Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

Abstract

Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of certain color and brightness but instead have their own ‘personalities’ regarding spectroscopic parameters, redox properties, size and water solubility, photostability and several more. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single molecule biophysical studies which was later overcome using Trolox through reducing/oxidizing system, but a boon for super-resolution imaging due to the controllable photoswitching. Understanding photophysics is critical in design and interpreting single molecule experiments.

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

“…This procedure yielded an immobilized RNAP density of approximately one molecule per 4 μm 2 . Afterward, the flow chamber was flushed with 0.5× TMNE containing 2 mM Trolox, 1% (wt/wt) glucose, 100 U/mL glucose oxidase, and 160 U/mL catalase (69). For experiments involving TFE or Spt4/5, high excess of these proteins was additionally added to the measuring buffer to ensure saturation of the RNAP with transcription factors during the measurement.…”

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

“…We then investigated whether we could improve the photon counts through the addition of antibleaching agents. We discovered that the addition of Trolox15 caused a dramatic performance improvement in oxygen‐deprived buffer, giving an image of the cellular actin network (Figure 2 a) with better localization precision (5.3 nm) than either previous experiment owing to substantially higher photon counts (median=14 944). This precision allowed high‐resolution imaging of actin‐rich structures, such as filopodia (Figure 2 b,c) and smaller features (Figure S3).…”

Fluorescent labeling and modification of proteins

“…During these cycles, approximately 1:1,000 excitations leads to a transition from the singlet state (S 1 /S 2 ) to the triplet state (T 1 ; Fig. 2) [26]. This state is long-lived (in millisecond) and the fluorophore will not emit again until it reaches the ground state and is re-excited.…”