Fluorophores, Dyes & Probes

Fluorescent dyes, or fluorophores, are compounds that absorb light at a given wavelength and emit light at a higher wavelength, producing fluorescence in various colors. These dyes can be grouped into categories such as organic dyes (e.g., fluorescein, rhodamine, AMCA), biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin) and quantum dots. Our use of fluorophores in biological labeling and staining began as early as the 1930s, and the research community has gone a long way since then. Biologists now enjoy a plethora of different fluorescent colors. The amount of choices permits not only flexibility, but also more opportunities in what can be detected and measured in research applications such as fluorescence microscopy, flow cytometry, and high throughput screening. Some things to consider in choosing fluorescent dyes include: the fluorophore’s excitation and emission spectra, its relative intensity, the imaging instrument capabilities (such as excitation lasers and filters), and target’s abundance.

Recommended Reading:

Fluorophore Buyer’s Guide

With such an extensive range of fluorophores commercially available, the choice of a suitable molecule can be daunting. Yet, there are several key points to consider.
Setting Up a Multicolor Flow Cytometry Panel

To generate accurate and meaningful data, researchers must consider which fluorophores will be used together, which analytes these will target, and whether their instrumentation can detect their chosen readouts effectively.
Three Tips for Assembling a Multicolor Flow Cytometry Panel

As more fluorophores are assembled into a panel together, it is important that their assignments to cellular markers are designated strategically, because a suboptimally designed panel will negatively impact experimental results.