Fluorescent Dyes

An Overview


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.


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.


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.

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.


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.

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.

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

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 33258Hoechst 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 (s. Table 1) . 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 (s. Table 1).

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.

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.

Table 1

Sample Fluorescent DyesExcitationEmission
Indo-1, Ca saturated331 nm404 nm
Indo-1 Ca2+346 nm404 nm
Cascade Blue BSA pH 7.0401 nm419 nm
Cascade Blue398 nm420 nm
LysoTracker Blue373 nm421 nm
Alexa 405401 nm421 nm
LysoSensor Blue pH 5.0374 nm424 nm
LysoSensor Blue374 nm424 nm
DyLight 405399 nm434 nm
DyLight 350332 nm435 nm
BFP (Blue Fluorescent Protein)380 nm439 nm
Alexa 350343 nm441 nm
7-Amino-4-methylcoumarin pH 7.0346 nm442 nm
Amino Coumarin345 nm442 nm
AMCA conjugate347 nm444 nm
Coumarin360 nm447 nm
7-Hydroxy-4-methylcoumarin360 nm447 nm
7-Hydroxy-4-methylcoumarin pH 9.0361 nm448 nm
6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0358 nm450 nm
Hoechst 33342352 nm455 nm
Pacific Blue404 nm455 nm
Hoechst 33258352 nm455 nm
Hoechst 33258-DNA352 nm455 nm
Pacific Blue antibody conjugate pH 8.0404 nm455 nm
PO-PRO-1434 nm457 nm
PO-PRO-1-DNA435 nm457 nm
POPO-1433 nm457 nm
POPO-1-DNA433 nm458 nm
DAPI-DNA359 nm461 nm
DAPI358 nm463 nm
Marina Blue362 nm464 nm
SYTOX Blue-DNA445 nm470 nm
CFP (Cyan Fluorescent Protein)434 nm474 nm
eCFP (Enhanced Cyan Fluorescent Protein)437 nm476 nm
1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS)375 nm479 nm
Indo-1, Ca free346 nm479 nm
1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)375 nm480 nm
BO-PRO-1-DNA462 nm482 nm
BOPRO-1462 nm482 nm
BOBO-1-DNA461 nm484 nm
SYTO 45-DNA451 nm486 nm
evoglow-Pp1448 nm495 nm
evoglow-Bs1448 nm496 nm
evoglow-Bs2448 nm496 nm
Auramine O431 nm501 nm
DiO487 nm501 nm
LysoSensor Green pH 5.0447 nm502 nm
Cy 2489 nm503 nm
LysoSensor Green447 nm504 nm
Fura-2, high Ca336 nm504 nm
Fura-2 Ca2+sup>336 nm505 nm
SYTO 13-DNA488 nm506 nm
YO-PRO-1-DNA491 nm507 nm
YOYO-1-DNA491 nm509 nm
eGFP (Enhanced Green Fluorescent Protein)488 nm509 nm
LysoTracker Green503 nm509 nm
GFP (S65T)489 nm509 nm
BODIPY FL, MeOH502 nm511 nm
Sapphire396 nm511 nm
BODIPY FL conjugate503 nm512 nm
MitoTracker Green490 nm512 nm
MitoTracker Green FM, MeOH490 nm512 nm
Fluorescein 0.1 M NaOH493 nm513 nm
Calcein pH 9.0494 nm514 nm
Fluorescein pH 9.0490 nm514 nm
Calcein493 nm514 nm
Fura-2, no Ca367 nm515 nm
Fluo-4494 nm516 nm
FDA495 nm517 nm
DTAF495 nm517 nm
Fluorescein495 nm517 nm
Fluorescein antibody conjugate pH 8.0493 nm517 nm
CFDA495 nm517 nm
FITC495 nm517 nm
Alexa Fluor 488 hydrazide-water493 nm518 nm
DyLight 488493 nm518 nm
5-FAM pH 9.0492 nm518 nm
FITC antibody conjugate pH 8.0495 nm519 nm
Alexa 488493 nm520 nm
Rhodamine 110497 nm520 nm
Rhodamine 110 pH 7.0497 nm520 nm
Acridine Orange431 nm520 nm
Alexa Fluor 488 antibody conjugate pH 8.0499 nm520 nm
BCECF pH 5.5485 nm521 nm
PicoGreendsDNA quantitation reagent502 nm522 nm
SYBR Green I498 nm522 nm
Rhodaminen Green pH 7.0497 nm523 nm
CyQUANT GR-DNA502 nm523 nm
NeuroTrace 500/525, green fluorescent Nissl stain-RNA497 nm524 nm
DansylCadaverine335 nm524 nm
Rhodol Green antibody conjugate pH 8.0499 nm524 nm
Fluoro-Emerald495 nm524 nm
Nissl497 nm524 nm
Fluorescein dextran pH 8.0501 nm524 nm
Rhodamine Green497 nm524 nm
5-(and-6)-Carboxy-2', 7'-dichlorofluorescein pH 9.0504 nm525 nm
DansylCadaverine, MeOH335 nm526 nm
eYFP (Enhanced Yellow Fluorescent Protein)514 nm526 nm
Oregon Green 488498 nm526 nm
Oregon Green 488 antibody conjugate pH 8.0498 nm526 nm
Fluo-3506 nm527 nm
BCECF pH 9.0501 nm527 nm
SBFI-Na+336 nm527 nm
Fluo-3 Ca2+506 nm527 nm
Rhodamine 123, MeOH507 nm529 nm
FlAsH509 nm529 nm
Calcium Green-1 Ca2+506 nm529 nm
Magnesium Green507 nm530 nm
DM-NERF pH 4.0493 nm530 nm
Calcium Green506 nm530 nm
Citrine515 nm530 nm
LysoSensor Yellow pH 9.0335 nm530 nm
TO-PRO-1-DNA515 nm531 nm
Magnesium Green Mg2+507 nm531 nm
Sodium Green Na+507 nm531 nm
TOTO-1-DNA514 nm531 nm
Oregon Green 514512 nm532 nm
Oregon Green 514 antibody conjugate pH 8.0513 nm533 nm
NBD-X466 nm534 nm
DM-NERF pH 7.0509 nm537 nm
NBD-X, MeOH467 nm538 nm
CI-NERF pH 6.0513 nm538 nm
Alexa 430431 nm540 nm
Alexa Fluor 430 antibody conjugate pH 7.2431 nm540 nm
CI-NERF pH 2.5504 nm541 nm
Lucifer Yellow, CH428 nm542 nm
LysoSensor Yellow pH 3.0389 nm542 nm
6-TET, SE pH 9.0521 nm542 nm
Eosin antibody conjugate pH 8.0525 nm546 nm
Eosin524 nm546 nm
6-Carboxyrhodamine 6G pH 7.0526 nm547 nm
6-Carboxyrhodamine 6G, hydrochloride525 nm547 nm
Bodipy R6G SE528 nm547 nm
BODIPY R6G, MeOH528 nm547 nm
6 JOE520 nm548 nm
Cascade Yellow antibody conjugate pH 8.0399 nm549 nm
Cascade Yellow399 nm549 nm
mBanana540 nm553 nm
Alexa Fluor 532 antibody conjugate pH 7.2528 nm553 nm
Alexa 532528 nm553 nm
Erythrosin-5-isothiocyanate pH 9.0533 nm554 nm
6-HEX, SE pH 9.0534 nm559 nm
mOrange548 nm562 nm
mHoneydew478 nm562 nm
Cy 3549 nm562 nm
Rhodamine B543 nm565 nm
DiI551 nm565 nm
5-TAMRA-MeOH543 nm567 nm
Alexa 555553 nm568 nm
Alexa Fluor 555 antibody conjugate pH 7.2553 nm568 nm
DyLight 549555 nm569 nm
BODIPY TMR-X, SE544 nm570 nm
BODIPY TMR-X, MeOH544 nm570 nm
PO-PRO-3-DNA539 nm571 nm
PO-PRO-3539 nm571 nm
Rhodamine551 nm573 nm
Bodipy TMR-X conjugate544 nm573 nm
POPO-3533 nm573 nm
Alexa 546562 nm573 nm
BODIPY TMR-X antibody conjugate pH 7.2544 nm573 nm
Calcium Orange Ca2+549 nm573 nm
TRITC550 nm573 nm
Calcium Orange549 nm574 nm
Rhodaminephalloidin pH 7.0558 nm575 nm
MitoTracker Orange551 nm575 nm
MitoTracker Orange, MeOH551 nm575 nm
Phycoerythrin565 nm575 nm
Magnesium Orange550 nm575 nm
R-Phycoerythrin pH 7.5565 nm576 nm
5-TAMRA pH 7.0553 nm576 nm
5-TAMRA549 nm577 nm
Rhod-2552 nm577 nm
FM 1-43472 nm578 nm
Rhod-2 Ca2+553 nm578 nm
Tetramethylrhodamine antibody conjugate pH 8.0552 nm578 nm
FM 1-43 lipid473 nm579 nm
LOLO-1-DNA568 nm580 nm
dTomato554 nm581 nm
DsRed563 nm581 nm
Dapoxyl (2-aminoethyl) sulfonamide372 nm582 nm
Tetramethylrhodamine dextran pH 7.0555 nm582 nm
Fluor-Ruby554 nm582 nm
Resorufin571 nm584 nm
Resorufin pH 9.0571 nm584 nm
mTangerine568 nm585 nm
LysoTracker Red578 nm589 nm
Lissaminerhodamine572 nm590 nm
Cy 3.5578 nm591 nm
Rhodamine Red-X antibody conjugate pH 8.0573 nm591 nm
Sulforhodamine 101, EtOH578 nm593 nm
JC-1 pH 8.2593 nm595 nm
JC-1592 nm595 nm
mStrawberry575 nm596 nm
MitoTracker Red578 nm599 nm
MitoTracker Red, MeOH578 nm599 nm
X-Rhod-1 Ca2+580 nm602 nm
Alexa Fluor 568 antibody conjugate pH 7.2579 nm603 nm
Alexa 568576 nm603 nm
5-ROX pH 7.0578 nm604 nm
5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt)578 nm604 nm
BO-PRO-3-DNA574 nm604 nm
BOPRO-3574 nm604 nm
BOBO-3-DNA570 nm605 nm
Ethidium Bromide524 nm605 nm
ReAsH597 nm608 nm
Calcium Crimson589 nm608 nm
Calcium Crimson Ca2+590 nm608 nm
mRFP585 nm608 nm
mCherry587 nm610 nm
Texas Red-X antibody conjugate pH 7.2596 nm613 nm
HcRed590 nm614 nm
DyLight 594592 nm616 nm
Ethidium homodimer-1-DNA528 nm617 nm
Ethidiumhomodimer528 nm617 nm
Propidium Iodide538 nm617 nm
SYPRO Ruby467 nm618 nm
Propidium Iodide-DNA538 nm619 nm
Alexa 594590 nm619 nm
BODIPY TR-X, SE588 nm621 nm
BODIPY TR-X, MeOH588 nm621 nm
BODIPY TR-X phallacidin pH 7.0590 nm621 nm
Alexa Fluor 610 R-phycoerythrin streptavidin pH 7.2567 nm627 nm
YO-PRO-3-DNA613 nm629 nm
Di-8 ANEPPS469 nm630 nm
Di-8-ANEPPS-lipid469 nm631 nm
YOYO-3-DNA612 nm631 nm
Nile Red-lipid553 nm636 nm
Nile Red559 nm637 nm
DyLight 633624 nm646 nm
mPlum587 nm649 nm
TO-PRO-3-DNA642 nm657 nm
DDAO pH 9.0648 nm657 nm
Fura Red, high Ca434 nm659 nm
Allophycocyanin pH 7.5651 nm660 nm
APC (allophycocyanin)650 nm660 nm
Nile Blue, EtOH631 nm660 nm
TOTO-3-DNA642 nm661 nm
Cy 5646 nm664 nm
BODIPY 650/665-X, MeOH646 nm664 nm
Alexa Fluor 647 R-phycoerythrin streptavidin pH 7.2569 nm666 nm
DyLight 649652 nm668 nm
Alexa Fluor 647 antibody conjugate pH 7.2653 nm668 nm
Alexa 647653 nm669 nm
Fura Red Ca2+435 nm670 nm
Atto 647644 nm670 nm
Fura Red, low Ca472 nm673 nm
Carboxynaphthofluorescein pH 10.0600 nm674 nm
Alexa 660664 nm691 nm
Alexa Fluor 660 antibody conjugate pH 7.2663 nm691 nm
Cy 5.5673 nm692 nm
Alexa Fluor 680 antibody conjugate pH 7.2679 nm702 nm
Alexa 680679 nm703 nm
DyLight 680678 nm706 nm
Alexa Fluor 700 antibody conjugate pH 7.2696 nm719 nm
Alexa 700696 nm720 nm
FM 4-64, 2% CHAPS506 nm751 nm
FM 4-64508 nm751 nm

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