Photophysical Properties of Fluorescent Labels: A Meta‐Analysis to Guide Probe Selection Amidst Challenges with Available Data

Abstract

Fluorescent labels have been paramount in the advancement of molecular biology and related fields, for they unveil Nature's unknowns. Numerous labels have been developed that can be attached to most biomolecules via various labeling chemistries. Practitioners are expected to navigate through the growing libraries of fluorescent probes to select the best one for their target application. It is precisely this vast collection of labels that makes the selection process challenging, often leading to non‐ideal choices. Here, we present a meta‐analysis of fluorescent labels data with the intention of facilitating the selection process. We classify tags into molecular (dyes), macromolecular (proteins), and nanoparticles. Groups are then organised into subgroups by chemical structure. Entries were structured based on cost, absorption and emission wavelengths, Stokes shifts, molar extinction coefficients, quantum yields, lifetimes, and brightness. We correlate chemical composition to available photophysical properties of the fluorophores in their bound‐whenever possible‐and unbound states. The limited access to complete, comparable, and meaningful photophysical data for fluorescent labels and to reliable fluorescence standards, where calibration is still challenging and benchmarks are still ill‐defined, are highlighted to raise critical awareness of the current issues hampering further development of the field.

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“…Fluorescence spectra were measured at 25 and 37 °C. The ATTO 520 dye used for determination of relative quantum yields 40 was purchased from ATTO tech GmbH (Siegen, Germany).…”

“…Samples were allowed to equilibrate for 2 min before each measurement. ATTO 520 dye used for the determination of relative quantum yields 40 was purchased from ATTO-TEC GmbH (Siegen, Germany).…”

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