荧光探针技术测定细胞内离子浓度课件.ppt

1、cytoplasmNa+Cl-Ca2+K+FeFe2+2+细胞结构的组成成分细胞结构的组成成分 血红蛋白中血红蛋白中Fe2+、超氧化物歧化酶中、超氧化物歧化酶中Cu2+维持细胞膜电位维持细胞膜电位 K+、Cl-、Na+、Ca2+维持胞内外渗透压维持胞内外渗透压 Na+协同转运葡萄糖等营养物质协同转运葡萄糖等营养物质排泄排泄代谢产物代谢产物高血压高血压骨质疏松骨质疏松癫痫癫痫心律失常心律失常猝死猝死惊厥惊厥阵发性舞蹈手足徐动症阵发性舞蹈手足徐动症不同脲素浓度下,通过监测1H,15N信号强度变化监控 MMP-12 折叠过程Luis A.Rivas,Luis A.Rivas,et.al.et.al.

2、A 200-Antibody Microarray Biochip for Environmental Monitoring:Searching for Universal Microbial A 200-Antibody Microarray Biochip for Environmental Monitoring:Searching for Universal Microbial Biomarkers through ImmunoprofilingBiomarkers through ImmunoprofilingGuoliang KeGuoliang Ke,et.al.A Cell-Su

3、rface-Anchored Ratiometric Fluorescent Probe for Extracellular pH Sensinget.al.A Cell-Surface-Anchored Ratiometric Fluorescent Probe for Extracellular pH SensingAbhik Mallick,et.al.Dual Drug Conjugated Nanoparticle for Simultaneous Targeting of Mitochondria and Nucleus in Cancer Cells Abhik Mallick,

4、et.al.Dual Drug Conjugated Nanoparticle for Simultaneous Targeting of Mitochondria and Nucleus in Cancer Cells Michihiro Nakamura,Michihiro Nakamura,et.al.Thiol-Organosilica Particles Internally Functionalized with Propidium et.al.Thiol-Organosilica Particles Internally Functionalized with Propidium

5、 Iodide as a Multicolor Fluorescence and Xray Computed Tomography Probe and Application for Non-Iodide as a Multicolor Fluorescence and Xray Computed Tomography Probe and Application for Non-Invasive Functional Gastrointestinal Tract ImagingInvasive Functional Gastrointestinal Tract ImagingJihoonJih

6、oon Kim,et.alKim,et.al.Rapid Cytotoxicity Screening Platform for Amyloid Inhibitors Using a Membrane-Potential Sensitive Fluorescent Probe.Rapid Cytotoxicity Screening Platform for Amyloid Inhibitors Using a Membrane-Potential Sensitive Fluorescent ProbeBright-FieldBright-FieldF Fluo-4luo-4 fluoresc

7、encefluorescence MergeMergeChenye Yang,et.al.Chenye Yang,et.al.Continuous Fluorescence Imaging of Intracellular Calcium by Use of Continuous Fluorescence Imaging of Intracellular Calcium by Use of Ion-Selective Nanospheres with Adjustable Spectra Ion-Selective Nanospheres with Adjustable Spectra Chr

8、istophe M.Lamy,et.al.Sodium Sensing in Neurons with a Dendrimer-Based Nanoprobe Christophe M.Lamy,et.al.Sodium Sensing in Neurons with a Dendrimer-Based Nanoprobe ControlControlT TTXTXBaoli DongBaoli Dong,et.al.Dual Site-Controlled and Lysosome-Targeted Intramolecular Charge Transferet.al.Dual Site-

9、Controlled and Lysosome-Targeted Intramolecular Charge TransferPhotoinduced Electron Photoinduced Electron TransferTransferFluorescence Resonance Energy Transfer Fluorescent Probe for Monitoring pH Changes in Living Cells Fluorescence Resonance Energy Transfer Fluorescent Probe for Monitoring pH Cha

10、nges in Living Cells 光光学学检检测测-荧光显微镜/激光共聚焦优点：样本兼容性强，定性、定位分析缺点：分析速度慢，参数少，难以定量Edgar J.Paredes-GameroEdgar J.Paredes-Gamero,et.al.et.al.CellCell Permeable Gomesin Peptide Promotes Cell Death by Intracellular CaPermeable Gomesin Peptide Promotes Cell Death by Intracellular Ca2+2+Overload Overload 流流式式细细胞

11、胞术术的的概概念念流式细胞仪通过接收激光照射后液流内单个细胞产生的光信号，从而进行细胞功能分析或者分选的技术。流流式式细细胞胞术术的的概概念念流式细胞仪通过接收激光照射后液流内单个细胞产生的光信号，从而进行细胞功能分析或者分选的技术。荧光染料EX EM 应用荧光染料EX EM 应用 熟悉仪器的激光和检测器配置 清楚实验所用染料的激发波长(EX)及发射波长(EM)荧荧光光素素选选择择荧光染料EX EM 应用荧光染料EX EM 应用 熟悉仪器的激光和检测器配置 清楚实验所用染料的激发波长(EX)及发射波长(EM)荧荧光光素素选选择择Wan-Kyu Oh,et.alWan-Kyu Oh,et.al.

12、Fluorescent Polymer Nanoparticle for Selective Sensing of Intracellular Hydrogen Fluorescent Polymer Nanoparticle for Selective Sensing of Intracellular Hydrogen Peroxide Peroxide O H ET AL.V O L.6N O.108516 85242012w w w.acsnano.org8520RAW 264.7 cel l si ncubated w i th 5and 10 g m L1BPANnanoparti

13、cl es(Fi gure 5a and b;l ef tcol um n).Cel l sw i thBPAN nanoparti cl eshad no consi derabl e change i n cel lshape.Bl ue f l uorescence f rom the BPAN nanoparti cl esw as observed both i nsi de and outsi de the cel l s.These m acrophages produce superoxi de upon sti m u-l ati on w i th phorbol-12-m

14、 yri state-13-acetate(PM A),and super oxi de i s know n to be degraded t o H2O2bysuperoxi de di sm ut ase orby spontaneous di sm utat i on.29Consequent l y,BPAN-t r eat ed cel l s w er e st i m ul at ed w i th1 gm L1PM A f or20m i n(Fi gur e5aand b;ri ghtcol um n).The cel l s t r eat ed wi th PM A e

15、xhi bi t ed hi gh f l uor escence,whi l e m acr ophages notst i m ul at ed wi t h PM A di spl ayedonl y weak i nt r acel l ul ar f l uorescence.The m ean f l uor es-cence i nt ensi t y val ues di f f ered by a f act or of ca.4 5(Fi gur e 5aand b;ri ghtgr aph).The bl ue-f l uor escentBPANnanopar t i

16、cl es wer e capabl e of vi sual i zi ng endogenousH2O2gener at i on i n RAW 264.7 cel l sdue to ef f i ci entel ec-t r on t r ansf er af t er react i on w i t h H2O2;the f l uor escencef r om t heSchi f fbase on t heHPAN nanopar t i cl esappear edwi t h 360 nmexci t at i on,whi l e t he f l uor esce

17、nce f r om300 nm exci tati on decr eased.I n gener al,conf ocalm i cro-scopy hasa bl ue f i l t er(ex:360 nm;em:457 nm),w hi ch i sadopt abl ef ort hi sexper i m ent.Judgi ng f rom theseexper i-m ents,t he BPAN nanopar t i cl esar e t aken up by the cel l sand ar e nontoxi c,asw el las capabl e ofde

18、tect i ng el eva-t i on i n H2O2l evel s undercondi ti ons ofoxi dat i ve st r ess.The BPAN nanopart i cl es ar e sensi ti ve enough to i m ageH2O2produced i n RAW 264.7 m acr ophages.Thi s r esul ti s consi st entwi t h t he f i ndi ngs ofAbo etal.t hatm ono-bor onat er eport ersar ecapabl eofdet e

19、ct i ng H2O2i n PM A-t r eat ed m acr ophages.26Fi gure 6.Ti trati on pl otofPM A concentrati on-dependentand BPAN dose-dependentf l uorescence al terati on i n RAW 264.7cel l s.(a)Response of7.5 g m L1BPAN nanoparticl e-treated cel l si n thepresence ofPM A(1,2,3,and 4 g m L1).(b)ResponseofBPAN nan

20、oparticl e-i ncubated cel l s(2.5,5,7.5,10,15 g m L1)afteraddi ng 1 g m L1PM A.Fi gure 7.Fl owcytom etry anal yses of parti cl e uptake.Quanti f i cati on of cel l ul ar uptake of(a)negati ve control,(b)10 g m L1,and(c)100 g m L1FI TC-m odi f i ed PAN nano-parti cl es.Upperri ghtval uesare the num b

21、erofnanoparti cl e-contai ned RAW 264.7 cel l s,and upper l eft m edi an val uesi ndi cate the am ountofnanoparti cl es taken up.Fi gure 8.TEM i m agesofRAW 264.7 cel l si ncubated w i th BPANnanoparti cl esf or24h(10 gm L1).(a c)W hi tearrow si ndi catethe nanoparti cl es that are l ocated i n endo

22、som es(scal e bar:200 nm).(d)The nanoparti cl es are i nternal i zed i nto the cel l s(gray dotted l i ne i ndi catesi nvagi nati on ofpl asm a m em brane).ARTI CLEO H ET AL.V O L.6N O.108516 85242012w w w.acsnano.org8520RAW 264.7 cel l si ncubated w i th 5and 10 g m L1BPANnanoparti cl es(Fi gure 5a

23、 and b;l ef tcol um n).Cel l sw i thBPAN nanoparti cl eshad no consi derabl e change i n cel lshape.Bl ue f l uorescence f rom the BPAN nanoparti cl esw as observed both i nsi de and outsi de the cel l s.These m acrophages produce superoxi de upon sti m u-l ati on w i th phorbol-12-m yri state-13-ac

24、etate(PM A),and super oxi de i s know n to be degraded t o H2O2bysuperoxi de di sm ut ase orby spontaneous di sm ut at i on.29Consequentl y,BPAN-t r eat ed cel l s w er e st i m ul at ed w i th1 gm L1PM A f or20m i n(Fi gur e5aand b;ri ghtcol um n).The cel l s t r eat ed wi th PM A exhi bi t ed hi g

25、h f l uor escence,whi l e m acr ophages notst i m ul at ed wi t h PM A di spl ayedonl y weak i nt r acel l ul ar f l uorescence.The m ean f l uor es-cence i nt ensi t y val ues di f f ered by a f act or of ca.4 5(Fi gur e 5aand b;ri ghtgr aph).The bl ue-f l uor escentBPANnanopar t i cl es wer e capa

26、bl e of vi sual i zi ng endogenousH2O2gener at i on i n RAW 264.7 cel l sdue to ef f i ci entel ec-t ron t r ansf er af t er react i on w i t h H2O2;the f l uor escencef rom t heSchi f fbase on t heHPAN nanopar t i cl esappear edwi t h 360 nmexci t at i on,whi l e t he f l uor escence f r om300 nm e

27、xci tati on decr eased.I n gener al,conf ocalm i cro-scopy hasa bl ue f i l t er(ex:360 nm;em:457 nm),w hi ch i sadopt abl ef ort hi sexper i m ent.Judgi ng f rom t heseexper i-m ent s,t he BPAN nanopar ti cl esar e taken up by the cel l sand ar e nontoxi c,as w el lascapabl e ofdetect i ng el eva-t

28、 i on i n H2O2l evel s undercondi ti ons ofoxi dat i ve st r ess.The BPAN nanopart i cl es ar e sensi ti ve enough to i m ageH2O2produced i n RAW 264.7 m acr ophages.Thi s r esul ti s consi st entwi t h t he f i ndi ngs ofAbo etal.t hatm ono-bor onat er eporter sar ecapabl eofdetect i ng H2O2i n PM

29、A-t reat ed m acr ophages.26Fi gure 6.Ti trati on pl otofPM A concentrati on-dependentand BPAN dose-dependentf l uorescence al terati on i n RAW 264.7cel l s.(a)Response of7.5 g m L1BPAN nanoparti cl e-treated cel l si n thepresence ofPM A(1,2,3,and 4 g m L1).(b)ResponseofBPAN nanoparticl e-i ncubat

30、ed cel l s(2.5,5,7.5,10,15 g m L1)afteraddi ng 1 g m L1PM A.Fi gure 7.Fl owcytom etry anal yses of parti cl e uptake.Quanti f i cati on of cel l ul ar uptake of(a)negati ve control,(b)10 g m L1,and(c)100 g m L1FI TC-m odi f i ed PAN nano-parti cl es.Upperri ghtval uesare the num berofnanoparti cl e-

31、contai ned RAW 264.7 cel l s,and upper l eft m edi an val uesi ndi cate the am ountofnanoparti cl es taken up.Fi gure 8.TEM i m agesofRAW 264.7 cel l si ncubated w i th BPANnanoparti cl esf or24h(10 gm L1).(a c)W hi tearrow si ndi catethe nanoparti cl es that are l ocated i n endosom es(scal e bar:2

32、00 nm).(d)The nanoparti cl es are i nternal i zed i nto the cel l s(gray dotted l i ne i ndi catesi nvagi nati on ofpl asm a m em brane).ARTI CLEO H ET AL.V O L.6N O.108516 85242012w w w.acsnano.org8520RAW 264.7 cel l si ncubated w i th 5and 10 g m L1BPANnanoparti cl es(Fi gure 5a and b;l ef tcol um

33、 n).Cel l sw i thBPAN nanoparti cl eshad no consi derabl e change i n cel lshape.Bl ue f l uorescence f rom the BPAN nanoparti cl esw as observed both i nsi de and outsi de the cel l s.These m acrophages produce superoxi de upon sti m u-l ati on w i th phorbol-12-m yri state-13-acetate(PM A),and sup

34、er oxi de i s know n t o be degr aded t o H2O2bysuperoxi de di sm ut ase orby spontaneous di sm ut at i on.29Consequentl y,BPAN-t r eat ed cel l s w ere st i m ul at ed w i t h1 gm L1PM A f or20m i n(Fi gur e5aand b;ri ghtcol um n).The cel l s t r eat ed wi th PM A exhi bi t ed hi gh f l uor escence

35、,whi l e m acr ophages notst i m ul at ed wi t h PM A di spl ayedonl y w eak i nt r acel l ul ar f l uorescence.The m ean f l uor es-cence i nt ensi t y val ues di f f er ed by a f act or of ca.4 5(Fi gure 5aand b;r i ghtgr aph).The bl ue-f l uor escentBPANnanopar t i cl es wer e capabl e of vi sual

36、 i zi ng endogenousH2O2gener at i on i n RAW 264.7 cel l sdue t o ef f i ci entel ec-t ron t r ansf er af t er r eact i on w i t h H2O2;the f l uor escencef rom t heSchi f fbase on t heHPAN nanopar t i cl esappear edwi t h 360 nmexci t at i on,whi l e t he f l uor escence f r om300 nm exci tati on d

37、ecr eased.I n gener al,conf ocalm i cro-scopy hasa bl ue f i l t er(ex:360 nm;em:457 nm),w hi ch i sadopt abl ef ort hi sexperi m ent.Judgi ng f r om t heseexper i-m ent s,t he BPAN nanopar ti cl esar e taken up by t he cel l sand ar e nontoxi c,as w el las capabl e ofdetect i ng el eva-t i on i n H

38、2O2l evel s undercondi ti ons ofoxi dat i ve st r ess.The BPAN nanopart i cl es ar e sensi ti ve enough to i m ageH2O2produced i n RAW 264.7 m acr ophages.Thi s r esul ti s consi st entwi t h t he f i ndi ngs ofAbo etal.thatm ono-bor onat er epor ter sar ecapabl eofdetect i ng H2O2i n PM A-t r eat e

39、d m acr ophages.26Fi gure 6.Ti trati on pl otofPM A concentrati on-dependentand BPAN dose-dependentf l uorescence al terati on i n RAW 264.7cel l s.(a)Response of7.5 g m L1BPAN nanoparti cl e-treated cel l si n thepresence ofPM A(1,2,3,and 4 g m L1).(b)ResponseofBPAN nanoparticl e-i ncubated cel l s

40、(2.5,5,7.5,10,15 g m L1)afteraddi ng 1 g m L1PM A.Fi gure 7.Fl owcytom etry anal yses of parti cl e uptake.Quanti f i cati on of cel l ul ar uptake of(a)negati ve control,(b)10 g m L1,and(c)100 g m L1FI TC-m odi f i ed PAN nano-parti cl es.Upperri ghtval uesare the num berofnanoparti cl e-contai ned

41、 RAW 264.7 cel l s,and upper l eft m edi an val uesi ndi cate the am ountofnanoparti cl es taken up.Fi gure 8.TEM i m agesofRAW 264.7 cel l si ncubated w i th BPANnanoparti cl esf or24h(10 gm L1).(a c)W hi tearrow si ndi catethe nanoparti cl es that are l ocated i n endosom es(scal e bar:200 nm).(d)The nanoparti cl es are i nternal i zed i nto the cel l s(gray dotted l i ne i ndi catesi nvagi nati on ofpl asm a m em brane).ARTI CLE