Two-photon excitation (2PE) microscopy is established as a method for imaging deeper into scattering tissue because of its longer-wavelength excitation and confined focal volume. However, standard 2PE remains limited by diffraction, and prior attempts to combine two-photon excitation with super-resolution schemes (for example, 2PE‑STED and 2PE‑RESOLFT) have typically retained a single-photon step for depletion or photoswitching. The study described here addresses that limitation by implementing a microscopy scheme in which both excitation and the reversible photoswitching step are driven by two-photon absorption.
The authors present a fully two-photon-driven RESOLFT microscopy approach. In this implementation, both the excitation of fluorescence and the reversible photoswitching process required for RESOLFT contrast control are achieved using two-photon processes rather than mixing two-photon excitation with single-photon depletion or switching.
For fluorescent labeling the study used the positive photoswitchable protein Padron2, which served as the reporter enabling reversible photoswitching under two-photon excitation conditions. The source reports the use of Padron2 but does not provide additional experimental parameters, illumination wavelengths, timings, or optical setup details in the abstract.
Using the fully two-photon-driven RESOLFT scheme with Padron2, the authors report achieving a lateral resolution of 205 nm. This resolution indicates super-resolution performance beyond the diffraction limit of conventional two-photon microscopy and demonstrates the feasibility of driving both required photophysical transitions with two-photon absorption.
The report emphasizes that the approach can produce improved lateral resolution while retaining the tissue-penetration advantages of two-photon excitation.
As an application example, the authors applied the method to image pancreatic pseudoislets. They resolved membrane protrusions of β cells inside these tissue-like aggregates that had not been reported previously. The source attributes the visualization of these small membrane structures to the combination of two-photon penetration and RESOLFT-type photoswitching control driven entirely by two-photon processes.
No further experimental conditions, quantitative morphological measures of the protrusions, or sample preparation details are reported in the abstract.
The authors position the work against existing hybrid approaches such as 2PE‑STED and prior 2PE‑RESOLFT implementations. Those earlier methods still rely on single-photon depletion or photoswitching for the non-linear switching step, limiting how fully two-photon benefits can be leveraged. This study's contribution is a RESOLFT implementation in which both excitation and reversible switching are two-photon-driven, potentially extending super-resolution capabilities deeper into tissue while preserving the mechanism of RESOLFT.
The abstract does not provide head‑to‑head performance metrics versus specific prior implementations beyond the conceptual distinction concerning the photophysical driving mechanism.
This article is a bioRxiv preprint and has not been certified by peer review, as stated in the source. The abstract and front matter report no competing interests declared by the authors.
Declared funders include the Japan Science and Technology Agency, the Japan Society for the Promotion of Science, the Japan Agency for Medical Research and Development, the Cooperative Study Program of the National Institute for Physiological Sciences, Joint Research of Exploratory Research Center on Life and Living Systems (ExCELLS), ExCELLS Encouragement Research for Young Scientists, and the Toyoaki Scholarship Foundation.
The abstract does not report detailed limitations, experimental controls, phototoxicity assessments, imaging depth limits, or photobleaching data; those details were not provided in the source abstract.
The study lists Ryohei Ozaki‑Noma, Hirokazu Ishii, Tetsuichi Wazawa, Joe Sakamoto, Yuichi Kozawa, Yuta Kato, Eri Mukai, Tomomi Nemoto, and Takeharu Nagai as authors. Correspondence contact is provided for Takeharu Nagai in the source.
The preprint is available on bioRxiv with DOI: https://doi.org/10.64898/2026.09.13.751305 and was posted on September 18, 2026. The authors declared no competing interest in the source document.
The abstract reports a methodological advance: a RESOLFT microscopy approach where both fluorescence excitation and reversible photoswitching are driven by two-photon absorption, implemented using the positive photoswitchable protein Padron2. The method yielded a lateral resolution of 205 nm and enabled visualization of previously unreported β-cell membrane protrusions inside pancreatic pseudoislets. Because this is a preprint, the findings should be interpreted as preliminary until peer review and further methodological detail are available in the full manuscript or subsequent publications.