流式細胞技術(shù)熒光抗體

Fluorochromes23-13536-00Rev.01FluorochromePropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum

(lowspectraloverlapincombination)Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitationandemissionwavelengthsRelativebrightness223-13536-00Rev.01FluorochromeMoleculeTypesSmallorganicmolecules

examples:FITC,BDHorizonTMV450,Cy7Fluorescentproteins

examples:PE,APC,PerCPTandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptor

examples:PE-Cy7,PerCP-CyTM5.5Nanocrystals(Qdots)—inorganicsemiconductors

examples:Qdot565,Qdot605323-13536-00Rev.01SomeCommonFluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor?488SmallorganicPhycoerythrin(PE)ProteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor?647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallorganicBDHorizonTMV500SmallorganicAmCyanProtein423-13536-00Rev.01SmallOrganicFluorochromesAdvantagesLowmolecularweightEasytoconjugate—directattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50?100nm)Tendtobelessbright523-13536-00Rev.01ProteinFluorochromesAdvantagesGoodstabilityConsistentemissionspectraMediumStokesShift(75?200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugate—intermediariesneededtoattachtomAb623-13536-00Rev.01TandemDyeFluorochromesAdvantagesVerylargeStokesShift(150?300nm)Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Hardertoconjugate(sameasfluorescentprotein)Sometandemshavepoorstability723-13536-00Rev.01NanocrystalFluorochromesAdvantagesLargeStokesShift(100?500nm)TendtobeverybrightEmissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsourceHighlyresistanttophotobleachingNanocrystalssharebiophysicalandconjugationproperties

DisadvantagesDifficulttoconjugateInstabilityofbindingsCytotoxicityWideexcitationrangeproducescross-laserspillover823-13536-00Rev.01ExcitationandEmissionExcitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.EmissionwavelengthsdeterminefiltersandPMTsthatcanmeasuretheemissionsignal.923-13536-00Rev.01KnowYourCytometerCytometerConfigurationBDFACSCantoTMII4-2-2configurationisshownbelowBDTMLSRII4-2-2configurationissimilar4detectors

forbluelaser2detectors

forredlaser2detectors

forvioletlaser1023-13536-00Rev.01TypicalExcitationandEmission

BDFACSCantoII4-2-2(BDLSRII4-2-2issimilar)Detector

RangeVioletLaser

405nmBlueLaser

488nmRedLaser

633nm410–490nmBDHorizonV450

PacificBlueTM500–560nmBDHorizonV500

AmCyan515–545nmFITC

AlexaFluor?488564–606nmPE650–670nmAPC

AlexaFluor?647670–735nmPerCP-Cy5.5

PE-Cy5

PerCP750–810nmPE-Cy7BDAPC-H7

APC-Cy71123-13536-00Rev.01FluorochromeUseDependson

theCytometerConfiguration6-color8-color

Morethan

8colorsFITC,AlexaFluor?488FITC,AlexaFluor?488FITC,AlexaFluor?488PEPEPEPE-TexasRed?,PE-AlexaFluor?610,orPE-AlexaFluor?594PE-Cy5,PerCP,

orPerCP-Cy5.5PE-Cy5,PerCP,

orPerCP-Cy5.5PE-Cy5,PerCP,

orPerCP-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC,AlexaFluor?647APC,AlexaFluor?647APC,AlexaFluor?647APC-Cy5.5,AlexaFluor?680,

AlexaFluor?700APC-H7,APC-Cy7APC-H7,APC-Cy7APC-H7,APC-Cy7BDHorizonV500,AmCyanBDHorizonV500,AmCyanBDHorizonV450,PacificBlue?BDHorizonV450,PacificBlue?PacificOrange?,Q-dots1223-13536-00Rev.01FluorochromeBrightnessThebrightnessofafluorochromedependsontwofactors:MolarExtinctionCoefficient(

)measureshowwellafluorochromeabsorbsenergy.QuantumYield(Qy)istheratioofphotonsemittedtophotonsabsorbed.Brightness=

x

QyRelativeBrightness=BrightnessofPEBrightness1323-13536-00Rev.01SomeFluorochromesareMUCHBrighterPEis~50xbrighterthanFITCand~10xbrighterthanAPC.APCis~5xbrighterthanPacificBlue?.Extinctioncoefficientismoresignificantthanquantumyieldindeterminingbrightness.FluorochromeMolarExtinctionCoefficient(mol-1xcm-1)QuantumYieldRelativeBrightness(toPE)PE19600000.98100.00%PE-Cy51960000<0.881.63%PerCP320000NA16.66%APC2320000.688.21%FITC670000.501.74%PacificBlue?360000.801.5%1423-13536-00Rev.01DD=differencebetweenthemediansofthepositiveandnegativepopulationsW

=spread(2xrSD)ofthenegativepopulationStainIndexStainIndex=Stainindexisapracticalwaytocharacterizethebrightnessofamarkerwithrespecttoagivenopticalconfiguration.DWWStainindexcanalsobeusedtocharacterizethesensitivityofafluoresenceparameter.1523-13536-00Rev.01TypicalCD4StainIndexes—BDLSRIIFluorochromeCD4CloneFilterSetStainIndexRelativeBrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4695/403016.66%APCRPA-T4660/202638.21%FITCRPA-T4530/30431.74%PacificBlue?RPA-T4440/40631.5%APChasahigherCD4stainindexthanPE-Cy5,

butapproximately1/10ththerelativebrightness.1623-13536-00Rev.01TypicalCD4StainIndexes—BDFACSCantoIIFluorochromeCD4CloneFilterSetStainIndexRelativeBrightnessPESK3585/42467100.00%PE-Cy5———81.63%PerCPSK3695/405916.66%APCSK3660/203848.21%FITCSK3530/30581.74%PacificBlue?SK3450/50131.5%FITChasapproximatelythesameCD4stainindexasPerCP,butapproximately1/10ththerelativebrightness.1723-13536-00Rev.01StainIndexFactorsStainindexisdependentontheopticalconfigurationandadditionalperformancefactors.Factorsthatcanaffectstainindexinclude:LaserwavelengthandpowerDetectorrangeDetectorefficiencyBackgroundsignalDon’tdependonpublishedvalues—measurestainindexonyourownsystem.1823-13536-00Rev.01CD4StainIndexesAcrossCytometersStainIndexExercise1923-13536-00Rev.01FluorescenceSpilloverEmissionofFITC

inPEchannel2023-13536-00Rev.01SignificantSpilloverson4-2-2Configuration2123-13536-00Rev.01SpilloverDecreasesSensitivityWithoutCD45AmCyanWithCD45AmCyanCD19FITCSpillovercansignificantlyincreasethevariabilityofnegativeanddimpopulations,evenaftercompensationisapplied.2223-13536-00Rev.01LostPopulationduetoSpilloverLymphocytesstainedwithCD45FITCandCD4PECD45FITCcausesdimCD4+CD45+

tobedifficulttodistinguishdueto

significantFITCspilloverintoPE.LymphocytesstainedwithCD45PerCPandCD4PECD45PerCPallowsdimCD4+CD45+

tobedistinguishedfrombackground

duetominimalPerCP

spilloverintoPE.2323-13536-00Rev.01TandemDyeIssuesUsetandemdyesinresearchapplicationswithconsiderationoftheirtechnicallimitations.APC-Cy7(andtoalesserextentPE-Cy7)candegradeinthepresenceoflight,fixation,andelevatedtemperature.DegradationcausesloweremissionintheCy7detectorandhigheremissioninthedetectoroftheparentdye(APCorPE).APC-H7,APC-Cy7,andPE-Cy7performancecanbeaffectedbypolystyrenetubes.2423-13536-00Rev.01TandemDegradation—FalsePositivesFalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7WithCD8APC-Cy7WithoutCD8APC-Cy72523-13536-00Rev.01TandemDegradationOverTime0hours~2hours~20hoursPECD3PE-Cy5CD8PE-Cy7TimeSample

LeftinLightPEPE2623-13536-00Rev.01APC-H7IsMoreStableThanAPC-Cy7ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy72723-13536-00Rev.01TandemDyeRecommendationsWhenusingAPC-Cy7andPE-Cy7,bewareoffixativeandlightinstabilityissues.IfproblemsarisewhenusingpolystyrenewithAPC-H7,APC-Cy7,orPE-Cy7,tryswitchingtoK-resinorpolycarbonate.PerCP-Cy5.5islesssusceptibletoinstabilitythanAPC-Cy7andPE-Cy7.BDoffersAPC-H7conjugatedantibodiesthataremorestablethanAPC-Cy7conjugatesandhavelessspilloverintotheAPCdetector.2823-13536-00Rev.01NewVioletLaserFluorochromesBDHorizonV450Maximumexcitationat404nm,emissionpeakof448nm.BD

HorizonV450reagentsexhibitperformancecomparabletoPacificBlue?reagentsasmeasuredbyStainIndex.ImprovedstabilitywithfixationcomparedtoPacificBlue?.2923-13536-00Rev.01NewVioletLaserFluorochromesBDHorizonV500Maximumexcitationat415nm,emissionpeakof500nm.ProvidessignificantlyreducedspilloverintotheFITCchannelcomparedtoAmCyan—V500haslowexcitationwiththebluelaser.ImprovedstabilitywithfixationcomparedtoAmCyan.3023-13536-00Rev.01BDHorizonV500LowSpilloverintoFITCV500stainedcellsAmCyanstainedcellsDataisuncompensated.3123-13536-00Rev.01FactorsAffectingReagentPerformanceRelativebrightnessofthefluorochromeNumberoffluorochromesperantibodyExpressionlevelson(orin)thecellsofinterestBackgroundcontributionsSpilloverPhotostabilityReagentenvironmentCytometerconfiguration3223-13536-00Rev.01Non-ConjugatedFluorescentDyesThesedyesbinddirectlytocellcomponents.Viabilitydyesdiscriminatelivecellsfromdeadcells.examples:7-AAD,TO-PRO-3,PI,DAPI,andtheLIVE/DEADseries.Proliferationdyesareusedtostudycelldivision.examples:CSFE,PKH67,andBDHorizonVPD450.DNAdyesareusedforDNAcell-cycleanaylsis.examples:PI,DAPI,andHoechst33342.Reporterdyesareusedtostudygeneexpression.examples:mCherryandGFP.3323-13536-00Rev.01Non-ConjugatedFluorescenceDyeDetection

BDFACSCantoII4-2-2andBDLSRII4-2-2Detector

RangeVioletLaser

405nmBlueLaser

488nmRedLaser

635nm410–490nmHoechst33342,DAPI,

HorizonVPD450,LIVE/DEADViolet500–560nmLIVE/DEADAqua515–545nmCSFE,GFP,

LIVE/DEADGreen564–606nmPI650–670nmTO-PRO-3670–735nm7-AAD750–810nmLIVE/DEADNear-IRmCherryisbestdetectedwitha561-nmlaseroptionanda590–630detectorrange.3423-13536-00Rev.01BDFluorescenceSpectrumViewer/spectraUsetheFluorescenceSpectrumViewertohelpchoosefluorochromesbasedon:thenumberofcolorsyouneedinyourexperimentyourcytometerconfigurationspilloverissues3523-13536-00Rev.01第一節(jié)活塞式空壓機的工作原理第二節(jié)活塞式空壓機的結(jié)構(gòu)和自動控制第三節(jié)活塞式空壓機的管理復(fù)習(xí)思考題單擊此處輸入你的副標題，文字是您思想的提煉，為了最終演示發(fā)布的良好效果，請盡量言簡意賅的闡述觀點。第六章活塞式空氣壓縮機

piston-aircompressor壓縮空氣在船舶上的應(yīng)用：

1.主機的啟動、換向；

2.輔機的啟動；

3.為氣動裝置提供氣源；

4.為氣動工具提供氣源；

5.吹洗零部件和濾器。

排氣量:單位時間內(nèi)所排送的相當?shù)谝患壩鼩鉅顟B(tài)的空氣體積。單位：m3/s、m3/min、m3/h第六章活塞式空氣壓縮機

piston-aircompressor空壓機分類：按排氣壓力分：低壓0.2～1.0MPa；中壓1～10MPa；高壓10～100MPa。按排氣量分：微型<1m3/min；小型1～10m3/min；中型10～100m3/min；大型>100m3/min。第六章活塞式空氣壓縮機

piston-aircompressor第一節(jié)活塞式空壓機的工作原理容積式壓縮機按結(jié)構(gòu)分為兩大類：往復(fù)式與旋轉(zhuǎn)式兩級活塞式壓縮機單級活塞壓縮機活塞式壓縮機膜片式壓縮機旋轉(zhuǎn)葉片式壓縮機最長的使用壽命-

----低轉(zhuǎn)速（1460RPM），動件少（軸承與滑片），潤滑油在機件間形成保護膜，防止磨損及泄漏，使空壓機能夠安靜有效運作；平時有按規(guī)定做例行保養(yǎng)的JAGUAR滑片式空壓機，至今使用十萬小時以上，依然完好如初，按十萬小時相當于每日以十小時運作計算，可長達33年之久。因此，將滑片式空壓機比喻為一部終身機器實不為過。滑(葉)片式空壓機可以365天連續(xù)運轉(zhuǎn)并保證60000小時以上安全運轉(zhuǎn)的空氣壓縮機1.進氣2.開始壓縮3.壓縮中4.排氣1.轉(zhuǎn)子及機殼間成為壓縮空間，當轉(zhuǎn)子開始轉(zhuǎn)動時，空氣由機體進氣端進入。2.轉(zhuǎn)子轉(zhuǎn)動使被吸入的空氣轉(zhuǎn)至機殼與轉(zhuǎn)子間氣密范圍，同時停止進氣。3.轉(zhuǎn)子不斷轉(zhuǎn)動，氣密范圍變小，空氣被壓縮。4.被壓縮的空氣壓力升高達到額定的壓力后由排氣端排出進入油氣分離器內(nèi)。4.被壓縮的空氣壓力升高達到額定的壓力后由排氣端排出進入油氣分離器內(nèi)。1.進氣2.開始壓縮3.壓縮中4.排氣1.凸凹轉(zhuǎn)子及機殼間成為壓縮空間，當轉(zhuǎn)子開始轉(zhuǎn)動時，空氣由機體進氣端進入。2.轉(zhuǎn)子轉(zhuǎn)動使被吸入的空氣轉(zhuǎn)至機殼與轉(zhuǎn)子間氣密范圍，同時停止進氣。3.轉(zhuǎn)子不斷轉(zhuǎn)動，氣密范圍變小，空氣被壓縮。螺桿式氣體壓縮機是世界上最先進、緊湊型、堅實、運行平穩(wěn)，噪音低，是值得信賴的氣體壓縮機。螺桿式壓縮機氣路系統(tǒng)：

A

進氣過濾器

B

空氣進氣閥

C

壓縮機主機

D

單向閥

E

空氣/油分離器

F

最小壓力閥

G

后冷卻器

H

帶自動疏水器的水分離器油路系統(tǒng)：

J

油箱

K

恒溫旁通閥

L

油冷卻器

M

油過濾器

N

回油閥

O

斷油閥冷凍系統(tǒng)：

P

冷凍壓縮機

Q

冷凝器

R

熱交換器

S

旁通系統(tǒng)

T

空氣出口過濾器螺桿式壓縮機渦旋式壓縮機

渦旋式壓縮機是20世紀90年代末期開發(fā)并問世的高科技壓縮機，由于結(jié)構(gòu)簡單、零件少、效率高、可靠性好，尤其是其低噪聲、長壽命等諸方面大大優(yōu)于其它型式的壓縮機，已經(jīng)得到壓縮機行業(yè)的關(guān)注和公認。被譽為“環(huán)保型壓縮機”。由于渦旋式壓縮機的獨特設(shè)計，使其成為當今世界最節(jié)能壓縮機。渦旋式壓縮機主要運動件渦卷付，只有磨合沒有磨損，因而壽命更長，被譽為免維修壓縮機。

由于渦旋式壓縮機運行平穩(wěn)、振動小、工作環(huán)境安靜，又被譽為“超靜壓縮機”。

渦旋式壓縮機零部件少，只有四個運動部件,壓縮機工作腔由相運動渦卷付形成多個相互封閉的鐮形工作腔，當動渦卷作平動運動時，使鐮形工作腔由大變小而達到壓縮和排出壓縮空氣的目的?；钊娇諝鈮嚎s機的外形第一節(jié)活塞式空壓機的工作原理一、理論工作循環(huán)（單級壓縮）工作循環(huán)：4—1—2—34—1吸氣過程

1—2壓縮過程

2—3排氣過程第一節(jié)活塞式空壓機的工作原理一、理論工作循環(huán)（單級壓縮）

壓縮分類：絕熱壓縮：1—2耗功最大等溫壓縮：1—2''耗功最小多變壓縮：1—2'耗功居中功＝P×V（PV圖上的面積）加強對氣缸的冷卻，省功、對氣缸潤滑有益。二、實際工作循環(huán)（單級壓縮）1.不存在假設(shè)條件2.與理論循環(huán)不同的原因：1）余隙容積Vc的影響Vc不利的影響—殘存的氣體在活塞回行時，發(fā)生膨脹，使實際吸氣行程（容積）減小。Vc有利的好處—

（1）形成氣墊，利于活塞回行；（2）避免“液擊”（空氣結(jié)露）；（3）避免活塞、連桿熱膨脹，松動發(fā)生相撞。第一節(jié)活塞式空壓機的工作原理表征Vc的參數(shù)—相對容積C、容積系數(shù)λv合適的C：低壓0.07-0.12

中壓0.09-0.14

高壓0.11-0.16

λv＝0.65—0.901）余隙容積Vc的影響C越大或壓力比越高，則λv越小。保證Vc正常的措施：余隙高度見表6-1壓鉛法—保證要求的氣缸墊厚度2.與理論循環(huán)不同的原因：二、實際工作循環(huán)（單級壓縮）第一節(jié)活塞式空壓機的工作原理2）進排氣閥及流道阻力的影響吸氣過程壓力損失使排氣量減少程度，用壓力系數(shù)λp表示：保證措施：合適的氣閥升程及彈簧彈力、管路圓滑暢通、濾器干凈。λp

（0.90-0.98）2.與理論循環(huán)不同的原因：二、實際工作循環(huán)（單級壓縮）第一節(jié)活塞式空壓機的工作原理3）吸氣預(yù)熱的影響由于壓縮過程中機件吸熱，所以在吸氣過程中，機件放熱使吸入的氣體溫度升高，使吸氣的比容減小，造成吸氣量下