Commercially derived versatile optical architecture for two-photon STED, wavelength mixing and label-free microscopy

Multimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and often require on-site adjustment of optical components performed by trained personnel for optimal performance. Here, we describe a hybrid system we built based on a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo turbid samples. The system is stable and endowed with remote alignment hardware that ensures long-term operability also for non-expert users, using the alignment protocol described in this article and in the related material. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging. (c) 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement

Automated summary

Multimodal microscopy combines multiple non-linear techniques to enhance contrast and obtain more information from biological samples. A hybrid system based on a commercial upright microscope was built, enabling multimodal imaging in both ex vivo and in vivo turbid samples. The system is stable and equipped with remote alignment hardware, ensuring long-term usability even for non-expert users.