New Microscopy Method Resolves Structures Below 100 Nanometers in Deep Tissue

Seeing fine cellular detail while also looking deep into living tissue has been a long-standing challenge in biological imaging. A group at Texas A&M University, led by Aart Verhoef, now reports a microscopy method that does both, described in the Journal of Biomedical Optics.

The approach, called 2PE-ISM-SOFI, stands for two-photon excited image scanning microscopy combined with super-resolution optical fluctuation imaging. It layers two established techniques onto a standard two-photon microscope. The result is lateral resolution of about 75 nanometers, roughly five times sharper than conventional two-photon microscopy, which is among the most widely used tools for imaging living cells and tissue. The method can resolve structures smaller than 100 nanometers, about a thousand times thinner than a human hair, while still imaging deep into intact samples.

“This technique builds on two microscopy techniques that were limited by depth and detail, but this technique allows us to look deep into tissue and improves the resolution,” Verhoef said.

Two-photon microscopy became a lab staple because it can image deep into biological samples with little damage. That makes it well suited to studying living tissue in neuroscience, developmental biology, and soil, animal, and plant sciences. The trade-off is that capturing fine detail has usually meant staying close to a sample’s surface.

Super-resolution methods such as Stimulated Emission Depletion (STED) and Photoactivated Localization Microscopy (PALM), whose developers won the 2014 Nobel Prize in Chemistry, can resolve features well below 100 nanometers. They, too, generally work only near the surface. The new technique keeps the depth advantage of two-photon imaging while approaching that finer resolution. 

The team set the stage for this approach in a 2024 PRX Life study. Taken together, the two papers point toward imaging tools that could reach further into plant and soil biology than current methods, such as capturing root structures, plant-microbe interactions, or disease progression at the cellular level. 

Co-author Alma Fernandez Gonzalez noted that applying the method across fields from neuroscience to plant and animal biology will take time. “Our friends in the College of Medicine are looking at ways to look deeper and with more detail,” she said. “For this study, they cultured midbrain dopamine neurons and labeled the tubulin in the cells with quantum dots. We still have further to go to improve this technique, but we see a path to do that.”