What increases signal on apparently clean glass?
Over the years working with self-cleaned coverslips, I’ve noticed that when finding focus on a PBS blank, I can obtain a baseline signal value as low as 0.05 and up to over 0.10 on the same slide, even though the FOV in all wells appears to be over clean glass in the native view. This occurs in the absence of any obvious artifact, such as diagonal lines as from well leakage. In the corresponding ratiometric view, the FOV simply appears noisier at the higher signal values. As I am using the same PBS for all wells, I have to believe glass itself is causing the elevated signal, even in the absence of an obvious contaminant. Can you explain how the glass itself, in the absence of unwanted particles in solution or on the glass surface, can elevate background signal? What about the glass might be doing this?
Dear Chase,
I’m Benjamin Cappe, Product Manager at Refeyn.
Thanks for the question.
What you're seeing is most likely due to subtle variations at the glass-liquid interface rather than visible contamination. Mass photometry is highly sensitive, and even when a field of view appears clean in the native image, microscopic differences in glass surface properties, batch-to-batch variation, residual contamination, surface roughness, or other locally adsorbed material can increase the background signal and make the ratiometric image appear noisier.
When using self-cleaned coverslips from third parties, it can be difficult to pinpoint the exact source, as factors such as glass batch variability, cleaning procedures, handling, buffers, and plasticware can all contribute to background noise.
This is one reason we developed MassGlass™ UC. These slides are specifically selected by our team, cleaned, and quality-controlled to provide a more consistent glass surface for mass photometry measurements, thereby reducing variability between experiments.
If you'd like to discuss your specific observations in more detail, feel free to contact our support team at support@refeyn.com. We'd be happy to review the data and help troubleshoot.
Best regards,
Benjamin Cappe, Product Manager