What Are the Limitations of Multiphoton Excitation?

Despite its advantages, multiphoton excitation has some limitations:

Cost: The equipment required for multiphoton excitation, including pulsed lasers and advanced microscopes, is expensive, limiting accessibility for some laboratories.
Complexity: The technique requires specialized knowledge and expertise to operate and interpret results, making it less accessible to non-experts.
Fluorophore limitations: Not all fluorophores are suitable for multiphoton excitation, and the choice of fluorophores may be limited by their absorption spectra.