流式細(xì)胞儀原理講解學(xué)習(xí)

流式細(xì)胞儀

工作原理EPICSXL/MCLFC500MCL/MPL流式細(xì)胞術(shù)的原理流式細(xì)胞術(shù)（FCM）是一種能對(duì)單個(gè)細(xì)胞、分子和生物顆粒（微生物）進(jìn)行多參數(shù)快速檢測(cè)的新型分析和分選技術(shù)。近幾年隨著多種單克隆抗體的成功制備和多種熒光素的應(yīng)用。FCM逐步運(yùn)用于對(duì)疾病診斷和治療監(jiān)測(cè)的臨床檢驗(yàn)常規(guī)工作。流路單細(xì)胞懸液大多數(shù)儀器是在50-300μm大小的孔徑中,將細(xì)胞懸液注射進(jìn)入鞘液中這一過程,成為流體動(dòng)力學(xué)聚焦FLOWCELLInjector

TipFluorescencesignalsFocusedlaserbeamSheath

fluid光路光源檢測(cè)信號(hào)散射光信號(hào)

FS:前向角散射光

SS:側(cè)向角散射光(90度散射光)熒光信號(hào) 熒光染料 光學(xué)濾片

顏色補(bǔ)償電路光電倍增管

光路光源 與通過的細(xì)胞聚焦在同一點(diǎn)上激光 空冷 488nmblue,633nmred,325nmUV,514nmgreen,空冷/水冷EnterpriseII水冷Innova300系列 Innova70系列檢測(cè)信號(hào)散射光信號(hào)—前向散射光(FS)FSForward

(AngelLight)Scatter

在激光束正前方的檢測(cè)器,為前向散射光檢測(cè)器

FS的強(qiáng)度與細(xì)胞或其他顆粒的大小,形狀及opticalhomogeneity有關(guān)FS用于檢測(cè)細(xì)胞或其他粒子的表面屬性 如:大小前向散射光散射光信號(hào)–側(cè)向散射光SS-Side

(AngelLight)Scatter

與激光束垂直方向的檢測(cè)器為側(cè)向檢測(cè)器,也稱為90度散射光檢測(cè)器

SS的強(qiáng)度與細(xì)胞或其他顆粒的大小,形狀及opticalhomogeneity有關(guān)SS用于檢測(cè)細(xì)胞內(nèi)部結(jié)構(gòu) 如:胞漿內(nèi)顆粒多少,核質(zhì)比側(cè)向散射光90DegreeScatter02004006008001000815203040501002001000LymphocytesMonocytesNeutrophilsSideScatterProjectionLightScatterGatingScale光路-熒光信號(hào)每種熒光染料均有特定的激發(fā)波長(zhǎng),并激發(fā)后會(huì)有發(fā)射波長(zhǎng),流式細(xì)胞儀檢測(cè)的即是它特定的發(fā)射波長(zhǎng).利用各種不同波長(zhǎng)的光學(xué)濾片來收集(檢測(cè))這些光學(xué)信號(hào)熒光染料的特性激發(fā)波長(zhǎng)(EXCITING)發(fā)射波長(zhǎng)(EMISSION)常用熒光染料的特性熒光染料激發(fā)波長(zhǎng)(nm)發(fā)射波長(zhǎng)(nm)用途顏色FITC488525免疫熒光綠色PE(RD1)488575免疫熒光橙色ECD488620免疫熒光橙紅PeCy5488675免疫熒光紅PI488620DNA染色橙紅PECy7 488 755 免疫熒光 深紅PerCP 488 670APC633 670光路-濾片特性當(dāng)以45o

角放置濾片時(shí),該濾片可以通過一定波長(zhǎng)的光,同時(shí)也可以阻斷并折射該波長(zhǎng)以下的光

這種方式的濾片稱為二向色性濾片(dichroicfilter)

SourcelightPassedlightCertaincolorsabsorbedAbsorbancefilter標(biāo)準(zhǔn)帶通濾片bandpass光源通過的光譜630/30nmBP615-645nmlight

Unique4-ColorSingleLaserDesignAir-cooledArgonIonLaserBeamShapingLensesHighSensitivityQuartzFlowCellForwardScatterDetectorSideScatterDetector525BPFL1(FITC)575BPFL2(RD1)620BPFL3(ECD)675BPFL4(PC5)488DL488BK550DL600DL645DLPC7Air-cooledArgonIonLaserBeamShapingLensesHighSensitivityQuartzFlowCellForwardScatterDetectorSideScatterDetector525BPFL1(FITC)575BPFL2(RD1)675BP(PC5)750BPFL4(PC7)488DL488BK550DL600DL700DLOptics-DetectorsPhotomultipliertube(PMT)

將光學(xué)信號(hào)轉(zhuǎn)變?yōu)殡娒}沖(數(shù)字?jǐn)?shù)據(jù))信號(hào)

不同的脈沖信號(hào)被轉(zhuǎn)換成數(shù)字通道(CHANNEL)的過程

SingleParameterHistogram:CountvsMFI顏色補(bǔ)償各熒光染料發(fā)射的熒光信號(hào)有重迭,因此要進(jìn)行色補(bǔ)償以除去重迭部分?emissionintensitywavelengthFLBFLA光譜重疊需進(jìn)行色補(bǔ)償減法顏色補(bǔ)償減法補(bǔ)償去除疊加信號(hào),在使用雙色以上時(shí)會(huì)產(chǎn)生減得過多的錯(cuò)誤BDFACSCalibur補(bǔ)償試劑FITCPECorrectOver-compFITCPEUnder-compFITCPE全矩陣顏色補(bǔ)償全矩陣在每個(gè)PMT上對(duì)于每一個(gè)熒光計(jì)算“疊加熒光素”的比率通過矩陣去除或加上信號(hào)EPICSXLCytomicsFC500CD45-ECDEPICSXL臨床科研型流式細(xì)胞儀

EPICSXL合理的光路設(shè)計(jì)單激光激發(fā)四色熒光標(biāo)準(zhǔn)模式確保在同一個(gè)細(xì)胞上獲得最多的信息

EPICSXL直線光路設(shè)計(jì)，光損失少可互換式光學(xué)濾片，方便新染料的應(yīng)用Air-cooledArgonIonLaserBeamShapingLensesHighSensitivityQuartzFlowCellForwardScatterDetectorSideScatterDetector525BPFL1(FITC)575BPFL2(RD1)620BPFL3(ECD)675BPFL4(PC5)488DL488BK550DL600DL645DLAir-cooledArgonIonLaserBeamShapingLensesHighSensitivityQuartzFlowCellForwardScatterDetectorSideScatterDetector525BPFL1(FITC)575BPFL2(RD1)675BP(PC5)750BPFL4(PC7)488DL488BK550DL600DL700DLPC7WithThankstoDr.FrankSchniedersGentherapieGruppe–AbteilungMolekulareZellbiologieInstitutfürMedizinischeBiochemieundMolekularbiologieUniversit?tsklinikumHamburg-EppendorfEGFP,EYFPand7-AADWithEPICS?XLTMandEXPO32TMADC-SoftwareHEK293HumanEmbryonalKidneyCells293FilterSetBP510LAF21BP550LAF30DichroicMirror525DRLPCellViabilitywith7-AADHEK293-cellswithEYFPTransfectionHEK293-cellswithEGFPTransfectionMixtureofEGFP-andEYFP-transfectedHEK293-CellsEGFP...EnhancedGreenFluorescentProtein(Ex488;Em507nm)EYFP...EnhancedYellowFluorescentProtein(Ex513;Em527nm)7-AAD...7-Aminoactinomycin(ViabilityStain;Ex546;Em647nm)四色熒光中的補(bǔ)償傳統(tǒng)補(bǔ)償與ADC補(bǔ)償EPICSXL采用最先進(jìn)的通訊傳輸設(shè)計(jì)光纖通訊與數(shù)字信號(hào)傳輸EPICSXL中的PRISM功能

將多張直方圖濃縮在一張上HowardM.Shapiro,PracticalFlowCytometry,3rded.,page355“TheXLispresentlyuniqueamongproductionflowcytometerinitsuseofdigitalelectronicsfortransformationbetweenlinearandlogarithmicsignalsandofcomputeralgorithmforfluorescencecompensation.Ihavealreadypraisedthisapproachafewtimes(pp.19,166,189),whilealsonothingthatitprobablyrepresentedtheonlypracticalwaytoimplementanotherfeaturewhichpresentlydistinguishestheEPICSXLfromit’scompetitors,i.e.,4-colorfluorescenceanalysis.Bothfeatureswillhavetheothermanufacturerstryingtoplaycatch-upforawhile.”“XL因?yàn)橛腥珨?shù)字化的線性-數(shù)轉(zhuǎn)換及電腦軟體螢光補(bǔ)償而成為現(xiàn)今流式細(xì)胞分析儀系統(tǒng)中最獨(dú)特的產(chǎn)品.個(gè)人也多次表示欽佩(第19,166,189頁(yè)).EPICSXL(單一激光產(chǎn)生)的四個(gè)螢光分析性能,使其鶴立於其他競(jìng)爭(zhēng)者之中.這些功能將會(huì)讓其他廠商花很長(zhǎng)的時(shí)間來追趕”.摘自”流式細(xì)胞分析儀系統(tǒng)實(shí)務(wù)第三版,1995.”作者HowardM.Shapiro,PracticalFlowCytometry,3rded.,page355專家評(píng)述來自ISAC的報(bào)告產(chǎn)品介紹

XL設(shè)計(jì)的所有優(yōu)勢(shì)：?jiǎn)渭す饧ぐl(fā)四色熒光光纖通訊數(shù)字化信號(hào)傳遞可互換式光學(xué)濾片，無需調(diào)整光路具有620nm的濾片,最適合DNA倍體分析利用軟件進(jìn)行全矩陣優(yōu)化（ADC)顏色補(bǔ)償PRISM濃縮柱形圖快速調(diào)節(jié)電壓快速調(diào)整補(bǔ)償補(bǔ)償向?qū)Ю蒙飿颖具M(jìn)行顏色補(bǔ)償WINDOWS界面操作軟件Cytomics?FC500MCL進(jìn)樣MCL自動(dòng)模式.MCL直接(

或手動(dòng))

模式.單管固定位置進(jìn)樣.可以暫停/旋轉(zhuǎn)以方便隨時(shí)添加刺激劑等條碼識(shí)別樣本混勻產(chǎn)品介紹：硬件產(chǎn)品介紹：硬件可選MPL(研究用)24孔每孔樣本體積0.5