The following tables contain data about various fluorescence excitation and emission properties of over 75 commonly used fluorochromes. They are intended to provide a reference source that relates the spectral properties of these fluorochromes to commercially available filtering cubes. Much of this data was retrieved from F. H. Kasten's excellent reference on fluorescence microscopy and probes. Table 1 is provided as a reference listing of the codes used in Table 2 for filter types.

Filter Color Codes

Code Filter Type U Ultraviolet V Violet BV Blue-Violet B Blue IB Interference Blue Filter G Green IG Immuno Gold Staining Method BF Brightfield, Phase Contrast, and DIC only

Table 1

The table below (Table 2) contains data on the peak excitation and emission wavelengths of over 75 selected fluorochromes along with suggested applications and excitation filter cube type. The selection of a wideband, superwide band or narrow band filter cube will depend on the condition of the specimen and observation purpose.

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Fluorescence Filter Cubes Discover how variations in excitation and barrier filters allow certain wavelengths to pass.

In general, a wide band filter cube is suitable in most cases. For some specimens where more brightness is desired, a superwide filter cube may be applied at some expense to contrast. If more contrast for a specific wavelength band is required for a sufficiently bright specimen, a narrow band filter cube may be appropriate.

Fluorochrome Excitation/Emission/Cube/Application Data

Fluorochrome Excitation Wavelength Emission Wavelength Applicable Excitation Comment Fluorochrome Antibody Method   Allophyco- cyanin (APC) 650 660 IY Antibody labeling 7-Amino-4-Methyl- coumarin-3-Acetic Acid (AMCA) 350 450 U Antibody Labeling BODIPY FL 503 512 B, IB Antibody Labeling Cascade Blue 376 425 U Antibody Labeling Fluorescein- Isothiocyanate (FITC) 490 520 IB Antibody Labeling Phycoerythrin B (PE-B) 545 576 G Antibody Labeling Phycoerythrin R (PE-R) 490, 565 578 G, IB Antibody Labeling Rhodamine B-Iso- thiocyanate (RITC) 570 595 G Antibody Labeling Texas Red 596 620 IY Antibody Labeling Tetramethylrhodamine- Isothyocyanate (TRITC) 541 572 IG Antibody Labeling Protein Modification   8-Anilino-1-Napthalene Sulfonic Acid Mg-salt (ANS) 385 485 V, U, BV Hydrophobic Part Probe Coumarin Maleimide (CMP) 385 465 U Thiol Group Cy 3 552 565 IG Amino Group, Antibody Labeling Cy 5 650 667 IY Amino Group, Antibody Labeling Dimethylaminonaphthalene- 5-Sulfonic Acid (DANS) 380 520 U Thiol Group Dansyl Chloride 335 500 U Amine Labeling Dansyl Hydrazine 336 531 U Carbonyl Group Eosin-Isothiocyanate 524 548 IB Amine Labeling Fluorescamine 390 460 U, V Amine Labeling Lucifer Yellow CH 435 530 BV, B Aldehyde, Ketone Monochlorobimane 380 461 U Thiol Group o-Phtalaldehyde (OPT) 340 455 U Amine Labeling Resorufin 570 585 G Thiol Group SITS 350 420 U Amine Labeling Nuclei, Nucleic Acid   Acridine Orange 490 530, 640 B, IB Single/Double-stranded Nuclieic Acid Acridine Yellow 470 550 B Nucleic Acid Acriflavine-Feulgen 430 515 BV DNA Auramine O-Feulgen 460 550 B DNA 7-Amino- Actinomycin D 555 655 IY, IG G-C Range Bis-Aminophenyl- Oxabizole (BAQ)-Feulgen 380 470 U DNA Chromomycin A3 450 570 B, BV G-C Range 4, 6-Diamidino-2- Phenylindole HCl (DAPI) 372 456 U A-T Range Ethidium Bromide 545 605 G, IG DNA•RNA Hoechst 33258 365 465 U A-T Range Hoechst 33342 355 465 U A-T Range Mithramycin 395 570 BV G-C Range Olivomycin 430 545 BV G-C Range Pararosaniline- Feulgen 560 625 G DNA Propidium Iodide 530 615 IB, G, IG DNA•RNA Pyronin Y 540 570 IG DNA•RNA Quinacrine Mustard 385 525 BV A-T Range, Q Band Bone   Acidic Fuchsin (Pararosaniline) 540 630 G Calcification Calcein 495 520 IB Bone Growth Calcein Blue 375 435 U Bone Growth Tetracycline 390 560 BV, U Calcification Xylenol Orange 440, 570 610 G Bone Growth Carbonydrate Modification   Acriflavine SO2, PAS 430 515 BV Sugar Cell Viability Judgement & Living Cell Staining   Carboxyfluoresceindiacetate (CFDA) 495 520 IB Esterase Substrates Fluoresceindiacetate (FDA) 495 520 IB Esterase Substrates Calcein/AM 495 520 IB Esterase Substrates BCECF/AM 500 530 IB Esterase Substrates Acridine Orange 490 590 B, IB Acid Organanelle Dead Cell Staining   Ethidium Bromide 545 605 G, IB Cell membrane Impermeability Ethidium Homodimer 528 617 G, IB Cell membrane Impermeability Propidium Iodide 530 615 G, IB Cell membrane Impermeability Nerve Cell Tracers   Evans Blue 550 610 G Retrograde Labeling Fast Blue 360 410 U Retrograde Labeling Granular Blue 375 410 U Retrograde Labeling Lucifer Yellow CH 430 535 BV Tracer True Blue 373 404 U Retrograde Labeling Membranes   8-Anilino-Naphthalene Sulfonic Acid (ANS) 385 485 V Negative Ionicity DiI 550 565 G, IG Positive Ionicity, Tracer DiO 484 501 B, IB Positive Ionicity, Tracer Dipheny-1- Hexatriene (DPH) 351 430 U Neutral Filipin 350   U Cholesterol Fluorescamine 390 460 U, V Membrane Surface Protein Merocyanine 540 500 572 G Membrane Potential Organelles   Acridine Orange 490 590 B, IB Lysosome BODIPY FL 503 512 IB Golgi Apparatus DiOC6(3) 480 501 IB Endoplasmic Reticulum Nile Red 485-530 525-605 B, G Lysosome Rhodamine 123 500 540 IB Mitochondria Pyronin Y 540 570 IG Mitochondria Bacteria Staining   Acridine Orange 490 530, 640 B, IB Tubercle Bacilli Acridine Yellow 470 550 B Tubercle Bacilli, Acid-Fast Bacteria Auramine O 460 550 B Tubercle Bacilli, Acid-Fast Bacteria Berberine Sulfate Hoechst 33258 430 550 BV General Bacteria Hoechst 33258 365 465 U Mycoplasma Detection Other Applications   NBD-Phallacidin 465 530 B, IB Actin Fibers Rhodamine-Phalloidin 550 580 G, IG Actin Fibers Thioflavin T 430 550 BV Amyloid

Table 2

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