it has been carried out 3 minutes with 1000rpm Centrifugation.Cell granulations have been rinsed 2 times with the PBS of 1mL.Supernatant is removed, in addition the 0.1%Triton X-100/ of 1mL PBS has reacted 15 minutes at room temperature.At room temperature, centrifugation in 3 minutes has been carried out with 1000rpm.With the 1% of 1mL BSA/PBS has been rinsed 2 times.Supernatant is removed, in addition the 3%BSA/PBS of 1mL, has stood 30 minutes at room temperature.In 3%BSA/ An antibody of 1/1,600 400 μ L is added in PBS.It has reacted at room temperature 1 hour.At room temperature, it is carried out with 1000rpm Centrifugation in 3 minutes.It is rinsed with the 1%BSA/PBS of 1mL.The 400 of 1/1000 are added in 3%BSA/PBS The secondary antibodies of μ L.It has reacted at room temperature 30 minutes.It has been rinsed 2 times with the 1%BSA/PBS of 1mL.Supernatant is removed, is added to The 1%BSA/PBS of 50 μ L.

4. being detected using flow cytometer

As the flow cytometer that can obtain fluorescent image, ImageStreamX Mark II Imaging have been used Flow Cytometer(Merck Millipore).The sample modulated according to above-mentioned 3 is set to be flowed into the stream of the flow cytometer 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 correspond to.The injection power of the laser of wavelength 647nm is 10mW.Pass through the fluorchrome to identifying NF- κ B The laser of illumination wavelength 647nm, produces fluorescence.

In above-mentioned flow cytometer, via the filter member of transmission peak wavelength section 642nm~740nm, to passing through wavelength The fluorescence that the laser of 647nm generates is imaged, and the fluorescent image of high intensity is obtained.In addition, via transmission peak wavelength section 740nm The filter member of~800nm, the fluorescence generated to the laser by wavelength 647nm are imaged, are obtained low intensive glimmering Light image.In addition, 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 is suitably incident on acceptance part.

4 (a), (b) referring to Fig.1 illustrate the image obtained by above-mentioned detection.

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

Shown in Figure 14 (a) in the case of 3 cells, the intensity of the image based on 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 of the image of the fluorescence based on high intensity is appropriate, so can sentence Not Wei NF- κ B locally lie in cytoplasm.Shown in Figure 14 (b) in the case of 3 cells, the fluorescence based on high intensity The intensity of image is excessively high, so being difficult to differentiate locally lying in for NF- κ B.On the other hand, image based on low intensive fluorescence Intensity is appropriate, is locally lain in cytoplasm so NF- κ B can be determined as.

As described above, according to this verification it is found that even if as Embodiment 3 according to 2 different fluorescence of intensity In the case that image discriminating is locally lain in, locally lying in for NF- κ B can be also differentiated.In this way, according to embodiment 3, make from a kind The fluorescence that fluorchrome generates obtains 2 fluorescence by the filter member 21,22 for making the fluorescence of different wavelength periods pass through Image, thus, it is possible to locally lying in for NF- κ B is correctly obtained in primary measurement for 1 cell.

As shown in figure 15, in the apparatus structure of embodiment 3, compared to embodiment 1, optics shown in fig. 6 is omitted In test section 130, light source 302, collector lens 312 and dichroscope 321 are distinguished instead of filter member 412,422 Additional filter member 414,425.Filter member 414 makes to have transmitted in the light of filter member 411, wavelength period B4 light Reflection, makes the light transmission other than wavelength period B4.Filter member 425 only makes in the light reflected by filter member 414, wave The light transmission of long section B4 interdicts the light other than wavelength period B4.In this way, filter member 414,425 be configured to only to detach from In the light that flow cell 200 generates, wavelength period B4 fluorescence.Acceptance part 502 takes the photograph the low intensive fluorescence of wavelength period B4 Picture.

In embodiment 3, also in the same manner as embodiment 1 so that generate the fluorescence of high intensity and low intensive respectively Fluorescence can obtain the fluorescent image of high intensity and low intensive fluorescent image.Therefore, in the same manner as embodiment 1, Neng Gougen Fluorescent image according to high intensity and low intensive fluorescent image precisely parse the distribution in cell and measure 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 step that explanation is different from embodiment 1.

In step sl, as shown in figure 16, only include in 11 marked cells of fluorescent material same as embodiment 1 NF-κB.In step s 2, including the sample of 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 fluorescence is generated from fluorescent material 11,13.At this point, being shone with high power for cell The light of wavelength X 1 is penetrated, so the fluorescence of the wavelength period B1 of transmission filter component 21 becomes high intensity in the same manner as embodiment 1. In turn, so that the cell is moved in flow cell, to the light of mobile cell irradiation wavelength X 1, fluorescence is generated from fluorescent material 11. At this point, for cell with the light of low-power illumination wavelength lambda 1.Therefore, the fluorescence of the wavelength period B1 of transmission filter component 21 becomes Low-intensity.

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

In addition, Figure 17 is the figure for observing optical detection portion 130 on the direction parallel with X/Y plane.In fig. 17, it is For the sake of convenient, about illumination part 300, it is illustrated that in the state that Y-axis pros look up, about light collecting part 400 and acceptance part 505, it is illustrated that in the state that X-axis losing side looks up.

In the flow path 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 It is constituted 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 It is condensed to the position 212 of the Z axis positive side in flow path 210 positioned at position 211.Collector lens 471 makes to generate from position 212 glimmering Light optically focused.Filter member 472 only makes in the light by 471 optically focused of collector lens, wavelength period B1 light transmission.Acceptance part 505 Receive by the low intensive light of the wavelength period B1 of 473 optically focused of collector lens, using the image information based on the light received as taking the photograph As signal exports.

Time T of the cell from the contraposition of position 211 to position 212 is obtained in advance.As a result, if connect by acceptance part 501 After the light based on some cell, by time T, then received by acceptance part 505 based on homocellular light.Therefore, energy Enough images obtained by the image obtained according to acceptance part 501 and according to acceptance part 505 and the image pair obtained from same cell It should get up.

In embodiment 4, also in the same manner as embodiment 1 so that generate the fluorescence of high intensity and low intensive respectively Fluorescence can obtain the fluorescent image of high intensity and low intensive fluorescent image.Therefore, in the same manner as embodiment 1, Neng Gougen Fluorescent image according to high intensity and low intensive fluorescent image precisely parse the distribution in cell and measure 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 step that explanation is different from embodiment 1.

In step s 2, as shown in figure 18, including the sample of the cell identified with fluorescent material 11~13 is flowed into circulation Pond, the illumination of 1~λ of wavelength X 3 are mapped to the cell flowed in flow cell, and fluorescence is generated from fluorescent material 11~13.From fluorescence The fluorescence that matter 11,12 generates is incident on filter member 24 altogether.Filter member 24 is made of prism.From fluorescent material 11,12 fluorescence generated are divided into the fluorescence and wavelength period B2 of wavelength period B1 according to the difference of wavelength period by filter member 24 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.As a result, in the same manner as embodiment 1, the fluorescence of the wavelength period B1 of transmission filter component 21 becomes high-strength The fluorescence of degree, the wavelength period B2 of transmission filter component 22 becomes low-intensity.

In addition it is also possible to be that the fluorescence generated from fluorescent material 11~13 is incident on filter member 24 altogether, 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 uses, but filter member 24 can also be used in the structure of embodiment 2~4.

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

The fluorescence that the sample flowed from flow cell 200 generates is incident on filter member 481.According to the wave of fluorescence It is long, project the fluorescence for being incident on filter member 481 at different angles from filter member 481.Collector lens 431 and light Portion 501 be configured at from the corresponding direction of the fluorescence of wavelength period B1 in the fluorescence that filter member 481 projects.Light as a result, Portion 501 can image the fluorescence of the high intensity of wavelength period B1.Collector lens 432 and acceptance part 502 be configured at from filter The corresponding direction of fluorescence of wavelength period B2 in the fluorescence that optical sheet unit 481 projects.Acceptance part 502 can be to wavelength period as a result, The low intensive fluorescence of B2 is imaged.

In embodiment 5, also in the same manner as embodiment 1 so that generate the fluorescence of high intensity and low intensive respectively Fluorescence can obtain the fluorescent image of high intensity and low intensive fluorescent image.Therefore, in the same manner as embodiment 1, Neng Gougen Fluorescent image according to high intensity and low intensive fluorescent image precisely parse the distribution in cell and measure varied NF- κ B.

In embodiment 6, matrix is set to touch the tested substance that includes in cell to generate fluorescent material, it is right The fluorescent material irradiation light of generation differentiates the office of tested substance according to the fluorescence that the irradiation by light is generated from fluorescent material Portion's existence.In embodiment 6, tested substance is cytoplasm.Matrix includes being cut when touching tested substance Disconnected place of incision generates fluorescent material if place of incision is cut off.More specifically, matrix is used as quilt by touching The cytoplasm for detecting substance, the digestion being present in cytoplasm are disconnected.In embodiment 6, according to from marked cells matter The fluorescence of fluorescent material, judgement is cytoplasmic to be locally lain in.In addition, tested substance can also be the albumen in such as cytoplasm The substance other than the cytoplasm for including 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 step that explanation is different from embodiment 3.

In step sl, as shown in figure 20, cell and matrix 16a mixing.Matrix 16a when by including in cytoplasm The substance of fluorescent material 16b is generated when esterase hydrolyzable.If cell and matrix 16a mixing, have transmitted the base of cell membrane Matter 16a, by the esterase hydrolyzable for including in cytoplasm, generates fluorescent material 16b by being contacted with cytoplasm.In this way, sharp With fluorescent material 16b marked cells matter.

Next, in the same manner as embodiment 3, the later processing of step S2 is carried out.That is, in step s 2, including with glimmering The sample of the cell of stimulative substance 16b marks is flowed into flow cell, and as shown in figure 20, the illumination of wavelength X 1, λ 3 is mapped in flow cell The cell of middle flowing generates fluorescence from fluorescent material 16b, 13 respectively.Then, as shown in figure 20, generated from fluorescent material 16b Fluorescence is divided into 2, and a side is made to pass through filter member 21, and another party is made to pass through filter member 22.As a result, with embodiment 3 similarly, has transmitted the fluorescence of the 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.In addition, being constituted in the same manner as device and embodiment 3 based on embodiment 6.

In embodiment 6, also in the same manner as embodiment 3 so that generate 2 different fluorescence of intensity, can obtain The fluorescent image of high intensity and low intensive fluorescent image.Therefore, it is possible to according to the fluorescent image of high intensity and low intensive glimmering Light image precisely differentiates cytoplasmic locally lie in.In this way, if it is possible to precisely differentiate cytoplasmic part In the presence of then capable of precisely defining cytoplasmic range.It as a result, for example, can be by cytoplasmic range and other parsings It combines and is parsed in more detail.In addition, for example, cytoplasmic range can be made to play a role in the research of cell etc..

Next, illustrating the verification for the embodiment 6 that inventor carries out.

1. preparing

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 include and figure The corresponding substances of matrix 16a shown in 20.Cell qualitative character reagent has hydrophobicity.Cell qualitative character reagent by cell membrane, Due to generating fluorescent material by intracellular esterase hydrolyzable.Fluorescence shown in the fluorescent material and Figure 20 generated herein Matter 16b is corresponded to.As nuclear staining pigment, 33342 solution (DOjinDO of Cellstain Hoechst 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 is modulated

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

3. step

HMVEC-C is to recommend agreement in accordance with manufacturer, utilizes EGM-2MV medium cultures.In this verification, use Subculture number is the cell within 6 times after buy.About culture medium, during the service life behind Kaifeng is set as 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 removed.The Track kit working solution that 3 μ L are added to the suspension of the 3mL of recycling, in 37 DEG C of CO₂ 30 minutes have been stood in insulating box.After having stood 30 minutes, at room temperature, centrifugation point in 3 minutes is carried out with 1000rpm From.The cell granulations PBS of 5mL is rinsed 3 times.Supernatant is removed, the 1%BSA/PBS of 50 μ L is added to.

4. being detected using flow cytometer

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

In above-mentioned flow cytometer, via the filter member of transmission peak wavelength section 480nm~560nm, to passing through wavelength The fluorescence that the laser of 488nm generates is imaged, and the fluorescent image of high intensity is obtained.In addition, via transmission peak wavelength section 560nm The filter member of~595nm, the fluorescence generated to the laser by wavelength 488nm are imaged, are obtained low intensive glimmering Light image.Via the filter member of transmission peak wavelength section 420nm~505nm, the fluorescence that the laser by wavelength 405nm is generated It is imaged, obtains fluorescent image corresponding with core.It is set as in addition, having irradiated wavelength to the sample flowed in flow cell Laser between 420nm~480nm.Via the filter member of transmission peak wavelength section 420nm~480nm, which is transmitted Light after cell is imaged, and bright field image is obtained.In addition, in above-mentioned flow cytometer, pass through filter member etc. The light of unwanted wavelength period is eliminated, to become the light of wavelength period of object is suitably incident on acceptance part.

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

" bright field " indicates 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 generates and based on from identifying cytoplasmic iridescent The image for the low intensive fluorescence that element generates." fluorescence from core " is based on from the nuclear staining pigment dyed 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 grayscale to acquired coloured image.Image other than bright field image In, white part indicates that the intensity of fluorescence is strong.

Shown in lower 1 section of cell and uppermost shown in uppermost in the case of cell, 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, the image of the fluorescence based on high intensity Intensity it is appropriate, so can precisely differentiate cytoplasmic locally lie in.The cell shown in lowermost and most lower In the case of cell shown in upper 1 section of section, the intensity of the image of the fluorescence based on high intensity is excessively high, so being difficult to differentiate cell Matter is locally lain in.On the other hand, the intensity of the image based on low intensive fluorescence is appropriate, so can precisely differentiate It is cytoplasmic to locally lie in.

As described above, according to this verification it is found that if as Embodiment 6 based on 2 different fluorograms of intensity Picture can then differentiate cytoplasmic locally lie in.In this way, according to embodiment 6 it is found that making a kind of fluorescence from marked cells matter The fluorescence that pigment generates obtains 2 fluorescent images by the filter member 21,22 for making the fluorescence of different wavelength periods pass through, Thus, it is possible to cytoplasmic locally lie in correctly is obtained in primary measurement for 1 cell.

In embodiment 7,2 kinds of matrix are made to touch the tested substance that includes in cell to generate 2 kinds of fluorescence Substance differentiates quilt to 2 kinds of fluorescent material irradiation lights of generation according to the fluorescence that the irradiation by light is generated from 2 kinds of fluorescent materials Detection substance locally lies in situation.That is, in embodiment 7,1 light is not used as Embodiment 6, but is used The light of mutually different wavelength obtains the fluorescence of mutually different intensity.In addition it is also possible to which it is detected so that a kind of matrix is touched Substance come make generate 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 step that explanation is different from embodiment 6.

In step sl, as shown in figure 22, cell and the mixing of matrix 17a, 18a.Matrix 17a, 18a is to work as to pass through cytoplasm In include esterase hydrolyzable when generate the substance of fluorescent material 17b, 18b respectively.Fluorescent material 17b, 18b are respectively structured as When illuminated wavelength X 1, λ 2 light when encourage the fluorescence of mutually different wavelength period.If cell and the mixing of matrix 17a, 18a, Matrix 17a, the 18a for then having transmitted cell membrane are contacted with cytoplasm, to pass through the esterase hydrolyzable for including in cytoplasm, production Raw fluorescent material 17b, 18b.In this way, utilizing fluorescent material 17b, 18b marked cells matter.

In step s 2, including flow cell is flowed into the sample of the cell of fluorescent material 17b, 18b mark, such as Figure 22 institutes Show, the illumination of wavelength X 1, λ 2, λ 3 is mapped to the cell flowed in flow cell, is generated respectively from fluorescent material 17b, 18b, 13 glimmering Light.At this point, with high power to the laser of cell irradiation wavelength X 1, with low-power to the laser of cell irradiation wavelength X 2.By making The fluorescence generated 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 generated becomes the fluorescence of wavelength period B2 by filter member 22.In this way, having passed through the wave of filter member 21 The fluorescence of long section B1 becomes high intensity, has passed through the fluorescence of the wavelength period B2 of filter member 22 and has become low-intensity.In addition, being based on It is constituted in the same manner as the device of embodiment 7 and embodiment 1.

In embodiment 7, also in the same manner as embodiment 6,2 different fluorescence of intensity can be generated, are obtained high-strength The fluorescent image of degree and low intensive fluorescent image.It therefore, can be according to the fluorogram of high intensity in the same manner as embodiment 6 Picture and low intensive fluorescent image, precisely differentiate cytoplasmic locally lie in.

Claims

1. a kind of cellular informatics acquisition methods, which is characterized in that

The multiple fluorescent materials tested substance identical with include in cell for keeping wavelength of fluorescence mutually different is combined,

To the cell irradiation light so that from the different fluorescence of the multiple fluorescent material generation wavelength and intensity,

According to each fluorescence of generation, multiple fluorescence informations are obtained.

2. cellular informatics acquisition methods according to claim 1, which is characterized in that

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

3. cellular informatics acquisition methods according to claim 1, which is characterized in that

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

4. cellular informatics acquisition methods according to claim 1, which is characterized in that

According to the multiple fluorescence information, differentiates that the tested substance is locally lain in and still locally lain in cytoplasm in core In.

5. cellular informatics acquisition methods according to claim 1, which is characterized in that

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

6. cellular informatics acquisition methods according to claim 1, which is characterized in that

2nd light weaker than the 1st light to the 1st light of the cell irradiation and intensity.

7. cellular informatics acquisition methods according to claim 1, which is characterized in that

The wavelength and intensity for the fluorescence that the multiple fluorescent material is generated by the light of illuminated Same Wavelength are mutually different.

8. cellular informatics acquisition methods according to claim 1, which is characterized in that

By each fluorescence generated from the multiple fluorescent material be separated into described in the 1st fluorescence and intensity ratio that the 1st fluorescence is weak it is the 2nd glimmering Light.

9. cellular informatics acquisition methods according to claim 1, which is characterized in that

The 1st fluorescent image of the 1st wavelength of fluorescence generated from the cell of illuminated 1st light is obtained,

Obtain the 2nd wavelength of fluorescence generated from the cell of illuminated intensity 2nd light weaker than the 1st light the 2nd is glimmering Light image.

10. cellular informatics acquisition methods according to claim 1, which is characterized 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 made to generate the fluorescence.

11. cellular informatics acquisition methods according to claim 3, which is characterized in that

In the differentiation of situation being locally lain in described in the tested substance, including at 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, which is characterized in that

According to from it is in the fluorescence information that the different multiple fluorescence of intensity obtain, be contained in from the intensity it is scheduled The fluorescence information that the fluorescence of range obtains carries out the differentiation for locally lying in situation of the tested substance.

13. cellular informatics acquisition methods according to claim 3, which is characterized in that

According to from the analysis object position of cell in the fluorescence information that the different multiple fluorescence of intensity obtain, described The difference of fluorescence intensity in the fluorescence intensity at place and cellular portions other than the analysis object position is than scheduled 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, which is characterized in that

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

15. cellular informatics acquisition methods according to claim 1, which is characterized in that

According to the multiple fluorescence information, calculates in the cell for including in sample, described tested substance and locally lie in The ratio or quantity of cell in specific position.

16. a kind of cellular informatics acquisition methods, which is characterized in that

Making matrix, identical tested substance contacts and to generate mutually different multiple of wavelength of fluorescence with include in cell Fluorescent material,

17. a kind of cellular informatics acquisition methods, which is characterized in that

Fluorescent material tested substance identical with include in cell is set to be combined,

To the cell irradiation light so that generating fluorescence from the fluorescent material,

The multiple fluorescence different from the fluorescence of generation acquisition wavelength and intensity,

According to each fluorescence of acquisition, multiple fluorescence informations are obtained,

18. cellular informatics acquisition methods according to claim 17, which is characterized in that

19. cellular informatics acquisition methods according to claim 17, which is characterized in that

By the fluorescence generated from the fluorescent material be separated into described in the 1st fluorescence and intensity ratio that the 1st fluorescence is weak it is the 2nd glimmering Light.

20. a kind of cellular informatics acquisition methods, which is characterized in that

So that matrix tested substance identical with include in cell is contacted and to generate fluorescent material,

21. a kind of cellular informatics acquisition device, which is characterized in that have：

Illumination part, to including the thin of the identical tested substance for combining the mutually different multiple fluorescent materials of wavelength of fluorescence Born of the same parents' irradiation light so that the fluorescence different from the multiple fluorescent material generation wavelength and intensity；

Acceptance part receives each fluorescence generated from the multiple fluorescent material；And

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

22. cellular informatics acquisition device according to claim 21, which is characterized in that

The illumination part has：

1st light source irradiates the 1st light；And

2nd light source, the 2nd light that illumination wavelength is different from the 1st light and intensity is weaker than the 1st light.

23. cellular informatics acquisition device according to claim 21, which is characterized in that

Has the filter member of each fluorescence separation for making to generate from the multiple fluorescent material.

24. cellular informatics acquisition device according to claim 21, which is characterized in that have：

1st acceptance part receives the 1st fluorescence；And

2nd acceptance part receives intensity 2nd fluorescence weaker than the 1st fluorescence.

25. cellular informatics acquisition device according to claim 21, which is characterized in that

The acquisition unit obtain the 1st wavelength of fluorescence generated from the cell of illuminated 1st light the 1st fluorescent image and from 2nd fluorescent image of the 2nd wavelength of fluorescence that the cell of illuminated intensity 2nd light weaker than the 1st light generates.

26. a kind of cellular informatics acquisition device, which is characterized in that have：

Illumination part, to including the cell irradiation light for the identical tested substance for combining fluorescent material so that from described glimmering Stimulative substance generates fluorescence；

Acceptance part receives the wavelength that is generated from the fluorescent material and the different multiple fluorescence of intensity；

Acquisition unit obtains multiple fluorescence informations according to the multiple fluorescence received；And

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

27. cellular informatics acquisition device according to claim 26, which is characterized in that

Have weak for the fluorescence generated from the fluorescent material to be separated into the 1st fluorescence described in the 1st fluorescence and intensity ratio The 2nd fluorescence filter member.

28. a kind of cellular informatics acquisition device, which is characterized in that have：

Illumination part, to including the cell photograph for making to generate the identical tested substance of fluorescent material by contacting with matrix Penetrate light so that generate fluorescence from the fluorescent material；