Immunofluorescence
In fluorescence microscopy there are two ways to visualize your protein of interest. Either with the help of an intrinsic fluorescent signal – that means by cloning and therefore 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, were 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 watch 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 (1st antibody). But a second one (2nd antibody) which is binding the 1st 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 2nd antibody binds to one 1st antibody. On the other hand it is not necessary to label each antibody against your favorite protein all the time with a fluorescent dye but to use commercial fluorescently labeled 2nd antibodies. Broadly used fluorescent dyes for immunofluorescence are FITC, TRITC or several Alexa Fluor® dyes which are mentioned in the following.
FITC and TRITC
Fluorescein isothiocyanate (FITC) is an organic fluorescent dye which is still used in immunofluorescence and flowcytometry. 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. Its basic form – Fluorescein – has a molar mass of 332 g/mol and is often used as a fluorescent tracer. FITC (389 g/mol) was one of the first dyes which was used for fluorescence microscopy and served as an origin for further 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 the same breath with FITC is its similar sounding partner 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. In contrast to FITC, TRITC (479 g/mol) 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 if FITC and TRITC are still in use, they are rather weak fluorescent dyes and not recommended for state of the art microscopy. Their profit is based on their economical price.
Cyanines
This relatively small collection of fluorescent dyes was derived from cyanine which was also the origin for their names: Cy2, Cy3, Cy5 and Cy7. All of them can be linked to nucleic acids or proteins via their reactive groups. For proteinous labeling maleimide groups are used, for example. Interestingly – concerning fluorescence – Cy5 is sensitive to its electronic surrounding. This can be utilized for enzyme measurement. Conformational changes of the attached protein lead to positive or negative alterations in fluorescence emission. Furthermore Cy3 and Cy5 can be used for FRET experiments. Cyanine dyes are comparatively old fluorescent dyes and the basis for other fluorochromes with improved brightness, photostability, quantum yield etc.
Alexa Fluor® dyes
Alexa Fluor® dyes are a big group of negatively charged and hydrophilic fluorescent dyes which are used very often in fluorescence microscopy. Their appellation goes back to their inventor Richard Paul Haugland who named the dyes after his son Alex Haugland. The tag is a trademark of Molecular Probes (a subsidiary of life technologies). Furthermore the respective laser excitation wavelength is mentioned in their labeling. For example the very broadly used Alexa Fluor®488 has an excitation maximum at 493 nm, which allows excitation with a standard 488 nm laser. Alexa Fluor®488 has an emission maximum at 519 nm. With this characteristics, Alexa Fluor®488 has very similar properties to FITC. Although Alexa Fluor®488 is a fluorescein derivate, in contrast to FITC it has a better stability, brightness and lower pH sensitivity. All the Alexa Fluor® dyes are sulfonated forms of different basic fluorescent substances like fluorescein, coumarin, cyanine or rhodamin (e.g. Alexa Fluor®546, Alexa Fluor®633). Their molar mass ranges from 410 to 1,400 g/mol.
DNA staining
In fluorescence microscopy not only proteinaceous structures are of interest but also nucleic acids. Sometimes it is necessary 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. Therefore it can be used in fixed, as well as in living cells.
A second broadly used DNA stain option is the family of Hoechst dyes, which was originally produced by the chemical company Hoechst AG. Hoechst 33258, Hoechst 33342, and Hoechst 34580 are Bis-benzimides with intercalating tendency to A-T rich areas, whereupon the latter one is not used very often. 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. A difference to DAPI is their lower toxicity.
A membrane impermeable DNA stain is Propidium-Iodide. With it, it is often used to differentiate between living and dead cells in a cell culture, because it can`t 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 is able to enter acidic compartments like lysosomes. There the cationic dye is protonated. In this acidic surrounding Acridin Orange is excited by light in the blue spectrum, whereas emission is strongest in the orange region. Because apoptotic cells have a lot of engulfed acidic compartments this makes it an often used marker for those kinds of cells.
Compartment and organelle specific dyes
In fluorescence microscopy it is often reasonable to stain cell compartments like lysosomes or endosomes and organelles like mitochondria. For this purpose there is a palette of specific dyes available which will be mentioned in this section.
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. With it, it can bind to matrix proteins covalently by reacting with free thiol groups of cysteine residues. MitoTracker® exists in different colours and modifications (s. Table 1) and is a trademark of Molecular Probes. 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.
According to mitochondrial stains there are also dyes marking acidic compartments like lysosomes which are called LysoTracker. These are membrane permeable weak bases linked to a fluorophore. Most probably these bases have an affinity to acidic compartments because of protonation. LysoTrackers are also available in different colours (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®.
When it comes to protein secretion experiments the Endoplasmic Reticulum (ER) is of a special interest. One classical dye to stain this compartment is DiOC6(3). It 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 does not make them a very good tool for protein labeling but for lipid and membrane labeling.
The adjacent compartment to the ER – the Golgi appararatus – 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).
Besides using special non-proteinacous fluorescent dyes to label cellular compartments it is also possible to stain the area of interest with the help of proteins with preferences for distinct locations in the cell. These proteins can be linked to a fluorescent dye and visualized in the fluorescent microscope. One example for such an approach is the usage of wheat germ agglutinin (WGA) which binds specifically to sialic acid and N-acetylglucosaminyl present in the plasma membrane. WGA is coupled to a fluorescent dye. With it the plasma membrane can be observed.
Ion imaging
In the case of neuronal studies, gene activity or cellular movement – for example – it is of interest to know about 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, which change their spectral properties when bound to the appropriate ions. This principle is realized in the calcium indicators fura-2, indo-1, fluo-3, fluo-4 and Calcium-Green, for example.
For sodium detection, SBFI (sodium-binding benzofurzanisophthalate) or Sodium Green are commonly used. PBFI (potassium-binding benzofurzanisophthalate) detects potassium ions.
Interestingly there are also protein based calcium indicators. One of them is based on the jellyfish chemiluminescent protein aequorin. Interaction of aequorin, the luminophore coelenterazine, molecular oxygen and Ca2+ leads to the release of a blue light – a very prominent mechanism in the discovery of fluorescent proteins.
Fluorescent dyes and their excitation and emission wavelength peaks
All the dyes mentioned above are listed in the following table. Furthermore additional fluorescent dyes are mentioned together with their excitation and emission wavelength peaks.
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 |