Abstract
Advanced fluorescence imaging techniques such as single-molecule localization microscopy (SMLM) fundamentally rely on the photophysical behavior of the employed fluorophores. This behavior is generally complex and impacts data quality in a subtle manner. A simulation software named SMIS (Single-Molecule Imaging Simulator) is introduced that simulates a widefield microscope and incorporates fluorophores with their spectral and photophysical properties. With SMIS, data collection schemes combining 3D, multicolor, single-particle-tracking or quantitative SMLM can be implemented. The influence of advanced fluorophore characteristics, imaging conditions and environmental parameters can be evaluated, facilitating the design of real experiments and their proper interpretation.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Revised version of the manuscript, based on reviewer's comments. => New case-study #4 (Fig 4, Supplementary Figures S13-15, Supplementary Tables S10-11) => New Supplementary Table S15: Available SMLM simulators => New Supplementary Table S16: List of main SMIS features. => New Supplementary Table S17: Examples of available fluorophores in SMIS