148_12_AKR Fluorochromes Poster02:Layout 1 23/04/2012 15:10 Page 1
A Guide to
Fluorochromes
Laser line (488)
Excitation
Emission PE
350
400
Single
(PE)
450
500
550
600
650
700
750
800
Relative Intensity
Wavelength (nanometers)
Laser line (633)
Excitation
Emission Cy5®
350
400
Single
(Cy5®)
450
500
550
600
650
700
750
800
Relative Intensity
Wavelength (nanometers)
Laser line (488)
Excitation
Emission PE-Cy5®
350
400
Tandem
(PE-Cy5®)
450
500
550
600
650
700
750
800
Wavelength (nanometers)
Fluorochromes have unique and characteristic spectra for absorption (excitation) and emission. A single dye
is excited at a particular wavelength and emits a photon at a higher wavelength. A tandem dye consists of a
donor and acceptor fluorochrome molecule, placed in close proximity, allowing for energy transfer between
the two. The tandem dye is excited at the excitation wavelength of the donor molecule and emits a photon at
the emission wavelength of the acceptor molecule.
Energy Level Diagrams
Single dye
Es
2
Excitation
Laser
1
Fluorescence
(Emission)
3
Gs
Tandem dye
Es
D
Excitation
Laser
1
2
3
A
4
Fluorescence
(Emission)
Gs
For a single fluorescent dye, a laser set at the signature excitation wavelength for the dye provides
1
electromagnetic energy to an electron in that molecule (1).
The electron moves to an excitation state at the
2
next energy level (Es) (2).
Energy is then released in the form of a photon (fluorescence) and the electron
3
moves back down to the lower energy level (Gs) (3).
In the case of a tandem fluorescent dye, after excitation
2
D and absorbed
of the electron by a laser (11 - 2),
energy is released by an electron in the donor molecule (D)
A
3
by an electron in the acceptor molecule (A) (4). The electron in the acceptor molecule moves to an excitation
state at the next energy level (Es). Similar to a single dye, energy is then released in the form of the photon
4
(fluorescence) and the electron moves back down to the lower energy level (Gs) (5).
ab16051 - Beta Catenin
DRAQ5
ab7291 - Alpha Tubulin
Methoxycoumarin
DyLight® 405
Alexa Fluor® 405
Brilliant Violet 421™
HiLyte FluorTM 405
DyLight® 350
Alexa Fluor® 350
Aminocoumarin (AMCA)
BD Horizon™ V450
Pacific BlueTM
EviTagTM quantum dots-Lake Placid Blue
AMCyan
BD Horizon™ V500
Cy2®
ChromeoTM 488
DyLight® 488
Alexa Fluor® 488
FAM
Fluorescein Iso-thiocyanate (FITC)
EviTagTM quantum dots-Adirondack Green
ChromeoTM 505
HiLyte FluorTM 488
Alexa Fluor® 514
EviTagTM quantum dots-Catskill Green
Alexa Fluor® 430
Pacific Orange™
Alexa Fluor® 532
HEX
EviTagTM quantum dots-Hops Yellow
ChromeoTM 546
Cy3®
Alexa Fluor® 555
HiLyte FluorTM 555
5-TAMRA
Alexa Fluor® 546
DyLight® 550
Phycoerythrin (PE)
Tetramethyl Rhodamine Isothiocyanate (TRITC)
EviTagTM quantum dots-Birch Yellow
Cy3.5®
Rhodamine Red-X
PE-Dyomics® 590
EviTagTM quantum dots-Fort Orange
ROX
Alexa Fluor® 568
Red 613
Texas Red®
HiLyte FluorTM 594
PE-Texas Red®
Alexa Fluor® 594
DyLight® 594
EviTagTM quantum dots-Maple-Red Orange
Alexa Fluor® 610
ChromeoTM 494
Alexa Fluor® 633
SureLight® APC
DyLight® 633
Allophycocyanin (APC)
ChromeoTM 642
Quantum Red
SureLight® P3
Alexa Fluor® 647
Cy5®
PE-Cy5®
SureLight® P1
PE-Alexa Fluor® 647
PE-Dyomics® 647
DyLight® 650
HiLyte FluorTM 647
Peridinin Chlorophyll (PerCP)
IRDye® 700DX
Alexa Fluor® 660
PE-Cy5.5®
APC-Cy5.5®
Cy5.5®
TruRed
HiLyte FluorTM 680
Alexa Fluor® 680
DyLight® 680
Alexa Fluor® 700
APC-Cy7®
Alexa Fluor® 750
Cy7®
PE-Dyomics® 747
DyLight® 755
HiLyte FluorTM 750
PE-Cy7®
IRDye® 800RS
DyLight® 800
IRDye® 800CW
Alexa Fluor® 790
Nucleic Acid Dyes
Multicolor staining example
MERGED
Dye
DRAQ5™ (ab108410) allows rapid staining of
dsDNA/nuclei of LIVE or FIXED cells. It is
ideal for use in multi-color analysis and
compatible with GFP labels. As well as
offering half the scan times of Hoechst or
DAPI, DRAQ5™ can be used in a greater
variety of applications. The image on the left
shows HeLa cells stained with 5μM DRAQ5™
to label nuclear DNA (pseudocolor orange).
Pre-absorbed goat polyclonal secondary
antibodies to rabbit IgG - H&L were used to
visualise beta Catenin (DyLight® 488,
ab96899, green) and alpha Tubulin (DyLight®
594, ab96899, red).
DAPI
Hoechst 33258
Hoechst 33342
SYTOX Blue
YOYO-1
SYTOX Green
TOTO-1, TO-PRO-1
Mithramycin
SYTOX Orange
Chromomycin A3
CyTRAK Orange™*
Ethidium Bromide
Propidium iodide (PI)
DRAQ5™
DRAQ7™
Max. excitation
wavelength (nm)
Max.emission
wavelength (nm)
360
400
402
407
404
353
346
350
404
404
470
457
415
489
488
493
495
494
495
505
505
501
518
525
434
403
532
535
545
545
548
555
550
541
556
562
496,566
557
560
576
570
488
585
575
578
480,565
595
593
566
590
593
600
612
494
632
652
638
650
642
488
614
650
647
565
545
567
488
654
650
477
680
663
565
650
675
490,675
678
679
692
702
650
749
753
488
754
753
566
770
777
778
782
410
420
421
421
428
432
442
445
448
456
490
491
500
506
517
518
519
519
519
520
526
527
540
540
541
551
554
556
560
561
561
565
566
568
573
576
576
576
580
589
590
599
600
602
603
613
613
616
616
617
618
620
628
628
647
657
658
660
660
660
662
665
665
666
666
669
672
673
675
678
687
690
693
694
694
695
699
702
712
723
774
775
775
776
776
778
778
786
794
794
805
Max. excitation
wavelength (nm)
Max. emission
wavelength (nm)
Excitation laser
lines (nm)
359
352
350
431
491
504
509
450
547
445
457,488,549
493
305,540
646
599, 644
461
461
461
480
509
523
533
570
570
575
615
620
620
681,697
678,697
325,360,405,407
*CyTRAK Orange™ stains both nucleus and cytoplasm
Discover more at abcam.com/fluorochromes
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information/detail is correct at time of going to print. Color bars are an approximation of corresponding emission wavelength.
Dylight® is a trademark of Thermo Fisher Scientific and its subsidiaries. Cy® and CyDye® are trademarks of GE Healthcare Limited. Alexa Fluor® is a trademark of Molecular Probes Inc. HILYTE FLUOR™ is a trademark of AnaSpec, Inc. DRAQ5™, DRAQ7™ and CyTRAK Orange™are trademarks of Biostatus Ltd.
Excitation laser
lines (nm)
360,405,407
488
488
488
488,514
532
488
568,543
532
568,543,514
595,633,635,647
595,633,635,647
633,635
488
488
488
595,633,635,647
647
595,633,635,647
488
325,360,488
148_12_AKR
Relative Intensity
Excitation and Emission Spectral Profiles