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Fluorescence Microscopy

Fluorochrome Data Tables
Excitation/Emission Wavelengths
and Filter Cube Suggestions
Listed by Application

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.

Interactive Java Tutorial
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

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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