XiaO

Fluorescence Dye

XiaO / 2013-11-10

当我们在进行多色流式分析的时候，对分析成功的一个关键影响因素是每个抗体选择何种荧光染料标记。通常会有很多可行的结合，我们在做选择的时候有很多的因素需要我们去考虑。对任何单克隆抗体，其阴性和阳性的信噪比相差四到六倍都取决于荧光素的使用。相对的荧光强度也取决于使用的仪器。高密度表达的抗原我们可以应用任何荧光素标记的抗体来检测，低密度表达的抗原就需要我们应用高信噪比高的荧光素，譬如PE或者APC标记的抗体来充分检测分离出阳性表达的细胞与阴性表达的细胞。

荧光（Fluorescence）

各种常用荧光素

Alexa Fluor 488 has a spectrum almost identical to that of fluorescein isothiocyanate (FITC), but with extraordinary photostability. Because of this photostability, it has become a choice for fluorescent microscopy applications and has become popular in cytometry applications. It is detected in the FL1 detector of the FACSCalibur or FACScan. Unlike other fluorochromes with similar emission spectra, Alexa Fluor 488 is pH insensitive over a broad range.

Alexa Fluor 633 is a practical alternative to APC as well as Cy5. Alexa Fluor 633 conjugates can be used in multi-color flow cytometry with instruments equipped with a second red laser or red diode. It is detected in the FL4 detector of the FACSCalibur. The FACScan cannot be used to detect Alexa Fluor 633 conjugates because the FACScan lacks a red laser or diode. Like other Alexa Fluor dyes, Alexa Fluor 633 exhibits uncommon photostability, making it an ideal choice for fluorescent microscopy.

Allophycocyanin(APC) is an accessory photosynthetic pigment found in blue-green algae. APC has 6 phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in phycoerythrin or PE. APC tandem dyes, APC-Cy5.5 and APC-Cy7, are also available. APC has a 650-nanometer wavelength absorption maximum and a 660-nanometer fluorescence emission maximum. APC can be used in flow cytometers equipped with dual lasers for multi-color analysis. Like Alexa Fluor 633, APC is excited using the helium-neon red diode laser (633 nanometers) of the FACSCalibur and is detected on the FL4 detector. APC cannot be detected on the FACScan as that instrument is not equipped with a red laser.

APC-Cy7 is a tandem conjugate system that combines APC and a cyanine dye (Cy7) and has an absorption maximum at ~650 nanometers. This tandem uses the efficiency of the fluorescence light energy transfer between the two fluorochromes. When excited by light from a helium-neon laser, the excited fluorochrome (APC) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is in the deep red at approximately 767 nanometers. APC-Cy7 run on a FACSCalibur results in dim expression because the FL4 detector’s optical filter is centered for APC emission (660 nanometers) and not the longer red wavelengths excited with the helium-neon diode. It is recommended that special precautions be taken with this conjugate, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.

Carboxyfluorescein Diacetate (CFSE) can be used to track asynchronous cell division. Cell division results in sequential halving of the initial fluorescence, resulting in a cellular fluorescence histogram.

Cy3 and Cy5 are excited by the 488-nanometer line of an argon laser and the 633-nanometer line of a helium-neon diode or laser, respectively. These conjugates can be used in flow cytometry but typically do not give the fluorescence intensity comparable to that of PE or APC. Applications where a smaller dye is required are more appropriate for these dyes. These fluorochromes are well suited for fluorescent microscopy.

Enhanced Cyan Fluorescent Protein (eCFP) cannot be analyzed using the FACScan or FACSCalibur as this molecule requires excitation in the violet range. The protein is easily detected using the violet laser of the LSRII, or the violet lines of the MoFlo’s argon or krypton lasers. This molecule has an excitation maximum at 475 nanometers and is best excited using the MoFlo’s 457nm line of the argon or argon-krypton mixed gas laser. Use of the 407nm violet laser of the LSRII will result in weak eCFP expression. More detailed discussion of this molecule can be found in the next section of this web site, the section on Fluorescent Proteins.

Enhanced Green Fluorescent Protein (eGFP) can be excited at 488 nanometers with a peak emission at 509 nanometers and is detected in the FL1 detector on the FACSCalibur or FACScan. The MoFlo and LSRII are able to distinguish between concurrently expressing eGFP and eYFP cells if the proper optical filters and experimental controls exist. More detailed discussion of this molecule can be found in the next section of this web site, the section on Fluorescent Proteins.

Enhanced Yellow Fluorescent Protein (eYFP), a yellow-shifted variant of the eGFP molecule, is also excited at 488 nanometers with a peak emission at 535 nanometers and is also detected in the FL1 detector on the FACSCalibur or FACScan. More detailed discussion of this molecule can be found in the next section of this web site, the section on Fluorescent Proteins.

Fluorescein isothiocyanate (FITC) is currently the most commonly used fluorescent dye for flow cytometry analysis. When excited at 488 nanometers, FITC has a green emission that’s usually collected at 530 nanometers, the FL1 detector of the FACSCalibur or FACScan. FITC has a high quantum yield (efficiency of energy transfer from absorption to emission fluorescence) and approximately half of the absorbed photons are emitted as fluorescent light. For fluorescent microscopy applications, FITC is seldom used as it photobleaches rather quickly though in flow cytometry applications, its photobleaching effects are not observed due to a very brief interaction at the laser intercept. FITC is highly sensitive to pH extremes.

Peridinin chlorophyll protein (PerCP) has a 677 nanometer maximum emission, red, when excited at 488 nanometers and is detected on the FL3 detector of the FACSCalibur or FACScan. A PerCP tandem dye is also available (PerCP-Cy5.5). PerCP is not suited for the high-powered lasing (>150mW) applications, such as on the MoFlo, due to its photobleaching characteristics. PerCP conjugates can only be obtained from Becton Dickinson and its subsidiary, Pharmingen.

Phycoerythrin (PE or R-PE) has a huge absorption coefficient and almost perfect quantum efficiency. In vivo, it functions to transfer light energy to chlorophyll during photosynthesis. It is one of the brightest dyes used today and emits in the yellow/orange at about 570 nanometers. Those accustomed to fluorescent microscopy may not be familiar with this fluorochrome as it photobleaches rather quickly under a microscope.

Phycoerythrin-Cy5 (PE-Cy5) is a tandem conjugate where PE is coupled to the cyan dye, Cy5. When excited by 488-nanometer light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength in the red at 670 nanometers. This tandem dye is known by a confusing myriad of names to include Becton Dickinson’s CyChrome, Caltag and Sigma’s Tri-Color, GIBCO’s RED670, Coulter’s PC5, and probably others. Other PE conjugates exist, e.g., PE-Cy5.5 and PE-Cy7, that will not be discussed in this introductory fluorophore section. It is recommended that special precautions be taken with this conjugate, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.

Phycoerythrin-Texas Red (PE-Texas Red) is a tandem conjugate where PE is coupled to Texas Red. Similar to other tandem conjuates, when excited by 488-nanometer light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the Texas Red molecule, which then fluoresces at a longer wavelength with a peak in the orange at 612 nanometers. This tandem is also known by other names such as Red 612 and ECD (Electron Coupled Dye). Know that PE-Texas Red conjugates run on the FACScan or FACSCalibur will result in dull expression due to the extant optical filters. This is not observed on the LSRII or MoFlo when equipped with the appropriate optical filters for this conjugate. There is considerable overlap of emission when running PE and PE-Texas Red specimens.

Propidium Iodide (PI) is a membrane-impermeant dye that stains by nondiscriminately intercalating into every 4th or 5th nucleic acid base pair, binding both DNA and RNA. Once bound, PI undergoes a conformational change and becomes ~40 times brighter. Propidium iodide has a broad emission spectrum with a peak in the orange at 620 nanometers. A number of assays employ propidium iodide. Cells or nuclei with altered DNA content are identified by staining alcohol-fixed, RNAse-treated cells or nuclei with this dye (see below).

Propidium iodide has also been employed for many years as a marker for viability as the disrupted membranes of dead cells allow the dye to pass freely to the nucleic acids. However, this dye is very sticky; it will stick to sample tubing and, given sufficient time exposure to living cells, living cells will appear to be propidium iodide positive. Given this dye’s broad emission spectrum and its sticky properties, contemporary flow cytometry labs have replaced propidium iodide with other nucleic acid dyes, e.g., 7-AAD (listed below), TO-PRO-3, or DAPI, among many others, for viability measurements though propidium iodide remains the most commonly used dye for DNA content analysis.

**Texas Red **has an excitation maximum in the yellow-orange range of the color spectrum; consequently, Texas Red cannot be excited on any of the benchtop analyzers in the core facility. It can, however, be detected using one of green laser lines of the MoFlo’s argon laser but ideally using the yellow laser line of the MoFlo’s krypton laser. When excited, Texas Red has an excitation maximum in the orange at 612 nanometers.

7-Aminoactinomycin D (7-AAD), like propidium iodide, is a DNA intercalating dye but 7-AAD is specific for C-G base pairs. It is well suited for viability measurements and also for apoptosis experiments where it’s paired with Annexin V. Unlike propidium iodide, this dye has minimal emission bleed from the FL3 detector into the FL2 (PE) detector on the FACSCalibur or FACScan. Whereas PI can be detected in either FL2 or FL3, though it is typically detected in FL2, of the FACScan or FACSCalibur, 7-AAD is detected in FL3.

Notes