各种染料对应的激发波长和吸收波长
常用染料的激发与发射
常用荧光染料的激发和发射波长备注：发射波长的颜色及频率荧光染料的使用吖啶橙：吖啶橙是最经典的灵敏的荧光染料，它可对细胞中的DNA和RNA同时染色而显示不同颜色的荧光，DNA呈绿色荧光，RNA呈橙红色荧光。
EB:染色DNA和RNA 荧光素双醋酸酯(FDA)：FAD本身无荧光,无极性,可透过完整的原生质膜。
一旦进入原生质体后,由于受到酯酶分解而产生具有荧光的极性物质荧光素。
它不能自由出入原生质膜，因此有活力的细胞能产生荧光,无活力的原生质体不能分解FAD无荧光产生。
5mgFDA溶于1ml丙酮中,避光4℃下贮存,使用时取贮存液加入L甘露醇中.使用时,使最终浓度为%。
荧光染料Ho33342和若丹明123:活细胞双荧光染色观察细胞核和线粒体。
一般的生物染料不能穿透细胞膜，只有当细胞被固定后改变了细胞膜的通透性，染料才能进入细胞内。
但有些活体染料能进入活细胞，并对细胞不产生毒性作用。
荧光染料Ho33342和若丹明123都是活体染料。
Ho33342能与细胞中DNA进行特异的结合，若丹明123能与线粒体进行特异的结合。
采用两种荧光染料的混合染液可对一个活细胞的核和线粒体同时染色。
荧光组化实验中应注意的几个问题：1．每种荧光染料，均有自己的最适PH值，此时荧光最强。
当pH改变时，不仅荧光强度减弱，而且波长将有所改变，因此荧光检测时要在一定的PH值的缓冲液中进行。
2．一放荧光染色在20℃以下时荧光比较稳定，温度升高常出现温度猝灭。
3．在荧光观察中，常因激发光的增强而使样品荧光很快衰竭，造成观察和照相困难。
为此最好用能量小的长波长光进行观察，需照相时再适当增强激发光。
4．一般荧光染液的浓度在万分之一以下，甚至亿万分之一，也能使标本着色。
在一定的限度内，荧光强度可随荧光素的浓度增加而增强，但超过限度，荧光强度反而下降，这是由于荧光分子间的缔合而使自身荧光猝灭所致。
常用荧光染料的激发及发射波长
492
518
5-Carboxynapthofluorescein (pH 10)
512/598 563/668 Ratio Dye, pH
5-Carboxytetramethylrhodamine (5-TAMRA) 542
568
5-FAM (5-Carboxyfluorescein)
492
518
5-HAT (Hydroxy Tryptamine)
Anilin Blue Anthrocyl stearate APC (Allophycocyanin) APC-Cy7 APTRA-BTC = Ratio Dye, Zn2+ APTS Astrazon Brilliant Red 4G Astrazon Orange R Astrazon Red 6B Astrazon Yellow 7 GLL Atabrine ATTO-TAG™ CBQCA ATTO-TAG™ FQ Auramine Aurophosphine G Aurophosphine BAO 9 (Bisaminophenyloxadiazole) BCECF (high pH) BCECF (low pH) Berberine Sulphate Beta Lactamase BFP blue shifted GFP (Y66H)
荧光素名称
激发波长 发射波长 备注 (nm) (nm)
1,5 IAEDANS
336
490
1,8-ANS
372
480
4-Methylumbelliferone
385
502
5-carboxy-2,7-dichlorofluorescein
504
529
常用荧光染料的激发及发射波长
常用荧光染料的激发及发射波长FLUOROCHROME EXCITATION EMISSION3-Hydroxypyrene 5,8,10-Tri Sulfonic acid 403 513 5-Hydroxy Tryptamine 380-415 520-5305-Hydroxy Tryptamine (5-HT) 400 530Acid Fuchsin 540 630Acridine Orange (bound to DNA) 502 526Acridine Red 455-600 560-680Acridine Yellow 470 550Acriflavin 436 520AFA (Acriflavin Feulgen SITSA) 355-425 460 Alizarin Complexon 530-560 580Alizarin Red 530-560 580Allophycocyanin 650 661ACMA 430 474Aminoactinomycin D 555 655Aminocoumarin 350 445Anthroyl Stearate 361-381 446Astrazon Brilliant Red 4G 500 585Astrazon Orange R 470 540Astrazon Red 6B 520 595Astrazon Yellow 7 GLL 450 480Atabrine 436 490Auramine 460 550Aurophosphine 450-490 515Aurophosphine G 450 580BAO 9 (Bisaminophenyloxadiazole) 365 395BCECF 505 530Berberine Sulphate 430 550Bisbenzamide 360 600-610BOBO 1 462 481Blancophor FFG Solution 390 470Blancophor SV 370 435Bodipy Fl 503 512BOPRO 1 462 481Brilliant Sulphoflavin FF 430 520Calcien Blue 370 435Calcium Green 505 532Calcofluor RW Solution 370 440Calcofluor White 440 500-520Calcophor White ABT Solution 380 475Calcophor White Standard Solution 365 435Cascade Blue 400 425Catecholamine 410 470Chinacrine 450-490 515Coriphosphine O 460 575Coumarin-Phalloidin 387 470CY3.1 8 554 568CY5.1 8 649 666CY7 710 805Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid) 340 525 Dansa (Diamino Naphtyl Sulphonic Acid) 340-380 430Dansyl NH-CH3 in water 340 578DAPI 350 470Diamino Phenyl Oxydiazole (DAO) 280 460 Dimethylamino-5-Sulphonic acid 310-370 520 Diphenyl Brilliant Flavine 7GFF 430 520 Dopamine 340 490-520Eosin 525 545Erythrosin ITC 530 558Ethidium Bromide 510 595Euchrysin 430 540FIF (Formaldehyde Induced Fluorescence) 405 435 Flazo Orange 375-530 612Fluorescein Isothiocyanate (FITC) 490 525Fluo 3 485 503Fura-2 340-380 512Genacryl Brilliant Red B 520 590Genacryl Brilliant Yellow 10GF 430 485Genacryl Pink 3G 470 583Genacryl Yellow 5GF 430 475Gloxalic Acid 405 460Granular Blue 355 425Haematoporphyrin 530-560 580Hoechst 33258 (bound to DNA) 346 460Indo-1 350 405-482Intrawhite Cf Liquid 360 430Leucophor PAF 370 430Leucophor SF 380 465Leucophor WS 395 465Lissamine Rhodamine B200 (RD200) 575 595Lucifer Yellow CH 425 528Lucifer Yellow VS 430 535Magdala Red 524 600Maxilon Brilliant Flavin 10 GFF 450 495Maxilon Brilliant Flavin 8 GFF 460 495MPS (Methyl Green Pyronine Stilbene) 364 395 Mithramycin 450 570NBD Amine 450 530Nile Red 515-530 525-605Nitrobenzoxadidole 460-470 510-650 Noradrenaline 340 490-520Nuclear Fast Red 289-530 580Nuclear Yellow 365 495Nylosan Brilliant Flavin E8G 460 510 Pararosaniline (Feulgen) 570 625 Phorwite AR Solution 360 430 Phorwite BKL 370 430Phorwite Rev 380 430Phorwite RPA 375 430Phosphine 3R 465 565Phycoerythrin R 480-565 578 Pontochrome Blue Black 535-553 605 Primuline 410 550Procion Yellow 470 600Propidium Iodide 536 617Pyronine 410 540Pyronine B 540-590 560-650Pyrozal Brilliant Flavin 7GF 365 495 Quinacrine Mustard 423 503 Rhodamine 123 511 534Rhodamine 5 GLD 470 565Rhodamine 6G 526 555Rhodamine B 540 625Rhodamine B 200 523-557 595 Rhodamine B Extra 550 605 Rhodamine BB 540 580Rhodamine BG 540 572Rhodamine WT 530 555Rose Bengal 540 550-600Serotonin 365 520-540Sevron Brilliant Red 2B 520 595 Sevron Brilliant Red 4G 500 583 Sevron Brilliant Red B 530 590 Sevron Orange 440 530Sevron Yellow L 430 490SITS (Primuline) 395-425 450SITS (Stilbene Isothiosulphonic acid) 365 460Stilbene 335 440Snarf 1 563 639sulphO Rhodamine B Can C 520 595Sulpho Rhodamine G Extra 470 570Tetracycline 390 560TRITC (Tetramethyl Rhodamine Isothiocyanate) 557 576Texas Red 596 615Thiazine Red R 510 580Thioflavin S 430 550Thioflavin TCN 350 460Thioflavin 5 430 550Thiolyte 370-385 477-484Thiozol Orange 453 480Tinopol CBS 390 4.3TOTO 1 514 533TOTO 3 642 661True Blue 365 420-430Ultralite 656 678Uranine B 420 520Uvitex SFC 365 435Xylene Orange 546 580XRITC 582 601YO PRO 1 491 509、红640—780nm，橙640—610，黄610—530，绿505—525，蓝505—470，紫470—380。
DAPI和Hoechst-PI染料
DAPIDAPI即4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)，是一种能够与DNA强力结合的荧光染料，常用于荧光显微镜观测。
因为DAPI可以透过完整的细胞膜，它可以用于活细胞和固定细胞的染色。
DAPI介绍在荧光显微镜观察下，DAPI染剂是利用紫外光波长的光线激发。
当DAPI与双股DNA 结合时，最大吸收波长为358nm，最大发射波长为461nm，其发散光的波长范围含盖了蓝色至青绿色。
DAPI也可以和RNA结合，但产生的荧光强度不及与DNA结合的结果，其发散光的波长范围约在500nm左右。
DAPI的发散光为蓝色，且DAPI和绿色荧光蛋白(Green fluorescent protein, GFP)或Texas Red染剂(红色荧光染剂)的发散波长，仅有少部分重叠，研究员可以善用这项特性在单一的样品上进行多重荧光染色。
DAPI能快速进入活细胞中与DNA结合，因此DAPI对生物体而言，也被视为一种毒性物质与致癌物。
使用过程中应注意操作与抛弃的处理程序。
中文名：4，6-联脒-2-苯基吲哚(?)英文名：4',6-diamidino-2-phenylindole，2-(4-amidinophenyl)-1H -indole-6-carboxamidine，DAPI dihydrochloride分子式：C16H15N5分子量：277.324CAS number：28718-90-3光谱性质：DAPI的最大激发波长为340nm，最大发射波长为488nm与双链DNA结合时最大吸收/最大发射为358 nm/461 nm；与RNA结合时，最大发射移动到400 nm左右。
DAPI染色原理：DAPI 为一种荧光染料，可以穿透细胞膜与细胞核中的双链DNA结合而发挥标记的作用，可以产生比DAPI自身强20多倍的荧光，和EB相比，对双链DNA的染色灵敏度要高很多倍。
各种激发光和吸收光波长
各样激发光和汲取光波长此刻发现一个更全的相关激发光和发射光的波长图谱。
这对 CONFOCAL去做多重染色时是必不行少的参照资料。
所有文件Excitation and Emission Wavelengths of FluorophoresTavi\'s \"FluoroTable\": Common fluorophores(from )Fluorophore Absorption Emission Other info1,5 IAEDANS3364901,8-ANS3724804-Methylumbelliferone3855025-carboxy-2,7-dichlorofluorescein5045295-Carboxyfluorescein (5-FAM)4925185-Carboxynapthofluorescein (pH 10)512/598563/668Ratio Dye, pH5-Carboxytetramethylrhodamine (5-TAMRA)5425685-FAM (5-Carboxyfluorescein)4925185-HAT (Hydroxy Tryptamine)370-415520-5405-Hydroxy Tryptamine (HAT)370-415520-5405-ROX (carboxy-X-rhodamine)5786045675915-TAMRA (5-Carboxytetramethylrhodamine)5485525425686-Carboxyrhodamine 6G5185436-CR 6G5185436-JOE5205487-Amino-4-methylcoumarin3514307-Aminoactinomycin D (7-AAD)5466477-Hydroxy-4-methylcoumarin360449, 4559-Amino-6-chloro-2-methoxyacridine412, 430471, 474ABQ344445Acid Fuchsin540630ACMA (9-Amino-6-chloro-2-methoxyacridine)412, 430471, 474Acridine Orange + DNA502526Acridine Orange + RNA460650Acridine Orange, both DNA & RNA440-480520-650Acridine Red455-600560-680Acridine Yellow470550Acriflavin436520Acriflavin Feulgen SITSA355-425460Aequorin (Photoprotein)466PhotoproteinAFPs - AutoFluorescent Protein - (QuantumBiotechnologies) see sgGFP, sgBFPAlexa Fluor 350346442342441Alexa Fluor 430431540Alexa Fluor 488495, 492519, 520Alexa Fluor 532531, 532553, 554Alexa Fluor 546556, 557572, 573Alexa Fluor 568577, 578603Alexa Fluor 594590, 594617, 618Alexa Fluor 633632650Alexa Fluor 647647666Alexa Fluor 660668698Alexa Fluor 680679702Alizarin Complexon530-560,580,580624-645Alizarin Red530-560580Allophycocyanin (APC)630, 645655, 660AMC, AMCA-S345445AMCA (Aminomethylcoumarin)345425347444AMCA-X353442 Aminoactinomycin D555655Aminocoumarin346442350445 Aminomethylcoumarin (AMCA)345425347444Anilin Blue600Anthrocyl stearate360-381446APC (Allophycocyanin)630, 645655, 660APC-Cy7625-650755APTRA-BTC = Ratio Dye, Zn2+466/380520/530 Ratio Dye, Zn2+ APTS424505Astrazon Brilliant Red 4G500585Astrazon Orange R470540Astrazon Red 6B520595Astrazon Yellow 7 GLL450480Atabrine436490ATTO-TAG CBQCA465560ATTO-TAG FQ486591Auramine460550Aurophosphine G450580Aurophosphine450-490515BAO 9(Bisaminophenyloxadiazole)365395BCECF (high pH)492, 503520,528BCECF (low pH)482520Berberine Sulphate430550Beta Lactamase409447, 520BFP blue shifted GFP (Y66H)381,382,445, 447, blue shifted GFP (Y66H) Blue Fluorescent Protein383448Blue Fluorescent Protein BFP/GFP FRETBimane398490Bisbenzamide360461Bisbenzimide (Hoechst)360461bis-BTC = Ratio Dye, Zn2+455/405529/505Ratio Dye, Zn2+ Blancophor FFG390470Blancophor SV370435BOBO-1462481BOBO-3570602Bodipy 492/515490515Bodipy 493/503533549Bodipy 500/510509515Bodipy 505/515502510Bodipy 530/550528547Bodipy 542/563543563Bodipy 558/568558569Bodipy 564/570564570Bodipy 576/589579590Bodipy 581/591584592Bodipy 630/650-X625642Bodipy 650/665-X647665Bodipy 665/676605676Bodipy Fl504, 505511, 513Bodipy FL ATP505514Bodipy Fl-Ceramide505511Bodipy R6G SE528547Bodipy TMR542574Bodipy TMR-X conjugate544573Bodipy TMR-X, SE544570Bodipy TR589617Bodipy TR ATP591620Bodipy TR-X SE588616BO-PRO-1462481BO-PRO-3544570Brilliant Sulphoflavin FF430520BTC - Ratio Dye Ca2+464/401533/529Ratio Dye Ca2+BTC-5N - atio Dye, Zn2+459/417517/532Ratio Dye, Zn2+ Calcein494517Calcein Blue373440Calcium Crimson588, 589611, 615Calcium Green501, 506531Calcium Green-1 Ca2+ Dye506531Ca2+ DyeCalcium Green-2 Ca2+506/503536Ca2+Calcium Green-5N Ca2+506532Ca2+Calcium Green-C18 Ca2+509530Ca2+Calcium Orange549575576Calcofluor White385,395, 437,440,405445Carboxy-X-rhodamine (5-ROX)576601Cascade Blue377420398423399Cascade Yellow399550400552Catecholamine410470CCF2 (GeneBlazer)CFDA494520CFP - Cyan Fluorescent Protein430,433, 474,475, Cyan Fluorescent Protein436, (453)476, (501)CFP/YFP FRETChlorophyll480650Chromomycin A436-460470Chromomycin A445575CL-NERF (Ratio Dye, pH)504/514540Ratio Dye, pHCMFDA494520Coelenterazine Ca2+ Dye, bioluminescence(429)465Ca2+Dye,bioluminescence,native molecule Coelenterazine cp (Ca2+ Dye,)(430)442Ca2+Dye,bioluminescence Coelenterazine f(437)473Ca2+Dye,bioluminescence Coelenterazine fcp452Ca2+Dye,bioluminescence Coelenterazine h(437)464Ca2+Dye,bioluminescence Coelenterazine hcp(433)444Ca2+Dye,bioluminescence Coelenterazine ip441Ca2+Dye,bioluminescence Coelenterazine n(431)467Ca2+Dye,bioluminescence Coelenterazine O460575Coumarin Phalloidin387470C-phycocyanineCPM Methylcoumarin384469MethylcoumarinCTC400-450602CTC FormazanCy2489506Cy3.1 8554568Cy3.5581598Cy3514566552570554Cy5.1 8649666Cy5.5675695Cy5649666670Cy7710, 743767, 805Cyan GFP433 (453)475 (501)cyclic AMP Fluorosensor (FiCRhR)500517CyQuant Cell Proliferation Assay480520Cell Proliferation Assay Dabcyl453Dansyl340578Dansyl Amine337517Dansyl Cadaverine335518Dansyl Chloride372518Dansyl DHPE336517Dansyl fluoride356noneDAPI359461Dapoxyl403580Dapoxyl 2374574Dapoxyl 3373574DCFDA504529DCFH (Dichlorodihydrofluorescein Diacetate)505535DDAO463607DHR (Dihydorhodamine 123)505534Di-4-ANEPPS496705Di-8-ANEPPS (non-ratio)488605498713DiA (4-Di-16-ASP)456591 Dichlorodihydrofluorescein Diacetate (DCFH)505535DiD - Lipophilic Tracer644665Lipophilic TracerDiD (DiIC18(5))644665DIDS341415Dihydorhodamine 123 (DHR)505535DiI (DiIC18(3))549, 551565Dinitrophenol349DiO (DiOC18(3))484, 487501, 502DiR748780Lipophilic TracerDiR (DiIC18(7))750779DM-NERF (high pH)497/510540Ratio Dye, pHDNP349Dopamine340490-520DsRed558583Red fluorescent protein DTAF494520DY-630-NHS621660Hemicyanelabel for proteins and DNA DY-635-NHS634664Hemicyanelabel for proteins and DNA EBFP383447Enhanced BlueFluorescent ProteinECFP436474Enhanced CyanFluorescent ProteinEGFP488, 498507, 516Enhanced GreenFluorescent ProteinELF 97345530Eosin524545Erythrosin529, 532554, 555Erythrosin ITC529555Ethidium Bromide510, 523595, 605Ethidium homodimer -1 (EthD-1)528617Euchrysin430540EukoLightEuropium (III) chlorideEYFP513, 520527, 532Enhanced YellowFluorescent ProteinFast Blue360440FDA494520Feulgen (Pararosaniline)570625FIF (Formaldehyd Induced Fluorescence)405433FITC490, 494520, 525FITC Antibody493517Flazo Orange375-530612Fluo-3480506, 520, 527506Fluo-4494516Fluorescein (FITC)490, 494520, 525Fluorescein Diacetate494520Fluoro-Emerald495524Fluoro-Gold (Hydroxystilbamidine)361536Fluor-Ruby555582FluorX494520FM 1-43479598FM 4-46515640Fura Red(high pH)572657Fura RedFluo-3Fura-2, high calcium335505Excitation ratio dye Fura-2, low calcium363512Excitation ratio dye Fura-2/BCECFGenacryl Brilliant Red B520590Genacryl Brilliant Yellow 10GF430485Genacryl Pink 3G470583Genacryl Yellow 5GF430475GeneBlazer (CCF2)GFP (S65T)498516GFP red shifted (rsGFP)498516GFP wild type, non-UV excitation (wtGFP)475509GFP wild type, UV excitation (wtGFP)395509GFPuv385508Gloxalic Acid405460Granular Blue355425Haematoporphyrin530-560580Hoechst 33258345487Hoechst 33342347Filter sets installed on the Axiovert 200M:1)Brightfield filter cube contains DIC analyzerDIC:2)DAPI / BFP: Chroma set 31041, Ex at 380 nm (bandwidth 30 nm),Em at 460 nm (bandwidth 50 nm).3)FITC / GFP: Chroma set 41001, Ex at 480 nm (bandwidth 40 nm),Em at 535 nm (band width 50 nm).4)RHOD:Chroma set 41002c, Ex at 545 nm (bandwidth30nm), Em at 620 nm (bandwidth 60 nm).5)Cy5:Chroma set 41024, Ex at 620 nm (bandwidth60nm), Em 665 nm and above (long pass filter).Also available upon request:4)GFP:Chroma set 41017, Ex at 470 nm (bandwidth40nm), Em 525 nm (band width 50 nm).5)CFP:Chroma set 31044v2, Ex at 436 nm (bandwidth 20nm), Em 480 nm (bandwidth 40 nm).6)YFP:Chroma set 41028, Ex at 500 (bandwidth 20 nm), Em 535 (bandwidth 30 nm).7) CFP/YFP FRET:Chroma set 31052, Ex 436 (bandwidth 20 nm), Em535 (bandwidth 30 nm).。