PT  - JOURNAL ARTICLE
AU  - Altun, Hamza Yusuf
AU  - Secilmis, Melike
AU  - Caglar, Tuba Akgul
AU  - Vatandaslar, Emre
AU  - Öztürk, Gürkan
AU  - Vilain, Sven
AU  - Eroglu, Emrah
TI  - Visualizing H<sub>2</sub>O<sub>2</sub> and NO in endothelial cells: strategies and pitfalls
AID  - 10.1101/2023.02.15.528776
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.02.15.528776
4099  - http://biorxiv.org/content/early/2023/02/16/2023.02.15.528776.short
4100  - http://biorxiv.org/content/early/2023/02/16/2023.02.15.528776.full
AB  - The relationship between hydrogen peroxide (H2O2) and nitric oxide (NO) in the vasculature is multifaceted and remains controversial because the dynamic detection of these reactive molecules is challenging. Genetically encoded biosensors (GEBs) allow visualizing real-time dynamics in living cells and permit multiparametric detection of several analytes. Although robust, GEBs’ utility depends on several parameters that need fine-tuning for proper imaging and correct data analysis: i.e., camera binning, temperature, and the resolution power of the imaging instruments are some critical parameters that require optimization. We have generated a new double-stable transgenic endothelial cell line stably expressing the biosensors HyPer7 and O-geNOp and systematically tested different imaging modes and their impact on the performance of each biosensor. Ambient temperature and the type of imaging mode did not influence the results, while camera resolution settings significantly affected readouts of HyPer probes but not O-geNOp. Changing a single parameter in a co-imaging mode significantly altered the biosensor’s dynamic measurements, potentially causing misinterpretation. This study provides a general guide and the pitfalls of employing GEBs in a multispectral imaging mode.Competing Interest StatementThe authors have declared no competing interest.