Fluorescent Dyes

An Overview

Fluorescent Dyes

A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescent agent. This can be a fluorescent protein – for example GFP – genetically linked to the protein of interest. If cloning is impossible – for instance in histologic samples – techniques such as immunofluorescence staining are used to visualize the protein of interest. For this purpose, antibodies are utilized, which are linked to distinct fluorescent dyes and bind to the adequate target structure either directly or indirectly. With the help of fluorescent dyes, fluorescence microscopy is not only restricted to proteins but can also be used to detect nucleic acids, glycans and other structures. You can even use an application-specific variety of live cell dyes available that allow for organelle visualization with organelle-selective stains (e.g. ER, mitochondria, Golgi) or function assays like, e.g. live cell tracking, labeling, cell proliferation, or live dead assays, where fluorescence is the way of read-out. Even non-biological substances like Calcium ions can be detected. This article provides an introduction to the commonly used fluorescent agents.

Immunofluorescence

In fluorescence microscopy there are two ways to visualize your protein of interest. Either with the help of an intrinsic fluorescent signal - by genetically linking a fluorescent protein to a target protein - or with the help of fluorescently labeled antibodies that bind specifically to a protein of interest. For some biological questions it is more useful or even necessary to perform the latter one. In the case of histological samples, for example, it is not possible to use fluorescent proteins because in general the specimen is derived from an organism which does not hold any fluorescent proteins. Furthermore, if a functioning antibody is available, immunofluorescence is much faster than fluorescent protein techniques, where you have to clone the gene of interest and transfect DNA into the adequate cell. Another disadvantage of fluorescent proteins lies in their nature of being a protein themselves. With it, they have specific proteinous characteristics inside a cell which can lead to dysfunction or misinterpretations concerning the attached protein of interest. However, it should be considered that using fluorescent proteins is generally the method of choice to study living cells.

Immunofluorescence makes use of the very specific binding affinity of an antibody to its antigen. This can have two different appearances. The easiest way is to use one fluorescently labeled antibody which is binding to the protein of interest. This is called direct immunofluorescence.

In most cases there are two forms of antibodies used. The first one binds to the target protein and is not fluorescently labeled itself (primary antibody). But a second one (secondary antibody) which is binding the primary antibody specifically carries a fluorescent dye. This method is called indirect immunofluorescence and has several advantages. On the one hand there is an amplifying effect, because more than one secondary antibody binds to one primary antibody. On the other hand, it is generally easier to find secondary antibodies than the specific primary antibody labeled with a common fluorescent dye. Below, we review the most commonly used fluorescent dyes.

FITC and TRITC

Fluorescein isothiocyanate (FITC) is an organic fluorescent dye and probably one of the most commonly used in immunofluorescence and flow cytometry. It has an excitation/emission peak at 495/517 nm and can be coupled to distinct antibodies with the help of its reactive isothiocyanate group, which is binding to amino, sulfhydryl, imidazoyl, tyrosyl or carbonyl groups on proteins. FITC (was one of the first dyes which was used for fluorescence microscopy and served as a precursor for other fluorescent dyes like Alexa Fluor®488. Its fluorescence activity is due to its large conjugated aromatic electron system, which is excited by light in the blue spectrum.

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Fig. 1: Drosophila embryo development, Green: FITC, Red: TRITC.

A dye very often used in combination with FITC is TRITC (Tetramethylrhodamine-5-(and 6)-isothiocyanate). In contrast to FITC, TRITC is not a fluorescein but a derivate of the Rhodamine family. Rhodamines also have a large conjugated aromatic electron system, what leads to their fluorescent behavior. TRITC is excited with light in the green spectrum with a maximum at 550 nm. Its emission maximum is lying at 573 nm. The bond to proteins (e.g. antibodies) is also based on a reactive isothiocyanate group.

Even though FITC and TRITC are still widely used, they are rather weak fluorescent dyes and not recommended for state-of-the-art microscopy.

Cyanines

Alexa Fluor® dyes are a big group of negatively charged and hydrophilic fluorescent dyes, frequently used in fluorescence microscopy. All the Alexa Fluor® dyes are sulfonated forms of different basic fluorescent substances like fluorescein, coumarin, cyanine or rhodamine (e.g. Alexa Fluor®546, Alexa Fluor®633). The respective laser excitation wavelength is mentioned in their labeling. For example, Alexa Fluor®488, one of the most commonly used dyes, has an excitation maximum at 493 nm, which allows excitation with a standard 488 nm laser, and an emission maximum at 519 nm Alexa Fluor®488 is a fluorescein derivate and has similar properties than FITC. However, it shows better stability, brightness and lower pH sensitivity.

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Fig. 2: Mouse transgenic embryo, interlimb somites, Five interlimb somites of an E10.5 mouse transgenic embryo: EpaxialMyf5 eGFP; immuno-stained for GFP-Alexa 488; embryonic muscle fibers stained with Desmin-Cy3, the nuclei are revealed with Hoechst Size from top to bottom: 3.5 mm (a), 800 µm (b). Courtesy of: Aurélie Jory, Cellules Souches et Développement, Institut Pasteur, Paris, France and Imaging centre of IGBMC, IGBMC

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Fig. 3: Mouse fibroblasts, Green: F-Actin, FITC, Red: Tubulin, Cy5, Blue: Nuclei, DAPI. Courtesy of: Dr. Günter Giese, Max Planck Institute for Medical Research, Heidelberg, Germany.

DNA staining

You might want to study nucleic acids using fluorescent microscopy. For example, to define the exact position or number of cells by the detection of their nucleus. One of the most common DNA stains is DAPI (4',6-diamidino-2-phenylindole) which binds to A-T rich regions of the DNA double helix. DAPI fluorescence intensity increases if attached to DNA compared to its unbound state. It is excited by UV-light with a maximum at 358 nm. Emission spectrum is broad and peaks at 461 nm. A weak fluorescence can also be detected for RNA binding. In this case, emission shifts to 500 nm. Interestingly, DAPI is able to permeate an intact plasma membrane which makes it useful for fixed and living cells.

A second broadly used DNA stain option is the family of Hoechst dyes. Hoechst 33258, Hoechst 33342, and Hoechst 34580 are Bisbenzimides with intercalating tendency to A-T rich areas. Similar to DAPI they are excited by UV-light and have an emission maximum at 455 nm which is shifted to 510–540 nm in an unbound condition. Hoechst stains are also cell permeable and can therefore be used in fixed and living cells. They exhibit lower toxicity than DAPI.

A membrane-impermeable DNA stain is Propidium-Iodide which is often used to differentiate between living and dead cells in a cell culture because it cannot enter an intact cell. Propidium-Iodide is also an intercalating agent but with no binding preference for distinct bases. In the nucleic acid bound state, its excitation maximum is at 538 nm. Highest emission is at 617 nm. Unbound Propidium-Iodide excitation and emission maxima are shifted to lower wavelengths and lower intensity. It can also bind to RNA without changing its fluorescent characteristics. To distinguish DNA from RNA it is necessary to use adequate nucleases.

A dye which is capable to make a difference between DNA and RNA without previous manipulation is Acridine Orange. Its excitation/emission maximum pair is 502 nm/525 nm in the DNA bound version and turns to 460 nm/650 nm in the RNA bound state. Furthermore, it can enter acidic compartments like lysosomes where the cationic dye is protonated. In this acidic surrounding Acridine Orange is excited by light in the blue spectrum, whereas emission is strongest in the orange region. It is often used to identify apoptotic cells, as they have a lot of engulfed acidic compartments

Compartment and organelle specific dyes

There is a number of specific dyes to study cell compartments such as lysosomes, endosomes or organelles such as mitochondria.

One well known way to observe mitochondria is the utilization of MitoTracker®. This is a cell permeable dye with a mildly thiol-reactive chloromethyl moiety used to bind to matrix proteins covalently by reacting with free thiol groups of cysteine residues. MitoTracker® exists in different colours and modifications . In contrast to conventional mitochondria specific stains like rhodamine 123 or tetramethylrosamine, MitoTracker® is not washed out after destruction of the membrane potential with fixatives.

LysoTracker is a group of dyes available in different colors used to stain acidic compartments such as lysosomes. These are membrane permeable weak bases linked to a fluorophore. Most probably these bases have an affinity to acidic compartments because of protonation.

A comparable compartment to lysosomes is the vacuole in fungi like Saccharomyces cerevisiae. This membrane enclosed space is also of an acidic nature. One way to visualize it in fluorescence microscopy is the use of styryl-based dyes like FM 4-64® or FM 5-95®.

The Endoplasmic Reticulum (ER) is usually stained when studying protein secretion. One classical dye to stain this compartment is DiOC6(3) which has a preference for the ER but still binds to other membranes like those of mitochondria. Another way to specifically stain the ER is to use ER-Trackers like ER-Tracker Green and Red. Both are BODIPY based dyes which are linked to glibenclamide – a sulfonylurease – which binds to ATP sensitive Potassium channels exclusively resident in the ER membrane. BODIPY (boron-dipyrromethene) describes a group of relatively pH insensitive dyes which are almost all water insoluble. This makes them a very good tool for lipid and membrane labeling.

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Fig. 4: Purkinje cell, triple labeled parasagittal section of mouse cerebellar cortex. Red: anti-calbindin-D28k/Cy3, Green: anti-GFAP/Cy5, Blue: Hoechst 33258.

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Fig. 5: Bovine Pulmonary Endothelial cells. Red: Mitochondria labeled MitoTracker® Red CMXRos, Green: F-actin labeled with green-fluorescent BODIPY® FL phallacidin, Blue: DAPI labeled nuclei. This image was enhanced using 3D blind deconvolution.

The adjacent compartment to the ER – the Golgi apparatus – can be labeled with fluorescent ceramide analogs like NBD C6-ceramide and BODIPY FL C5-ceramide. Ceramides are Sphingolipids which are highly enriched in the Golgi apparatus.

With the help of further lipid-based dyes it is possible to stain special membrane regions like lipid-rafts. These cholesterol rich domains can be visualized by using NBD-6 Cholestrol or NBP-12 Cholesterol amongst others Avanti Polar Lipids.

It is also possible to stain the area of interest with the help of proteins with preferences for distinct locations in the cell. One example is the use of wheat germ agglutinin (WGA) which binds specifically to sialic acid and N-acetylglucosaminyl present in the plasma membrane.

Ion imaging

In the case of neuronal studies, gene activity or cellular movement it is of interest to study the ion concentration of the cell. Sodium, calcium, chloride or magnesium ions have a deep impact on many different cellular events. Typically, ions can be trapped with the help of fluorescently labeled chelators that change their spectral properties when bound to the appropriate ions. One example of labelled chelators are the calcium indicators fura-2, indo-1, fluo-3, fluo-4 and Calcium-Green. For sodium detection, SBFI (sodium-binding benzofurzanisophthalate) or Sodium Green are commonly used. PBFI (potassium-binding benzofurzanisophthalate) detects potassium ions.

Functional assays

“Functional assays” is the broad term used to cover standardized experiments to assess various functions that can be visualized with fluorescent markers. These markers can encompass but is not limited to any of the above-mentioned labeling techniques and fluorophores. For many of these functional assays, staining kits are commercially available and can easily be applied to a vast variety of samples. One example for a functional assay is the commonly known and widely used live dead assay. Two fluorophores are utilized to label live cells and dead cells. Having both values at hand the overall health status of the cells can be assessed. Correlating this information with additional markers may even increase the understanding of the underly process.

Fluorescent dyes and their excitation and emission wavelength peaks

The table below shows a comprehensive list of fluorescent dyes with their respective excitation and emission wavelength peaks. Please note that besides those peaks, every dye features distinct excitation and emission spectra. When selecting several dyes to use in combination in one experiment, researchers should be aware of overlapping excitation and emission spectra due to crosstalk (or bleedthrough), which can result in false negatives or positives, or otherwise obscure data. Another source that can distort fluorescence imaging is autofluorescence by naturally occurring fluorescent proteins in cells and tissues, which particularly needs to be considered in experiments with plants or algae. Good understanding of the spectra of the dyes used in the sample is also important to choose the right light source for excitation (e.g. LED, arc lamps, laser lines) and the right filters and detectors for emission.

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Fig. 6: Fluorescence emission profile of Alexa 488 (green curve) and Alexa 555 (yellow curve). The overlap of the two emissions spectra is plain to see. The red line indicates the band pass of a 488 emissions filter.

Table 1

Sample Fluorescent Dyes
Excitation
Emission
Indo-1, Ca saturated
331 nm
404 nm
Indo-1 Ca2+
346 nm
404 nm
Cascade Blue BSA pH 7.0
401 nm
419 nm
Cascade Blue
398 nm
420 nm
LysoTracker Blue
373 nm
421 nm
Alexa 405
401 nm
421 nm
LysoSensor Blue pH 5.0
374 nm
424 nm
LysoSensor Blue
374 nm
424 nm
DyLight 405
399 nm
434 nm
DyLight 350
332 nm
435 nm
BFP (Blue Fluorescent Protein)
380 nm
439 nm
Alexa 350
343 nm
441 nm
7-Amino-4-methylcoumarin pH 7.0
346 nm
442 nm
Amino Coumarin
345 nm
442 nm
AMCA conjugate
347 nm
444 nm
Coumarin
360 nm
447 nm
7-Hydroxy-4-methylcoumarin
360 nm
447 nm
7-Hydroxy-4-methylcoumarin pH 9.0
361 nm
448 nm
6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0
358 nm
450 nm
Hoechst 33342
352 nm
455 nm
Pacific Blue
404 nm
455 nm
Hoechst 33258
352 nm
455 nm
Hoechst 33258-DNA
352 nm
455 nm
Pacific Blue antibody conjugate pH 8.0
404 nm
455 nm
PO-PRO-1
434 nm
457 nm
PO-PRO-1-DNA
435 nm
457 nm
POPO-1
433 nm
457 nm
POPO-1-DNA
433 nm
458 nm
DAPI-DNA
359 nm
461 nm
DAPI
358 nm
463 nm
Marina Blue
362 nm
464 nm
SYTOX Blue-DNA
445 nm
470 nm
CFP (Cyan Fluorescent Protein)
434 nm
474 nm
eCFP (Enhanced Cyan Fluorescent Protein)
437 nm
476 nm
1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS)
375 nm
479 nm
Indo-1, Ca free
346 nm
479 nm
1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)
375 nm
480 nm
BO-PRO-1-DNA
462 nm
482 nm
BOPRO-1
462 nm
482 nm
BOBO-1-DNA
461 nm
484 nm
SYTO 45-DNA
451 nm
486 nm
evoglow-Pp1
448 nm
495 nm
evoglow-Bs1
448 nm
496 nm
evoglow-Bs2
448 nm
496 nm
Auramine O
431 nm
501 nm
DiO
487 nm
501 nm
LysoSensor Green pH 5.0
447 nm
502 nm
Cy 2
489 nm
503 nm
LysoSensor Green
447 nm
504 nm
Fura-2, high Ca
336 nm
504 nm
Fura-2 Ca2+sup>
336 nm
505 nm
SYTO 13-DNA
488 nm
506 nm
YO-PRO-1-DNA
491 nm
507 nm
YOYO-1-DNA
491 nm
509 nm
eGFP (Enhanced Green Fluorescent Protein)
488 nm
509 nm
LysoTracker Green
503 nm
509 nm
GFP (S65T)
489 nm
509 nm
BODIPY FL, MeOH
502 nm
511 nm
Sapphire
396 nm
511 nm
BODIPY FL conjugate
503 nm
512 nm
MitoTracker Green
490 nm
512 nm
MitoTracker Green FM, MeOH
490 nm
512 nm
Fluorescein 0.1 M NaOH
493 nm
513 nm
Calcein pH 9.0
494 nm
514 nm
Fluorescein pH 9.0
490 nm
514 nm
Calcein
493 nm
514 nm
Fura-2, no Ca
367 nm
515 nm
Fluo-4
494 nm
516 nm
FDA
495 nm
517 nm
DTAF
495 nm
517 nm
Fluorescein
495 nm
517 nm
Fluorescein antibody conjugate pH 8.0
493 nm
517 nm
CFDA
495 nm
517 nm
FITC
495 nm
517 nm
Alexa Fluor 488 hydrazide-water
493 nm
518 nm
DyLight 488
493 nm
518 nm
5-FAM pH 9.0
492 nm
518 nm
FITC antibody conjugate pH 8.0
495 nm
519 nm
Alexa 488
493 nm
520 nm
Rhodamine 110
497 nm
520 nm
Rhodamine 110 pH 7.0
497 nm
520 nm
Acridine Orange
431 nm
520 nm
Alexa Fluor 488 antibody conjugate pH 8.0
499 nm
520 nm
BCECF pH 5.5
485 nm
521 nm
PicoGreendsDNA quantitation reagent
502 nm
522 nm
SYBR Green I
498 nm
522 nm
Rhodaminen Green pH 7.0
497 nm
523 nm
CyQUANT GR-DNA
502 nm
523 nm
NeuroTrace 500/525, green fluorescent Nissl stain-RNA
497 nm
524 nm
DansylCadaverine
335 nm
524 nm
Rhodol Green antibody conjugate pH 8.0
499 nm
524 nm
Fluoro-Emerald
495 nm
524 nm
Nissl
497 nm
524 nm
Fluorescein dextran pH 8.0
501 nm
524 nm
Rhodamine Green
497 nm
524 nm
5-(and-6)-Carboxy-2', 7'-dichlorofluorescein pH 9.0
504 nm
525 nm
DansylCadaverine, MeOH
335 nm
526 nm
eYFP (Enhanced Yellow Fluorescent Protein)
514 nm
526 nm
Oregon Green 488
498 nm
526 nm
Oregon Green 488 antibody conjugate pH 8.0
498 nm
526 nm
Fluo-3
506 nm
527 nm
BCECF pH 9.0
501 nm
527 nm
SBFI-Na+
336 nm
527 nm
Fluo-3 Ca2+
506 nm
527 nm
Rhodamine 123, MeOH
507 nm
529 nm
FlAsH
509 nm
529 nm
Calcium Green-1 Ca2+
506 nm
529 nm
Magnesium Green
507 nm
530 nm
DM-NERF pH 4.0
493 nm
530 nm
Calcium Green
506 nm
530 nm
Citrine
515 nm
530 nm
LysoSensor Yellow pH 9.0
335 nm
530 nm
TO-PRO-1-DNA
515 nm
531 nm
Magnesium Green Mg2+
507 nm
531 nm
Sodium Green Na+
507 nm
531 nm
TOTO-1-DNA
514 nm
531 nm
Oregon Green 514
512 nm
532 nm
Oregon Green 514 antibody conjugate pH 8.0
513 nm
533 nm
NBD-X
466 nm
534 nm
DM-NERF pH 7.0
509 nm
537 nm
NBD-X, MeOH
467 nm
538 nm
CI-NERF pH 6.0
513 nm
538 nm
Alexa 430
431 nm
540 nm
Alexa Fluor 430 antibody conjugate pH 7.2
431 nm
540 nm
CI-NERF pH 2.5
504 nm
541 nm
Lucifer Yellow, CH
428 nm
542 nm
LysoSensor Yellow pH 3.0
389 nm
542 nm
6-TET, SE pH 9.0
521 nm
542 nm
Eosin antibody conjugate pH 8.0
525 nm
546 nm
Eosin
524 nm
546 nm
6-Carboxyrhodamine 6G pH 7.0
526 nm
547 nm
6-Carboxyrhodamine 6G, hydrochloride
525 nm
547 nm
Bodipy R6G SE
528 nm
547 nm
BODIPY R6G, MeOH
528 nm
547 nm
6 JOE
520 nm
548 nm
Cascade Yellow antibody conjugate pH 8.0
399 nm
549 nm
Cascade Yellow
399 nm
549 nm
mBanana
540 nm
553 nm
Alexa Fluor 532 antibody conjugate pH 7.2
528 nm
553 nm
Alexa 532
528 nm
553 nm
Erythrosin-5-isothiocyanate pH 9.0
533 nm
554 nm
6-HEX, SE pH 9.0
534 nm
559 nm
mOrange
548 nm
562 nm
mHoneydew
478 nm
562 nm
Cy 3
549 nm
562 nm
Rhodamine B
543 nm
565 nm
DiI
551 nm
565 nm
5-TAMRA-MeOH
543 nm
567 nm
Alexa 555
553 nm
568 nm
Alexa Fluor 555 antibody conjugate pH 7.2
553 nm
568 nm
DyLight 549
555 nm
569 nm
BODIPY TMR-X, SE
544 nm
570 nm
BODIPY TMR-X, MeOH
544 nm
570 nm
PO-PRO-3-DNA
539 nm
571 nm
PO-PRO-3
539 nm
571 nm
Rhodamine
551 nm
573 nm
Bodipy TMR-X conjugate
544 nm
573 nm
POPO-3
533 nm
573 nm
Alexa 546
562 nm
573 nm
BODIPY TMR-X antibody conjugate pH 7.2
544 nm
573 nm
Calcium Orange Ca2+
549 nm
573 nm
TRITC
550 nm
573 nm
Calcium Orange
549 nm
574 nm
Rhodaminephalloidin pH 7.0
558 nm
575 nm
MitoTracker Orange
551 nm
575 nm
MitoTracker Orange, MeOH
551 nm
575 nm
Phycoerythrin
565 nm
575 nm
Magnesium Orange
550 nm
575 nm
R-Phycoerythrin pH 7.5
565 nm
576 nm
5-TAMRA pH 7.0
553 nm
576 nm
5-TAMRA
549 nm
577 nm
Rhod-2
552 nm
577 nm
FM 1-43
472 nm
578 nm
Rhod-2 Ca2+
553 nm
578 nm
Tetramethylrhodamine antibody conjugate pH 8.0
552 nm
578 nm
FM 1-43 lipid
473 nm
579 nm
LOLO-1-DNA
568 nm
580 nm
dTomato
554 nm
581 nm
DsRed
563 nm
581 nm
Dapoxyl (2-aminoethyl) sulfonamide
372 nm
582 nm
Tetramethylrhodamine dextran pH 7.0
555 nm
582 nm
Fluor-Ruby
554 nm
582 nm
Resorufin
571 nm
584 nm
Resorufin pH 9.0
571 nm
584 nm
mTangerine
568 nm
585 nm
LysoTracker Red
578 nm
589 nm
Lissaminerhodamine
572 nm
590 nm
Cy 3.5
578 nm
591 nm
Rhodamine Red-X antibody conjugate pH 8.0
573 nm
591 nm
Sulforhodamine 101, EtOH
578 nm
593 nm
JC-1 pH 8.2
593 nm
595 nm
JC-1
592 nm
595 nm
mStrawberry
575 nm
596 nm
MitoTracker Red
578 nm
599 nm
MitoTracker Red, MeOH
578 nm
599 nm
X-Rhod-1 Ca2+
580 nm
602 nm
Alexa Fluor 568 antibody conjugate pH 7.2
579 nm
603 nm
Alexa 568
576 nm
603 nm
5-ROX pH 7.0
578 nm
604 nm
5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt)
578 nm
604 nm
BO-PRO-3-DNA
574 nm
604 nm
BOPRO-3
574 nm
604 nm
BOBO-3-DNA
570 nm
605 nm
Ethidium Bromide
524 nm
605 nm
ReAsH
597 nm
608 nm
Calcium Crimson
589 nm
608 nm
Calcium Crimson Ca2+
590 nm
608 nm
mRFP
585 nm
608 nm
mCherry
587 nm
610 nm
Texas Red-X antibody conjugate pH 7.2
596 nm
613 nm
HcRed
590 nm
614 nm
DyLight 594
592 nm
616 nm
Ethidium homodimer-1-DNA
528 nm
617 nm
Ethidiumhomodimer
528 nm
617 nm
Propidium Iodide
538 nm
617 nm
SYPRO Ruby
467 nm
618 nm
Propidium Iodide-DNA
538 nm
619 nm
Alexa 594
590 nm
619 nm
BODIPY TR-X, SE
588 nm
621 nm
BODIPY TR-X, MeOH
588 nm
621 nm
BODIPY TR-X phallacidin pH 7.0
590 nm
621 nm
Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2
567 nm
627 nm
YO-PRO-3-DNA
613 nm
629 nm
Di-8 ANEPPS
469 nm
630 nm
Di-8-ANEPPS-lipid
469 nm
631 nm
YOYO-3-DNA
612 nm
631 nm
Nile Red-lipid
553 nm
636 nm
Nile Red
559 nm
637 nm
DyLight 633
624 nm
646 nm
mPlum
587 nm
649 nm
TO-PRO-3-DNA
642 nm
657 nm
DDAO pH 9.0
648 nm
657 nm
Fura Red, high Ca
434 nm
659 nm
Allophycocyanin pH 7.5
651 nm
660 nm
APC (allophycocyanin)
650 nm
660 nm
Nile Blue, EtOH
631 nm
660 nm
TOTO-3-DNA
642 nm
661 nm
Cy 5
646 nm
664 nm
BODIPY 650/665-X, MeOH
646 nm
664 nm
Alexa Fluor 647 R-phycoerythrin streptavidin pH 7.2
569 nm
666 nm
DyLight 649
652 nm
668 nm
Alexa Fluor 647 antibody conjugate pH 7.2
653 nm
668 nm
Alexa 647
653 nm
669 nm
Fura Red Ca2+
435 nm
670 nm
Atto 647
644 nm
670 nm
Fura Red, low Ca
472 nm
673 nm
Carboxynaphthofluorescein pH 10.0
600 nm
674 nm
Alexa 660
664 nm
691 nm
Alexa Fluor 660 antibody conjugate pH 7.2
663 nm
691 nm
Cy 5.5
673 nm
692 nm
Alexa Fluor 680 antibody conjugate pH 7.2
679 nm
702 nm
Alexa 680
679 nm
703 nm
DyLight 680
678 nm
706 nm
Alexa Fluor 700 antibody conjugate pH 7.2
696 nm
719 nm
Alexa 700
696 nm
720 nm
FM 4-64, 2% CHAPS
506 nm
751 nm
FM 4-64
508 nm
751 nm

The original article was published by Leica Microsystems.