Fluorescence Anisotropy Reloaded—Emerging Polarization Microscopy Methods for Assessing Chromophores' Organization and Excitation Energy Transfer in Single Molecules, Particles, Films, and Beyond

Abstract

Fluorescence polarization is widely used to assess the orientation/rotation of molecules, and the excitation energy transfer between closely located chromophores. Emerging since the 1990s, single molecule fluorescence spectroscopy and imaging stimulate the application of light polarization for studying molecular organization and energy transfer beyond ensemble averaging. Here, traditional fluorescence polarization and linear dichroism methods used for bulk samples are compared with techniques specially developed for, or inspired by, single molecule fluorescence spectroscopy. Techniques for assessing energy transfer in anisotropic samples, where the traditional fluorescence anisotropy framework is not readily applicable, are discussed in depth. It is shown that the concept of a polarization portrait and the single funnel approximation can lay the foundation for alternative energy transfer metrics. Examples ranging from fundamental studies of photoactive materials (conjugated polymers, light‐harvesting aggregates, and perovskite semiconductors) to Förster resonant energy transfer (FRET)‐based biomedical imaging are presented. Furthermore, novel uses of light polarization for super‐resolution optical imaging are mentioned as well as strategies for avoiding artifacts in polarization microscopy.

Control of Intrachain Morphology in the Formation of Polyfluorene Aggregates on the Single‐Molecule Level

“…In π‐conjugated polymers the connection between the electronic properties and the morphology of the chain is prominent . A prime example is PFO, for which its morphology can even be separated into distinct phases (the α‐, β‐, and γ‐phase), where each phase has its own very distinct properties .…”

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

“…Therefore, we selected 50 wt % as the optimal dye loading, exhibiting a QY of 52 %. In addition, increasing the dye loading up to 50 wt % led to an over 100‐fold drop in the fluorescence anisotropy (Figure S10), which suggested strong coupling of closely packed dyes inside NPs and efficient excitation energy migration within donor dyes independently from the acceptor . This process should ensure long‐distance excitation energy transport to the acceptor leading to an overall FRET beyond the Förster radius (Scheme A) …”

“…A similar large number of acceptors are also needed to ensure FRET from upconverting NPs (UCNPs) and dye‐doped silica NPs . One way to go beyond the Förster radius is to use light‐harvesting nanomaterials in which the strongly coupled energy donors communicate through excitation energy migration, which allows transporting the energy through long distances up to the FRET acceptor. Previous works showed that energy migration can improve FRET efficiency in both dye‐doped silica NPs and UCNPs .…”

Polarization‐Sensitive Ultraviolet Detection from Oriented‐CdSe@CdS‐Dot‐in‐Rods‐Integrated Silicon Photodetector

“…As shown in Figure S4 (Supporting Information), the apparatus consisted of a linearly polarized semiconductor laser (λ = 405 nm), quarter‐wave plate, sample stage, lens, polarizer, and fiber‐optic spectrometer. The polarization ratio can be calculated according to Equation …”