Fluorochromes and Filters for Fluorescence Microscopyaix1.uottawa.ca/~sgee/protocols/Fluorochromes and Filters...1 1. Current filter sets from Carl Zeiss Filter set Catalogue number

M i c r o s c o p y f r o m C a r l Z e i s s

Fluorochromes and Filters for Fluorescence Microscopy

Spectral Data and

Recommended Filter Sets

The history of fluorescence microscopy

started at Carl Zeiss in 1904

when August Köhler described the

phenomenon of fluorescence emission

from biological objects under UV

irradiation. As early as 1913, Carl Zeiss

built a commercial fluorescence

microscope (with carbon arc lamp).

During recent years, fluorescence

microscopy has experienced an un-

expected renaissance. Today, the

development of complex dyes (fluoro-

chromes) permits very selective quali-

tative and quantitative information

to be gained about structures and

processes in the objects examined.

The technique can be applied in

a wide variety of areas in cell biology,

molecular biology, neurobiology,

cytogenetics, histology, pathology and

clinical cytology.

The rapid development of application-

specific fluorochromes requires suit-

able filter sets for the respective

absorption and emission ranges.

In this publication, we would like

to recommend filters for the most

common of these fluorochromes.

Filter set in modern filter moduleswith “light trap”Contents

1. Current filter sets from Carl Zeiss

2. Standard fluorochromes and recommended filter sets

3. Minimization of pixel shift in multiple fluorescence exposures

using fluorescence filters from Carl Zeiss

4. Spectral data of the filter sets

To ensure the constant enhancement of our products, we attach majorimportance to the opinions and feedback we receive from our users. Please contact us (address on back page) with regard to any amendments,special requests for filter sets, information on new fluorochromes or other subjects.

1

1. Current filter sets from Carl Zeiss

Filter set Catalogue number Diameter Diameter Size of beam splitter SpecialitiesExcitation Emission

00 488000-0000.000 25 mm 25 mm 36 x 26 x 1 mm single excitation














21 488021-0000.000 18 mm 25 mm 36 x 26 x 1 mm2 excitation filters,

alternating excitation

23 488023-0000.000 25 mm 25 mm 36 x 26 x 1 mmdouble simultaneousmultiband excitation


25 488025-0000.000 25 mm 25 mm 36 x 26 x 1 mmtriple simultaneous

multiband excitation




31 1031-350 25 mm 25 mm 36 x 26 x 1 mm single excitation






40 488040-0000.000 18 mm 25 mm 36 x 26 x 1 mmtriple simultaneous


40 1021-600 25 mm 25 mm 36 x 26 x 1 mmtriple simultaneous


Filter set 23 contains a double bandpass filter set for synchronous double fluorescence: FITC + TRITC.

Filter set 24 contains a double bandpass filter set for synchronous double fluorescence: FITC + Texas red.

Filter set 25 contains a triple bandpass filter set for synchronous triple fluorescence: FITC + Texas red + DAPI.

This filter set contains specifically paired filters which cannot be combined individually.

Filter set 40 contains a triple bandpass filter set for synchronous triple fluorescence.

Filter set 21 contains two excitation filters for the filter changer for dual wavelength fluorometry.

3

2. Fluorochromes and recommended filter sets

The filter sets 13, 37 and 38 also

support current applications with

the GFP (Green Fluorescent Protein)

fluorochrome.

This fluorescent protein is used

to mark even living objects with

a fluorochrome (see micro info

40-508 / 8.97).

GFP modifications, such as

enhanced GFP (EGFP), CFP or YFP,

have also been taken into

consideration.

XX ð recommended filter combination(XX)ð filter combination applicable

to a limited extent

*) ð filter combination available on request

(combined of non-standard filters)

Fluorochrome Excit. Emiss. Single excitation Double excitation Triple excitation(nm) (nm)

1,8 ANS 372 480 02

5-TAMRA548 552

(15, 20)542 568

5ROX578 604

31567 591

6 JOE 520 548 (17)

Acridin yellow 470 550 09; 10

Acridin orange + DNA 502 526 09; 10; (16, 17)

Acridin orange + RNA 460 650 09

Acriflavin-Feulgen 480 550-600 09; 16

Adrenaline 05

Alexa FluorTM 350 346 442 02; (01) 40

Alexa FluorTM 430 431 541 05

Alexa FluorTM 488 495 519 09; 10; (16; 17) 40

Alexa FluorTM 532 531 554 (09; 20)

Alexa FluorTM 546 556 573 20 40

Alexa FluorTM 568 578 603 14; 15

Alexa FluorTM 594 590 617 00 24 25

Alizarin complexon 530-560 580 14; 15

Allophycocyanin (APC) 630 660 26; (00)

AlphaRedTM 490 670 on request*) (16)

AMCA / AMCA-X 345 425 02; (01) 40

7-Amino-4-Methyl cumarin 354 441 01; 02

7-Amino-Actinomycin D(7-AAD) 555 655 00; 14; 15

Amino Coumarin 346 442 01; 02

6-Amino-Quinolin 360 443 01; 02

Aniline Blue 370 509 18

1,8-ANS 372 480 01; 02

APC (aliophycocyanin) 650 660 26

Atebrin 436 490 06

ATTO-TAGTM CBQCA 465 560 09

ATTO-TAGTM FQ 486 591 09; 16

Auramin 460 550 09; 10

Auraphospinmin 460 550 09; 10

BAO (Ruch) 380 460 01; 02; 34

BCECF 430/480 520 on request*)

4


BCECF (fluoresces 505 531 10; 17

at alkaline pH)

Berberine sulfate 430 550 06; 09

BFP(Blue Fluorescent Protein) 380 440 34

Bimane 398 490 05; 18; 34

BOBO-1 462 481 05; 06; 37

BOBO-3 570 602 00; 14; 15

BODIPY®-FL 503 512 09; 10; (16; 17) 40

BODIPY® FL-Ceramide 505 511 09; 10; (16; 17)

BODIY®-FL conjugate 504 511 09; 10; (16; 17)

BODIPY® 493/503 493 503 09; 10; (16; 17)

BODIPY® 530/550 530 550 (14; 15)

BODIPY® 558/568 558 568 20, 14, 15

BODIPY® 564/570 564 570 20, 14, 15

BODIPY 581/591 Phalloidin 584 592 31; (00)

BODIPY® 581/591 582 590 00

BODIPY® 630/650X 630 650 26

BODIPY® 650/665X 650 665 26; (32)

BODIPY® phosphatidylcholine(phospholipase) 503 512 09; 10; (16; 17)

BODIY® 630 625 642 26

BODIY® R6G SE 528 547 - - -

BODIY® TMR-X conjugate 544 573 20; 31

BODIY® TMR-X, SE 544 570 20; 31

BODIY® TR-X SE 588 616 00

BO-PRO-1 462 481 13; 37

BO-PRO-3 544 570 20; 31

BTC 401/464 529 05

Calcein 495 500-550 38; (09; 10)

Calcein blue 375 420-450 34; (01; 02) 28

Calcium CrimsonTM 588 611 00; 14; 15

Calcium GreenTM 501 53109; 10;

14; 15; (16; 17)

Calcium OrangeTM 506 531 14; 15; (20)

Calcofluor white 440 500-520 05; 37; (06)

5-Carboxyfluorescein (5FAM) 492 518 09; 10; 16; 17

5-Carboxynapthofluorescein(pH 10) 598 668 26

6-Carboxyrhodamine 6G 525 555 - - -

5-Carboxytetramethylrhodamine(5TAMRA) 542 568 14; 15; 20

Carboxy-X-rhodamine (5-ROX) 574 602 00; 31

Cascade blue® 376,399 423 02; (18)

Cascade Yellow® 399 552 05; 18

Catecholamine 410 470 05; 18 28

CBQCA 485 530 16; 17; 38 27; 28

CCF2 (GeneBLAzerTM) 402 520 05; 18

CFDA 494 520 16; 17; 38 27; 28

CFP - Cyan Fluorescent Protein 430 476 05; 18; (36)

Chinacrin 450-490 515 06; 37

5


Chlorotetracyclin 05; 09

Chromomycin/Mitramycin 436-460 470 05

CL-NERF (low pH) 504 540 10

CMFDA 494 520 16; 17; 38 27; 28

Coriphosphin 460 575 09

Coumarin phallacidin 355 443 01; 02

CPM 385 471 01; 02

C-phycocyanin 620 648 26

6-CR 6G 518 543 - - -

CTC 5-cyano-2,3-ditolyl-602 05; (18)

Tetrazolium-Chlorid

CTC Formazan 450 630 (05; 06)

Cyanine Cy2TM 489 505 09; 10; (16; 17) 40

Cyanine Cy3TM 550 570 15; 20; (14; 31;) 40

Cyanine Cy 3.5TM 580 598 00; 31

Cyanine Cy5TM 649 670 26

Cyanine Cy5.5TM 675 697 32

Cyanine Cy7TM 743 767 - - -

DABCSH 449 494 37; 38

Dansylamid 340 578 01; 02

Dansyl cadaverine 335 518 01; 02

Dansylchlorid 380 475 01; 02; 18; 34

DAO 01; 02

DAPI + DNA 359 461 01; 02 28 25; 40

Dapoxyl 373 574 01

DASPMI 05

DCM 481 644 09; 16

DDAO galactoside(ß-galactosidase) 633 655 26

DDAO GlcU(ß-glucuronidase) 633 655 26

DiA (4-Di-16-ASP) 491 613 09; 16

DiBAC4 Dibutylbarbitursäure Trimethinoxonol 439 516 06; 37

DiD (DilC18(5)) 644 665 26

DIDS Thiocyanato-stilbene 342 418 01; 02

Di-4 ANEPPS 488 605 09; 16

Di-8 ANEPPS 498 713 09; 16

DiIC3/4/12/22 Indocarbocyanine 540-560 556-575 14; 15; (00; 26)

DiI (DilC18(3)) 549 565 14; 15; 20

DiI 551 565 20; (15) 25

DiO (DiOC18(3)) 484 501 17, 09, 16, 10

DiR (DilC18(7)) 750 779 - - -

Dimethylacridinone (DDAO) phosphate (alkaline phosphatase) 633 655 26

DiO 487 501 16; 17; 38 27; 28

DiOC2/7/16 Carbocyanine 550 580 15; 20; (00; 14)

DiSC1/2/3/6 Thiacarbocyanine 559 585 14; 15; (26)

DTAF 494 520 16; 17; 38 27; 28

6


Diamino-naphtyl sulfonic acic 340 525 01; 02;

DM-NERF (high pH) 510 536 (17)

Dopamin 340 490-520 05

DPH 350 452 01; 02;

DsRed (Red Fluorescent Protein) 558 583 15; 20 23

DTAF 495 528 09; 10; 38

ECF substrate(alkaline phosphatase) 440 560 06

EGFP - Enhanced Green Fluorescent Protein 498 516 09; 10; 16; 17; 38 27; 28 40

ELF®-97 alcohol 345 530 02; 01

EnzChek DNase Kit(DNase I) 494 520 09; 10; 16; 17; 38 27; 28

EnzChek Protease Kit(Proteases) 503 512 09; 10;

EnzChek RNase Kit(RNase A) 497 513 09; 10; 16; 17; 38 27; 28

Eosin 517 537 - - -

Erythrosin 529 554 - - -

Ethidiumbromid + DNA 510 595 14; 15

Ethidium homodimer 528 616 14

Euchrysin 430 540 05; 06; 36

Evans blue 550 611 14; 15

EYFP - Enhanced YellowFluorescent Protein 520 532 - - -

5-FAM (5-Carboxyfluorescein) 492 518 09; 10; 16; 17; 38 27; 28

FAMTM 495 535 09; 10; 16; 17; 38 27; 28

Fastblue 365 420 18; (02; 05)

FAST CAT® Green (chloramphenicol 503 512 09; 10; (16; 17)acetyl transferase)

FDA 494 520 09; 10; 16; 17; 38 27; 28

Feulgen 480 560 14; 15

FIF - Falk 405 425 18 28

Fluo-3 480 520 09; 10; 38

Fluo-4 494 516 10; 38

Fluo 3(Ca2+ increases fluorescence) 506 526 09; 10;

Fluorescamin FLURAM 01; 02

Fluorescein (fluoresces at neutral and alkaline pH) 490 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluoresceindigalactopyranoside

(ß-galactosidase) 490 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluorescein diglucuronide (ß-glucuronidase) 490 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluorescein diphosphate(alkaline phosphatase) 490 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluorescein di-ß-D-glucopyrano-side (ß-glucosidase) 490 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluoresceindiacetat FDA 499 09

Fluoresceinisothiocyanat FITC 490 525 09; 10; 16; 17; 38 23; 24; 27 25; 40

Fluo-3 506 527 09; 17; (38)

7


Fluo-4 494 516 09; 10; 16; 17; 38 23; 24; 27; 28

Fluoro-Emerald 495 524 09; 10; 16; 17; 38 23; 24; 27; 28

Fluoro-GoldTM (low pH) 323 408 02; (01)

Fluoro-GoldTM (high pH) 350-395 530-600 on request*)

Fluoro-Jade 425 510 05; 18; 36

Fluor-Ruby 555 582 14; 15; 20

FluorXTM 494 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluorescein 494 520 09; 10; 16; 17; 38 23; 24; 27; 28

Fluorescein phalloidin 496 51609; 10;

(13; 16; 17)

FM® 1-43, 479 598 09; 16

Blue FluoSpheres 360 415 01; 02

Crimson FluoSpheres 625 645 00; 26

Red FluoSpheres 580 605 00; 14

Dark Red FluoSpheres 650 690 26; 32

Yellow-Green FluoSpheres 490 515 09; 10; 38

Fura RedTM (Ca2+ decreases fluorescence) 458 660 09

FURA-2 340/380 500/530 --- 21; 30

Gene BLAzerTM (CCF2) 402 520 05; 18

Generic blue/green combination 09; 10

GFP blue (Green Fluoresc. Protein) 382 448 34

GFP green 471 503 09; 10; 13; 16; 17

GFP Wild TypeUV excitation 396 508 05; 18;

GFP Wild Type,non UV excitation 470 508 10

GFP wt 450 450 510 37

7-Hydroxy-4-methylcoumarin (pH 9), 360 449 02; 01

Haematoporphyrin 530-560 580 14; 15; (31)

Hexidium iodide 518 600 09

Hoechst 33258 (Bisbenzimid) 365 480 01; 02

Hoechst 33342 (Bisbenzimid) 352 461 02 40

HEXTM 529 560 (14)

HNPP/Fast Red TR(alkaline phosphatase) 550 562 20

1,5 IAEDANS 336 482 02

ImaGene Green substrates 09; 10; 14; 15

ImaGene Red substrates 14; 15

Indo-1 330/360 410/480 on request*)

Indodicarbocyanine 644 665 ---

Indotricarbocyanine 739 765 ---

JC-1 498/593 525/595 16; 17

JOETM 525 557 (14)

JOJOTM-1 529 545 (14)

JOPROTM-1 530 546 (14)

Lambda Green 450-480 510-540 38

8


Lambda Red 664 600-650 664 26

Life/Dead Viability/Cytotoxity Kit(Calcein) 494 517 09; 10; 38

Life/Dead Viability/Cytotoxity Kit(Ethididium homodimer-1) 528 617 14; 15

Lissamin-Rodamin B 572 591 14; 15; 31

LIVE/DEAD®/EukoLightTM 485 520/620 09; 16 24

Lucifer Yellow 428 540 05

LysoSensorTM Blue (pH 5) 374 424 01

LysoSensor Green 442 505 37

LysoSensorTM Yellow/Blue(pH 3) 384 540 18

LysoTrackerTM Blue DND-22 373 422 01; 34

LysoTrackerTM Blue-White DPX 380 460-576 01; (34)

LysoTrackerTM Green DND 26 504 511 09; 10; (16; 17)

LysoTrackerTM Red DND 99 577 590 00; 14

LysoTrackerTM YellowHCK-123 466 536 37

LysoTrackerTM Yellow DND-68 534 551 14

Magnesium GreenMag Green 507 531 09; 10; (16; 17)

Magnesium Orange 550 575 14; 15

Mag-Fura-2 330 491 02; (21)

Mag-Inclo-1 330 417 02

Magdal red 540 570 14; 15

Marina BlueTM 365 460 02; 01

Mepacrin 05

Merocyanin 555 578 14; 15

MEQ, methoxyethylquinolin 344 442 02

4-Metylumbelliferon 360 450 01; 02

Methylgrün/Pyronin Stilben 01; 09

Mithramycin 420 575 05; 06; 18; (36)

MitoFluorTM Green 489 51709; 10; 13;

(16; 17)

MitoTracker Green 490 516 17; 09; 16; 10

MitoTracker Orange 551 576 14; 15; 20

MitoTracker Red 578 599 00

Monobromobimane 398 490 05; 18; 34

NanoOrange® 475 575 09; 16

NBD-Amine 460-485 534-542 09; 10

NBD - Chlorid 480 510-545 09; 10; 16; 17; 38

NBD phallacidin 465 535 06; 09

NBD phosphatidylcholine 460 534 37; 38

NBD-X 466 539 38

Nile Blue 690 624 660 26

Nile Red 515-560 590-640 14(Molecular Dynamics) 485 525 09; 14; 38

(Source: Chroma) 515-555 590(Source: Omega) 549 628(Source. Biorad) 559 640

Noradrenalin 340 490-520 05

9


Oli GreenTM 494 522 09; 10; 16; 17; 38 23; 24; 27; 28

Olivomycin 350-480 470-630 (05)

Oregon Green 514 511 530 09; 10; (16; 17)

Oregon GreenTM 488 496 524 09; 10; 16; 17

Oregon GreenTM 500 503 522 09; 10; (16; 17)

Oxytetracyclin 05; 09

Paciffic BlueTM 410 455 18

Pararosanilin-Feulgen 560 625 00; 14; 15

PBFI 340/380 420 21

Pep-TagTM

(kinases C and cAMP) 560 590 14; 15; 20; 31

Phosphin 465 565 09

C-Phycocyanin 605 645 00; 26

R-Phycocyanin 00

R-Phycoerythrin 480/550 578 09; 16

B-Phycoerythrin 546/565 575 20; (14; 15)

PicoGreen® 498 520 09; 10; (16; 17)

PKH 26 (Stigma) 551 567 20

POPOTM-1 434 456 02

POPROTM-1 iodide 434 456 05

POPOTM-3 534 570 14; 15

PO-PROTTM-3 iodide 539 567 14; 15

Primulin 410 550 05; 18 28

Propidiumjodid 536 617 14; (15)

PyMPO 415 570 05; 18

Pyren 343 380-400 01; (02)

Pyrolidin-Methyltetracyclin 05

Pyronin 490-580 530-610 (14; 15)

Pyrromethene 546 493 519 09; 10; (16; 17)

Pyrromethene 556 492 533 09; 10; (16; 17)

Pyrromethene 5567 517 546 09; 10; (16; 17)

QUIN 2 340/365 490 (01; 02)

Quinacrine Mustard 440 510 05; 06; 37

Red 613TM 480,565 613 auf Anfrage*)

Red 670TM 480,565 670 auf Anfrage*)

Red Fluorescent Protein (DsRed) 561 585 20; 31

Resorufin 571 585 00, 14; 15; 31

RH 414 500 635 09; 16

Rhod-110 497 520 09; 10; (16; 17)

Rhod-123 507 529 09; 10; (16; 17)

Rhod-2 552 576 14; 15; 20

Carboxyrhodamine (R6G) 519 543 09; 10

Rhodamin 540-560 580 14; 15

Rhodamine B 555 580 20; 31

Rhodamine 110 496 520 09; 10; 16; 17; 38 23; 24; 27; 28

Rhodamine 123 507 529 09; 10; (16; 17)

Rhodamine GreenTM 502 527 09; 10; (16; 17)

Rhodamine RedTM 570 590 31

Rhodamin Phalloidin 550 575 20; 14; 15; 31

10


Rhodamine phallacidin558 575 14; 15; 20542 565 14; 15; 20

5-ROX (carboxy-X-rhodamine) 574 602 31

Rhodol green 500 52509; 10;

(16; 17; 38)

RiboGreen® 500 520 09; 10; (16; 17)

R-Phycoerythrin 480,565 578 14; 15; 20

R-phycocyanin 618 642 26

Acid fuchsin 540 630 00; 14; 15

SBFI 340/380 420 21

SITS 337 436 02

Serotonin 365 520-540 01; 02

SNARF 480 600/650 auf Anfrage*)

SNAFL® (in acidic conditions; spectrum shifts to longer wave- 514 540 09; 10lengths in alkaline conditions)

SNAFL®-1 (high pH) 576 635 31

SNAFL®-2 525 546 (10; 14)

Spectrum Aqua® 433 480 05; 06; 18; 36

SodiumGreenTM 507 535 09; 10

SpectrumGreen® #1 497 524 05; 09; 10; 17 40

SpectrumGreen® #2 509 538 09; 10

SpectrumOrange® 559 588 14; 15; 20 40

SpectrumRed® 587 612 00

Stilben SITS, SITA 365 460 01; 02

Sulfaflavin 380-470 470-580 01; 02

SYBR® Gold 495 537 09; 10

SYBR® Green I 494 520 09; 10; (16; 17)

SYBR® Green II 497 513 09; 10; (16; 17)

SYPRO® Orange 472 570 09

SYPRO® Red 530 625 14

Syt011 508 527 09; 10

Syto® 13live-cell nucleic acid stain 488 509 16; 17

Syto® 16live-cell nucleic acid stain 488 518 16; 17

Syto® 17 621 634 26

SYTO® 40 416 445 02; 05; 34

SYTO® 45 452 484 06;

SYTO® 59 red fluorescentnucleic acid stain 640 660 26

SYTOX® Blue 445 470 05; 06; 18

SYTOX® Green 504 523 09; 10

SYTOX® Orange 547 570 14; 15; 20

5-TAMRA(5-Carboxy-tetramethlrhodamine) 542 568 14; 15; 20

TAMRATM 555 580 31

TETTM 519 545 09; 10; 16; 17

Tetracyclin 390 560 05; 18

Tetrametylrhodamin (TMR) 555 576 14; 15; 20

11


Texas red 595 620 00 24 25

Thiadicarbocyanine 651 671 26

Thiazin red 510 580 14

Thioflavin S 430 550 05; 06

TMA-DPH 355 430 01; 02

TO-PRO-1® iodide 515 531 09; 10; 16; 17

TO-PRO-3® iodide 642 661 26

TO-PRO-5® iodide 748 768 - - -

TOTO-1® + DNA 509 533 09; 10

TOTO-3® + DNA 642 661 26

TRITC(TetramethylRhodamine 540 580 14; 15; 20 23 28; 40

IsoThioCyanate)

WW 781 605 639 (26)

X-Rhodamine (XRITC) 580 605 00

XRITC 560 620 00; 14; 15 23

Vistra Green® 490 520 09; 10; (16; 17)

YFP (Yellow Fluorescent Protein) 520 532 09; 10; 16; 17

YO-PROTM-1 iodide 491 509 09; 10; (16; 17)

YO-PROTM-3 iodide 613 629 (26; 31)

YOYO-1® 491 509 09; 10; (16; 17)

YOYO-3® 642 660 26

Xylenolorange 377 610 14; 15

Filter set 40 contains a triple band-

pass filter set for synchronous triple

fluorescence. The fluorochromes

listed in the table for this filter set

are only some examples of a possible

selection.

Please bear in mind that the specified

spectral data are intended as

a guide and do not provide any

information about the full characte-

ristics of the absorption and fluores-

cence spectrum (secondary maxima,

shouldering, tailing of spectra).

This means that dyes can be excited

and observed outside the maximum

without overly high losses in intensity.

In the event of doubt, the absorption

and fluorescence spectra should

be used.

The spectra of the fluorochromes are

assessed by the manufacturer in

liquid solutions under defined

conditions. However, dyes are used

in inhomogeneous media (cells).

Therefore, it must be assumed that

the spectral characteristics may

be changed by the molecular and ion

interaction within this environment

(example: pH or Ca2+-sensitive dyes).

12

Multiple fluorescence exposures

using various filter sets (A, B) require

optimum congruence of the object

image during the different exposures

in order to avoid pixel shift. As is

often the case, the limits are set

by technology. The different wedge

angles of the emission filters

(AEM, BEM) and of the beam splitters

in the filter combinations required

for fluorescence applications cause

a slight pixel shift. Due to the tight

tolerances of our filters, the pixel

shift of filter sets from Carl Zeiss is

particularly small, i.e. these sets are

ideal for fluorescence applications.

Furthermore, expert integration

(in the factory or by service staff)

optimizes the filters for requests at

the very limits of technology. The

emission filters are aligned as shown

in figures 1 and 2.

3. Minimization of pixel shift in multiple fluorescenceexposures using fluorescence filters from Carl Zeiss

Fig. 1 Emission filters AEm and BEm which are not optimally aligned to each other The light rays a1 and b1 strike the emission filters AEm and BEm of the corresponding filter sets A and B.On account of the existing wedge angle of the filters, the ray is deflected in more or less oppositedirections, depending on the position of integration (a2 and b2 – are shown in an exaggerated form to make the matter clearer).Therefore, the pixels striking the image plane E do not exactly lie one on the other, but are displacedrelative to each other by the pixel shift PAPB .

13

AEm Emission filter of filter set A

BEm Emission filter of filter set B

a1 Light ray striking AEmb1 Light ray striking BEma2 Light ray deflected by AEmb2 Light ray deflected by BEmαA Angle between the incident light ray a1 and the deflected light ray a2 of filter AEMαB Angle between the incident light ray b1 and the deflected light ray b2 of filter BEME Image plane

PAPB Distance (pixel shift) between pixels striking the image plane E

PAPB’ Distance (pixel shift) between pixels striking the image plane E after alignment of the emission filters AEM and BEM

Fig. 2 Emission filters AEm and BEm , optimally aligned to each other, with minimized pixel shiftAfter insertion of the emission filters AEm a and BEm (see Fig. 1), the incident light rays a1 and b1are deflected in the same direction (a2 and b2 ). This minimizes the already slight pixel shift, or evencompensates for it in the ideal case (Pixel shift PAPB’).

14

4. Spectral data of filter sets

Filterset 00488000 - 0000

T [%]

excitation beamsplitter emission λ [nm]

Filterset 01488001 - 0000

T [%]


15

Filterset 02488002 - 0000

T [%]


Filterset 05488005 - 0000

T [%]


16

Filterset 06488006 - 0000

T [%]


Filterset 09488009 - 0000

T [%]


BP 450-490

17

Filterset 10488010 - 0000

T [%]


Filterset 13488013 - 0000

T [%]


18

Filterset 14488014 - 0000

T [%]


Filterset 15488015 - 0000

T [%]


19

Filterset 16488016 - 0000

T [%]


Filterset 17488017 - 0000

T [%]


20

Filterset 18488018 - 0000

T [%]


Filterset 20488020 - 0000

T [%]


21

Filterset 21488021 - 0000

T [%]


Filterset 23488023 - 0000

T [%]


22

Filterset 24488024 - 0000

T [%]


Filterset 25488025 - 0000

T [%]


23

Filterset 26488026 - 0000

T [%]


Filterset 27488027 - 0000

T [%]


BP 575-625

24

Filterset 28488028 - 0000

T [%]


Filterset 311031 - 350

T [%]


25


T [%]



T [%]


26


T [%]



T [%]


27


T [%]


Filterset 40488040 - 0000

T [%]

excitation I excitation II excitation III λ [nm]beamsplitter emission

excitation diameter 18 mm

28

Filterset 401021 - 6000

T [%]

excitation I excitation II excitation III λ [nm]beamsplitter emission

excitation diameter 25 mm

30Subject to change. micro special 40-627 e/4.00

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Carl Zeiss MicroscopyD-07740 JenaPhone: ++49-3641/64-16 16Telefax: ++49-3641/64-31 44E-mail: [email protected]: www.zeiss.de/micro

Documents

Fluorochromes and Filters for Fluorescence Microscopyaix1.uottawa.ca/~sgee/protocols/Fluorochromes and Filters...1 1. Current filter sets from Carl Zeiss Filter set Catalogue number