Fluorochromes are fluorescent dyes used in various biological applications such as cellular imaging and flow cytometry. They absorb light energy of a specific wavelength and re-emit it at a longer wavelength. The brightness of fluorochromes will depend on their ability to absorb light and the efficiency of converting absorbed light into emitted light. Different fluorochromes have distinct excitation and emission spectra, making them useful for multiplex experiments.
Fluorochromes are mostly compounds containing benzene rings or heterocycles and conjugated double bonds, which can absorb light of a specific wavelength. After absorbing the excitation light, the molecular energy of fluorochromes increases and they are in an excited state (S1'). The excited state has a high energy level and is an unstable state, so organic molecules generally release energy through chemical bond rotation or vibration to return to the ground state (S0). However, the molecular structure of the fluorochromes is special. After releasing part of the energy through the mechanical motion to reach a specific energy level (S1), they jump directly to the ground state, and at the same time release the remaining energy in the form of photons, i.e., emitting fluorescence. Since the fluorescent photon has lost part of its energy through molecular motion before emission, the energy of the fluorescent photon is lower than that of the excitation light, and the final performance is that the wavelength is longer than the excitation light (the shorter the wavelength, the higher the energy).
Fig. 1 Fluorescence principle. (Ishikawa-Ankerhold H C,et al., 2012)
Fluorochromes are used in many different biological research applications, including cellular imaging, flow cytometry, and fluorescence microscopy. In cellular imaging, fluorochromes are used to visualize specific cellular structures or molecules. For example, fluorescently labeled antibodies can be used to detect specific proteins in cells or tissue sections. In flow cytometry, fluorochromes are used to label cells for analysis. The cells are passed through a flow cytometer, where the fluorescence emitted by the cells is measured. This allows researchers to analyze large numbers of cells quickly and accurately. Fluorescence microscopy is another important application of fluorochromes. In this technique, fluorescently labeled molecules are visualized using a microscope. The high sensitivity and specificity of fluorochromes make them indispensable tools in biological research.
| Fluorochrome | Ex-Max | Em-Max | Property |
| FITC | 494 nm | 520 nm | Fluorescein isothiocyanate is the most widely used green fluorescein derivative in biology. Its isothiocyanate group can react with the amino-terminal and the first amine on the protein, thereby labeling fluorescein on the protein, including antibodies and agglutination. The biological applications of FITC include fluorescent labeling of proteins, fluorescent tracking of proteins, etc. |
| Alexa Fluor 488 | 495 nm | 519 nm | Alexa Fluor 488 and FITC have almost the same excitation and emission maxima, but Alexa Fluor 488 is more photostable and less prone to self-quenching. Additionally, Alexa Fluor 488 is pH insensitive and typically appears brighter than FITC on most instruments. These characteristics, including better sensitivity and environmental stability, make Alexa Fluor 488 a better choice for intracellular staining than FITC. |
| CFSE | 494 nm | 521nm | Carboxyfluorescein diacetate (CFSE) is a dye that can enter cells and is primarily used to track cell division and proliferation. Once inside the cell, CFSE is cleaved by intracellular esterases, generating a fluorescent product that binds to primary amines. CFSE remains stable in non-dividing cells for several days and is equally divided between daughter cells during cell division. As a result, the initial fluorescence of CFSE decreases proportionally with each cell division, making it a useful tool for tracking cell proliferation. |
| PE | 496 nm | 578 nm | R-phycoerythrin (PE) is a pigment that is naturally found in red algae, where it plays a role in transferring light energy to chlorophyll during photosynthesis. In biological research, PE is used as a fluorochrome because it has one of the highest absorption coefficients of all fluorochromes, which makes it one of the brightest available. This high brightness makes PE a popular choice for applications where high sensitivity and signal-to-noise ratios are critical, such as in flow cytometry experiments. |
| PE-Texas Red | 496 nm | 615 nm | PE-Texas Red is a tandem conjugate, where the excited PE molecule transfers its fluorescent energy to Texas Red, resulting in a fluorescent signal with a longer wavelength. However, special care must be taken when using PE-Texas Red conjugates with PE since there is considerable overlap in their emission profiles. This can lead to fluorescence spillover and inaccurate results, so researchers must use appropriate filters and controls to correct this spectral overlap. |
| PI | 536 nm | 617 nm | Propidium iodide (PI) is a fluorescent intercalating agent that binds non-specifically to nucleic acids. To distinguish between DNA and RNA, cells must be treated with nucleases. Once bound to nucleic acid, PI fluorescence increases 20-30 fold. PI is excluded from viable cells due to its inability to cross cell membranes but can penetrate the disrupted membranes of dead or dying cells, making it a useful marker of cell viability. PI is commonly used to stain DNA for cell cycle analysis, but cells must be permeabilized before exposure to the dye. |
| APC | 650 nm | 660 nm | Allophycocyanin is a bright fluorochrome found in blue-green algae that contains six chromophores per molecule, making it highly suitable for flow cytometry. It is excited by the red diode laser and can excite other tandem dyes, including APC-Cy5.5 and APC-Cy7. |
| PE-Cy5 | 496 nm | 667 nm | PE-Cy5 is a tandem conjugate. Due to its broad absorption range and emission spectrum that's equivalent to APC, PE-Cy5 is not recommended for simultaneous use with APC. Cyanine dyes are known to exhibit non-specific binding to Fc-receptors, which can be particularly noticeable in monocyte populations. As a result, researchers must use appropriate controls and gating strategies to minimize the impact of this non-specific binding on their experimental results. |
| PE-Cy7 | 496 nm | 785 nm | PE-Cy7 is a tandem fluorochrome that combines PE and a cyanine dye and is as bright as PE. However, it is highly sensitive to photo-induced degradation and fluorescence loss, so it's important to avoid light exposure and prolonged fixation in paraformaldehyde. Fixed cells should be analyzed within 4 hours or transferred to a paraformaldehyde-free buffer for overnight storage to maintain optimal fluorescence. |
| Alexa Fluor 647 | 650 nm | 668 nm | Alexa Fluor 647 is highly photostable and has a broad pH range. Its excitation and emission maxima are similar to APC, although APC is usually brighter. Alexa Fluor 647 is preferred for intracellular applications. |
When selecting a fluorochrome for a biological experiment, there are several factors to consider. One important factor is the excitation and emission spectra of the fluorochrome. The ideal fluorochrome will have a high degree of specificity and minimal spectral overlap with other fluorochromes being used in the experiment. This will help to minimize potential problems such as fluorescence spillover.
Another important factor to consider when selecting a fluorochrome is its brightness. Bright fluorochromes are more easily detected and can provide better signal-to-noise ratios in experiments. Additionally, some fluorochromes are more photostable than others, meaning they are less likely to photobleach or lose their fluorescence over time.
Creative Diagnostics is a leading provider of fluorochromes and antibodies for biological research. Our company offers a wide range of products, including conjugation kits, and primary and secondary antibodies. Our conjugation kits allow researchers to label their own antibodies with fluorescent dyes, making them ideal for custom experiments. In addition, we offer a wide range of primary and secondary antibodies that are labeled with fluorescent dyes, making them ready to use for many different applications.
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