Flow Cytometry (FCM) /FACS | Fluorochrome Selection

Excitation and Emission

In flow cytometry, laser light is usually used to excite the fluorochromes. These lasers produce light in the UV and/or visible range. Fluorochromes are selected based on their abilities to fluoresce with the wavelengths of light produced by the lasers. Therefore, if a flow cytometer has only one laser that produces only 488 nm light, then only fluorochromes that are excited by 488 nm light can be used. The chemical properties of the fluorochrome determine whether its electrons can be excited to the higher energy state by a specific wavelength of laser light. If the electrons can be excited to the higher energy state, the chemical properties of the fluorochrome will also determine the amount of energy lost as heat when the electrons drop back down to the lowest singlet excited state and the wavelength of light produced when the electrons return to their ground state.

The electrons of a fluorochrome can be excited by a range of wavelengths of light. For example, the fluorochrome, fluorescein, will fluoresce when hit by light with a wavelength between 430 nm and 520 nm. However, the closer the excitation wavelength is to 495 nm, the more fluorescence will be produced. This optimal wavelength is called the excitation peak. Similarly, the light produced by fluorochromes has a range of wavelengths. The emission of light from fluorescein, ranges from 490 nm to 630 nm, and the emission peak is approximately 520 nm.

Fluorochrome Selection

Knowing the excitation and emission properties of fluorescent compounds makes it possible to select combinations of fluorochromes that will work together optimally on a specific flow cytometer with specific lasers. However, for a fluorochrome to be useful in a biological application, it must attach to or be contained within a particle of biological significance. Some fluorochromes are useful because they bind to specific chemical structures, such as antibodies or the nucleic acids in DNA or RNA.

Fluorochromes that are used most often in flow cytometry are ones that attach in some way to biologically significant molecules and are excitable by the lasers that are commonly found on commercial flow cytometers. Many fluorochromes can be attached to antibodies, which will then bind to specific chemical structures on or inside of cells. If these chemical structures are unique to a specific type of cell, then the fluorochrome will identify that cell type. This technique of identifying cells using fluorescent antibodies is called immunophenotyping.

A list of the fluorochromes used most often in immunophenotyping is shown in Table 1 with their peak excitation and emission wavelengths and the laser wavelengths most often used to excite them on a flow cytometer. Table 2 shows the lasers that can generate the required wavelengths of light to excite the various fluorochromes. Some other common applications of fluorochromes in flow cytometry include the detection of intracellular calcium, measurement of the relative amount of cellular DNA or RNA, and measurement of transcription levels using a fluorescent protein as a reporter gene. Fluorochromes used for these applications are shown in Table 4.

Table1. Fluorochromes for Immunophenotyping

Fluorochrome Excitation Peak (nm) Emission Peak (nm) Laser Wavelengths (nm)
AMCA 345 440 334-364, 351-356
Alexa 350 350 445, 334-364, 351-356
Marina Blue 365 460 334-364, 405, 407
Cascade Blue 395 420 405, 407
Cascade Yellow 400 550 405, 407
Pacific Blue 405 455 405, 407
Alexa 430 435 540 458
Per-CP 490 670 488
FITC 495 520 488
Alexa 488 500 520 488
Alexa 532 532 555 514
TRITC 545 580 568
Alexa 546 560 570 568
Phycoerythrin (PE) 565 575 488, 514, 568
PE-Texas Red 565 615 488, 514
PE-Cy5 565 670 488, 514
PE-Cy5.5 565 695 488, 514
PE-Cy7 565 770 488, 514
Alexa 568 568 605 568
Alexa 594 594 620 568
Texas Red 595 615 568
Alexa 633 630 650 633, 635, 647
Alexa 647 647 670 633, 635, 647
Allophycocyanin (APC) 650 660 633, 635, 647
Cy5 650 665 633, 635, 647
APC-Cy7 650 770 633, 635, 647
Alexa 660 660 690 633, 635, 647
Cy5.5 675 695 633, 635, 647
Alexa 680 680 700 633, 635, 647
Alexa 700 700 720 633, 635, 647

Table2. Common Laser Wavelengths in Flow Cytometry

Laser UV Violet Blue Blue-Green Green Yellow Red
Argon 334-364   458 488 514    
Solid-State Violet Laser   405          
Krypton 351-356 407       568 647
Helium-Neon             633
Red Diode             635

Table3. Laser and Channel Set in BD FACSCalibur

Laser Channel Filter Info Common Fluorophores Alternate Fluorophores
Calcium FL-1 515-545nm FITC Alexa 488, GFP, YFP
Calcium FL-2 564-601nm PE DsRed(RFP)
Calcium FL-3 670LP PerCP PE-Alexa 700, PE-Cy5.5
Calcium FL-4 653-669nm APC Cy5-Alexa 647, TOTO-3

Table4. Fluorochromes for Other Flow Cytometry Applications

Application Fluorochrome Excitation Peak (nm) Emission Peak (nm) Laser Wavelengths (nm)
Calcium Indo-1 (calcium) 325 400 334-364, 351-356
Calcium Indo-1 (nocalcium) 345 485 334-364, 351-357
Calcium Fura Red 485 675 458, 488
Calcium Fluo-3 500 540 488
DNA Content Hoechst 33342 355 455 334-364, 351-356
DNA Content DAPI 360 460 334-364, 351-356, 405, 407
DNA Content Acridine Orange 495 535 488
DNA Content Propidium Iodide 305 620 334-364, 351-356
535 620 488, 514, 568, 633, 647
DNA Content 7-AAD 545 650 488, 514, 568
DNA Content To-Pro-3 640 655 633, 635, 647
Reporter Gene eCFP 430 475 458
Reporter Gene eGFP 495 510 488
Reporter Gene eYFP 520 535 514
Reporter Gene Ds-Red 555 585 514, 568
Reporter Gene HcRed 590 620 568

References

1. Haugland RP. Handbook of fluorescent probes and research products. 9th ed. Eugene, OR: Molecular Probes; 2002.
2. Shapiro HM. Practical flow cytometry. 4th ed. New York: Wiley-Liss; 2003.