Single-Molecule Characterization of mStayGold Variants for Fluorescence Microscopy

Abstract

Fluorescent proteins (FPs) are essential in modern biophysical and biochemical research, particularly in fluorescence microscopy of living cells. In combination with single-molecule microscopy techniques, they enable visualization of specific organelles or receptors beyond the diffraction limit, allowing for detailed insight into biological processes at the nanoscale. This work explores recent advancements in the development of more photostable and bright FPs, focusing on their potential to enhance the tracking ability and expand the range of applications where traditional monomeric enhanced green flourescent protein (EGFP) has reached its limitations.This study investigates the three newly developed monomeric StayGold variants (mStayGold-E138D, mBaoJin and mStayGold) and compares their performance to conventional mEGFP. The mStayGold variants are reported to be brighter and much more photostable than all currently available alternatives. While previous studies on the new mStayGold variants tested these properties in various bulk experimentsin live-cell applications, this work will provide a detailed characterization of their brightness and photostability at the single-molecule level. To achieve this, a cell-mimicking platform was established using supported lipid bilayers containing DGS-NTA-Ni lipids for the attachment of the Histidine-tagged FPs. To ensure accurate single-molecule localization and tracking, two different software approaches were applied: ThunderSTORM, an ImageJ localization plugin coupled with a customMATLAB script for tracking and DaoSTORM, a Python plugin in combination withTrackpy and std-python. The resulting tracking data underwent extensive Pythonbased analysis, generating statistics on brightness, on-times (duration a FP is active), off-times (duration a FP is inactive), total number of localizations and total numberof gaps (blinking events).The results on immobile FPs reveal that, compared to mEGFP, mStayGold demonstrates a marked improvement in single-molecule brightness (+25%), on-time (+55%) and total number of localizations (+145%) per recording while reducing the off-times(-25%), leading to a substantial overall enhancement in photostability and fluorescent output. The mBaoJin variant displayed similar properties as mStayGold, with marginal reductions across all metrics. However, the mStayGold-E138D variant did not show significant improvements in brightness or on-times, a small decrease in off-times and an marginal total increase in localizations. All results were validated for both mobile and immobile proteins, under varying excitation doses (150-1200W/cm2 and 80 to 1 ms illumination times) and in bulk bleaching experiments to create a comprehensive assessment of the FPs single-molecule performance. Additionally, we explored approaches to further improve the photostability without the need for additional hardware by introducing stroboscopic illumination. Splitting the continuous illumination into two microsecond bursts resulted in increased single-molecule half-times compared to mEGFP of up to 30% for mBaoJin and up to 76% for mStayGold under optimized settings. In total, the findings support that mBaoJin and mStayGold are strong candidates for single-molecule applications, offering substantially improved single-molecule brightness and photostability across various experimental settings compared to mEGFP.