Improving certainty in single molecule imaging

The spatiotemporal organization of biological entities and the complex network of interactions they sponsor has proven challenging to visualize. Modern biophysics has brought a wealth of techniques for probing cellular structure and dynamics of which fluorescence-based single molecule detection has emerged as a powerful tool. In this review, we summarize notable breakthroughs in biological research based on single molecule imaging, identify prevailing shortcomings in single molecule detection and present current opinion on ameliorating some of its limitations for wider applicability.