Circular Dichroism Spectroscopy of Single Objects: Problems, Artifacts, and Corrections

Abstract

Molecular aggregates are at the basis of a broad range of functional systems, including photosynthetic light-harvesting complexes, organic photovoltaics, optoelectronic devices, and molecular sensors. Their structural organization, however, is often difficult to resolve because intrinsic heterogeneity. In this context, circular dichroism (CD) spectroscopy is particularly powerful because of its sensitivity to the three-dimensional organization of molecular building blocks. Extending CD measurements from ensembles to individual objects therefore offers a unique approach to probing supramolecular architecture in heterogeneous, non-crystallizable systems. However, without spatial and orientational averaging, measurements are highly sensitive to imbalances between left- and right-circularly polarized excitation, deviations from ideal circular polarization, and polarization imperfections coupled to linear dichroism, which creates spurious CD signals. Here, we present a systematic theoretical and experimental framework for understanding, quantifying, and controlling artifacts in single-object CD spectroscopy. We develop a mathematical description of the principal artifact mechanisms, identify their origins in practical implementations, including Pockels-cell-based polarization modulation, and establish strategies for their minimization. We validate this framework using an experimental implementation capable of acquiring CD spectra from individual molecular aggregates over 690-785 nm. Our results demonstrate that reliable single-object CD measurements are achievable when polarization-related artifacts are rigorously characterized and controlled. This Tutorial Review provides a unified theoretical framework and practical guide for single-object CD spectroscopy and its application to single-particle chirality, supramolecular photonics, and biophotonics.