Labels and Probes for Live Cell Imaging: Overview and Selection Guide

Abstract

Fluorescence imaging is an important tool for molecular biology research. There is a wide array of fluorescent labels and activatable probes available for investigation of biochemical processes at a molecular level in living cells. Given the large number of potential imaging agents and numerous variables that can impact the utility of these fluorescent materials for imaging, selection of the appropriate probes can be a difficult task. In this report an overview of fluorescent imaging agents and details on their optical and physical properties that can impact their function are presented.

Photobleaching Lifetimes of Cyanine Fluorophores Used for Single‐Molecule Förster Resonance Energy Transfer in the Presence of Various Photoprotection Systems

“…In some cases, the fluorophore quantum yield fluctuates due to transient interactions with residues on the biomolecule to which it is attached. [13][14][15][16] Much more commonly, temporary photoblinking and irreversible photobleaching restrict photon collection. Photoblinking is a temporary dark state for a fluorophore and is usually due to the molecule entering a long-lived, low-emissive triplet state.…”

“…[3,17] Ameliorating unwanted photophysical and photochemical processes has been pursued by designing new fluorophores and introducing chemical extensions and blockers into biomolecules in order to reduce unwanted interactions. [8,13,21,22] The still ubiquitous photoblinking and photobleaching must be addressed; this has motivated the development of a variety of photoprotection systems (PSs), involving different combinations of photostabilizing solutions and oxygen-scavenging solutions to optimize the relaxation pathways and ultimately to extend the photobleaching lifetime. [17,23,24] Typically, photostabilization solutions reduce the time the fluorophore spends in the triplet state.…”

“…[23,28,29] The appropriate choice of PS elements depends on the fluorophore species and the surrounding environment. [13] smFRET measurements, in particular, are even more complex, because there are at least two separate fluorophores that both need to respond well to a PS without the PS interfering with the energy transfer between the fluorophores. In PSs, the most commonly used oxygen scavenger is glucose/glucose oxidase (GGO) in conjunction with catalase.…”

Luminescent probes for the bioimaging of small anionic species in vitro and in vivo

“…The ideal anion sensor functioning in vitro or in vivo must satisfy a demanding set of criteria (outlined in brief in Table 1) [62][63][64] and it is clear from this list that an imaging agent must 'do more' than a sensor. Key challenges include (i) selecting a suitable fluorophore, (ii) choosing an effective switching mechanism and (iii) catering for the biological environment in which the probe must function (see additional discussion for these three points below).…”

Developing Visible Fluorogenic ‘Click-On’ Dyes for Cellular Imaging

“…Fluorescent dyes are a family of optical reporters widely used in studying biomolecules in cells 1 and animals 2 . With an appropriate fluorescent tag, the location and quantity of the target biomolecules or bioprocess can be tracked conveniently.…”