From Molecules to Classrooms: A Comprehensive Guide to Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) has revolutionized our ability to visualize cellular structures, offering unprecedented detail. However, the intricate biophysical principles that underlie SMLM can be daunting for newcomers, particularly undergraduate and graduate students. To address this challenge, we introduce the fundamental concepts of SMLM, providing a solid theoretical foundation. In addition, we have developed an intuitive graphical interface APP that simplifies these core concepts, making them more accessible for students. This APP clarifies how super-resolved images are fitted and highlights the crucial factors determining image quality. Our approach deepens students' understanding of SMLM by combining theoretical instruction with practical learning. This development equips them with the skills to carry out single-molecule super-resolved experiments and explore the microscopic world beyond the diffraction limit.

Advancements and Practical Considerations for Biophysical Research: Navigating the Challenges and Future of Super-resolution Microscopy.

The introduction of super-resolution microscopy (SRM) has significantly advanced our understanding of cellular and molecular dynamics, offering a detailed view previously beyond our reach. Implementing SRM in biophysical research, however, presents numerous challenges. This review addresses the crucial aspects of utilizing SRM effectively, from selecting appropriate fluorophores and preparing samples to analyzing complex data sets. We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM. Emphasizing the integration of SRM with other analytical methods, we aim to overcome inherent limitations and expand the scope of biological insights achievable. By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives, we aim to empower researchers to explore complex biological processes with enhanced precision and clarity, thereby advancing the frontiers of biophysical research.

Variations in Intracellular Organometallic Reaction Frequency Captured by Single-Molecule Fluorescence Microscopy.

Studies of organometallic reactions in living cells commonly rely on ensemble-averaged measurements, which can obscure the detection of reaction dynamics or location-specific behavior. This information is necessary to guide the design of bioorthogonal catalysts with improved biocompatibility, activity, and selectivity. By leveraging the high spatial and temporal resolution of single-molecule fluorescence microscopy, we have successfully captured single-molecule events promoted by Ru complexes inside live A549 human lung cells. By observing individual allylcarbamate cleavage reactions in real-time, our results revealed that they occur with greater frequency inside the mitochondria than in the non-mitochondria regions. The estimated turnover frequency of the Ru complexes was at least 3-fold higher in the former than the latter. These results suggest that organelle specificity is a critical factor to consider in intracellular catalyst design, such as in developing metallodrugs for therapeutic applications.

Dissection of Interaction Kinetics through Single-Molecule Interaction Simulation.

The ability to extract kinetic interaction parameters from single-molecule fluorescence resonance energy transfer trajectories without the need for solving complex single-molecule differential equations has the potential to address some of the critical biophysical questions. Here, we provide a noise-free single-molecule interaction simulation (SMIS) tool to give the expected dwell-time distributions and relative populations of each FRET level based on the assigned kinetic model and to dissect kinetic interaction parameters from single-molecule FRET trajectories. The method provides the expected dwell-time distributions, average transition rates, and relative populations of each FRET level based on the assigned kinetic model. By comparing with ground truth data and experimental data, we demonstrated that SMIS is useful to quantify the interaction kinetic rate constants without using the traditional single-molecule analytical solution approach.

Seoul virus and hantavirus disease, Shenyang, People's Republic of China.

An outbreak of hemorrhagic fever with renal syndrome (HFRS) occurred among students in Shenyang Pharmaceutical University in 2006. We conducted a study to characterize etiologic agents of the outbreaks and clarify the origin of hantaviruses causing infections in humans and laboratory animals. Immunoglobulin (Ig) M or IgG antibodies against Seoul virus (SEOV) were detected in the serum samples of all 8 patients. IgG antibodies against hantavirus were also identified in laboratory rats, which were used by these students for their scientific research. Phylogenetic analysis showed that partial small segment sequences recovered from humans, laboratory rats, and local wild rats belonged to SEOV. Hantavirus sequences recovered from humans and laboratory rats clustered within 1 of 3 lineages of SEOV circulating among local wild rats in Shenyang. These results suggest that the HFRS outbreak in Shenyang was caused by SEOV that was circulating among local wild rats and had also infected the laboratory rats.