Fluorescent Dyes

An Overview

June 11, 2012

A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescing agent. This can be a fluorescing protein – for example GFP – genetically linked to the protein of interest. If cloning is impossible – for instance in histologic samples – it is required to use other techniques like immunofluorescence staining 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. Moreover, with the help of fluorescent dyes fluorescence microscopy is not only restricted to proteins but it gives also the chance to stain nucleic acids, glycans and other structures. 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 – 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.

 

Fig. 1: Drosophila embryo development, Green: FITC, Red: TRITC
Fig. 2: Mouse fibroblasts, Green: F-Actin, FITC, Red: Tubulin, Cy5, Blue: Nuclei, DAPI

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 name goes back to their inventor Alex Haugland and 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.

 

Fig. 3: 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
Fig. 4: 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

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

Fig. 5: Pukinje cell, triple labeled parasagittal section of mouse cerebellar cortex. Red: anti-calbindin-D28k/Cy3, Green: anti-GFAP/Cy5, Blue: Hoechst 33258
Fig. 6: 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

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

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