ByPeter Rupprecht, Lab of Rainer Friedrich
[From the website]
Two-photon microscope users who are keen on understanding more about the technical details and especially about what can go wrong with two-photon microscopes.
This case report describes how a two-photon microscope was found to come with a fluorescence yield that was lower than expected; how the underlying cause was found out in a systematic manner; and how the problem was solved. All approaches and solutions are specific for the microscope under question. However, I hope that this report (1) will inspire other people who are troubleshooting or optimizing their microscopes, (2) will help other people better understand two-photon microscopes and the relevance of technical details.
First, I will describe how the problem (too low photon yield) was identified. Before identification of the problem, the same microscope had been used without obvious hints that there was indeed an issue.
Next, I will describe the possible reasons underlying the problem and how the most likely cause (laser pulse dispersion) could be singled out.
Then, I will explain the tests that allowed to understand which optical component caused the laser pulse dispersion (degraded dielectric mirrors).
Finally, I will describe what was done to circumvent the problem and what else can be learnt from this experience.