Recent advancements in super-resolution microscopy have enabled unprecedented insights into the spatial organization of cellular structures. In this talk, I will present a series of methodological innovations that synergistically integrate fluorescence-lifetime single-molecule localization microscopy (FL-SMLM) [1,2], image scanning microscopy (ISM) [3,4], and metal-/graphene-induced energy transfer (MIET/GIET) imaging [5–7]. These approaches collectively offer isotropic three-dimensional resolution at the nanometer scale, multiplexed imaging capabilities, and robustness against chromatic aberrations.

First, I will discuss our work on MIET and GIET microscopy, which exploit distance-dependent quenching phenomena near metallic or graphene interfaces to determine the axial position of single emitters with sub-10 nm accuracy. The combination of MIET with dSTORM or DNA-PAINT provides truly isotropic 3D resolution, extending the reach of localization microscopy into the axial dimension without interferometric complexity.

Second, I will highlight the development of fluorescence lifetime DNA-PAINT (FL-PAINT), a technique that enables multi-target super-resolution imaging through fluorescence lifetime multiplexing without fluid exchange. By utilizing orthogonally designed imager strands conjugated to fluorophores with distinct lifetimes, we achieve simultaneous imaging of multiple targets in the dense intracellular environment.

Lastly, I will introduce our latest development of fluorescence-lifetime image scanning microscopy SMLM (FL-iSMLM), which achieves a near twofold enhancement in lateral resolution by integrating a single-photon detector array into a confocal laser scanning microscope. This method combines the localization precision of ISM with the multiplexing power of fluorescence-lifetime detection, enabling sub-5 nm resolution in fixed cells while simultaneously allowing discrimination of targets based solely on their fluorescence lifetimes.

References

[1] J. C. Thiele, D. A. Helmerich, N. Oleksiievets, R. Tsukanov, E. Butkevich, M. Sauer, O. Nevskyi, and J. Enderlein, Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy, ACS Nano 14, 14190 (2020).

[2] J. C. Thiele, O. Nevskyi, D. A. Helmerich, M. Sauer, and J. Enderlein, Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy, Front. Bioinform. 1, 740281 (2021).

[3] C. B. Müller and J. Enderlein, Image Scanning Microscopy, Phys. Rev. Lett. 104, 198101 (2010).

[4] N. Radmacher, O. Nevskyi, J. I. Gallea, J. C. Thiele, I. Gregor, S. O. Rizzoli, and J. Enderlein, Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy, Nat. Photon. 18, 1059 (2024).

[5] A. I. Chizhik, J. Rother, I. Gregor, A. Janshoff, and J. Enderlein, Metal-induced energy transfer for live cell nanoscopy, Nature Photon 8, 124 (2014).

[6] A. Ghosh, A. Sharma, A. I. Chizhik, S. Isbaner, D. Ruhlandt, R. Tsukanov, I. Gregor, N. Karedla, and J. Enderlein, Graphene-based metal-induced energy transfer for sub-nanometre optical localization, Nat. Photonics 13, 860 (2019).

[7] J. C. Thiele, M. Jungblut, D. A. Helmerich, R. Tsukanov, A. Chizhik, A. I. Chizhik, M. Schnermann, M. Sauer, O. Nevskyi, and J. Enderlein, Isotropic Three-Dimensional Dual-Color Super-Resolution Microscopy with Metal-Induced Energy Transfer, Science Advances 8, 14190 (2021).