Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization microscopy by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical and biochemical processes, including the organization of actin networks, movement of molecular motors, conformations of DNA strands, growth and remodeling of amyloid aggregates, and composition changes within lipid membranes. In this review, we discuss recent innovations in SMOLM and cover three key aspects: (1) biophysical insights enabled by labeling strategies that endow fluorescent probes to bind totargets with orientation specificity; (2) advanced imaging techniques that leverage the physics of light-matter interactions and estimation theory to encode orientation information with high fidelity into microscope images; and (3) computational methods that ensure accurate and precise data analysis and interpretation, even in the presence of severe shot noise. Additionally, we compare labeling approaches, imaging hardware, and publicly available analysis software to aid the community in choosing the best SMOLM implementation for their specific biophysical application. Finally, we highlight future directions for SMOLM, such as the development of probes with improved photostability and specificity, the design of “smart” adaptive hardware, and the use of advanced computational approaches to handle large, complex datasets. This review underscores the significant current and potential impact of SMOLM in deepening our understanding of molecular dynamics, paving the way for future breakthroughs in the fields of biophysics, biochemistry, and materials science.

Fluorescence correlation spectroscopy (FCS) is a well-known and established non-invasive method for quantification of physical parameters that preside over molecular mechanisms and dynamics. It combines maximum sensitivity and statistical confidence for the analysis of speed, size, and number of fluorescent molecules and interactions with surrounding molecules by time-averaging fluctuation analysis in a well-defined volume element. The narrow compass of this study is to acquaint the basic principle of diffusion and the FCS method in general regarding variable magnitudes and standardization adjustment. In this review, we give a theoretical introduction, examples of experimental applications, and utensils in solution systems with future perspectives.

The parallel and synergistic developments of atomic resolution structural information, new spectroscopic methods, their underpinning formalism, and the application of sophisticated theoretical methods have led to a step function change in our understanding of photosynthetic light harvesting, the process by which photosynthetic organisms collect solar energy and supply it to their reaction centers to initiate the chemistry of photosynthesis. The new spectroscopic methods, in particular multidimensional spectroscopies, have enabled a transition from recording rates of processes to focusing on mechanism. We discuss two ultrafast spectroscopies – two-dimensional electronic spectroscopy and two-dimensional electronic-vibrational spectroscopy – and illustrate their development through the lens of photosynthetic light harvesting. Both spectroscopies provide enhanced spectral resolution and, in different ways, reveal pathways of energy flow and coherent oscillations which relate to the quantum mechanical mixing of, for example, electronic excitations (excitons) and nuclear motions. The new types of information present in these spectra provoked the application of sophisticated quantum dynamical theories to describe the temporal evolution of the spectra and provide new questions for experimental investigation. While multidimensional spectroscopies have applications in many other areas of science, we feel that the investigation of photosynthetic light harvesting has had the largest influence on the development of spectroscopic and theoretical methods for the study of quantum dynamics in biology, hence the focus of this review. We conclude with key questions for the next decade of this review.