In single-molecule localization microscopy (SMLM), three-dimensional (3D) imaging is commonly realized by engineering the point spread function (PSF) to unambiguously determine the axial position of the individual molecule along with their in-plane coordinates. A wide variety of methods for engineering PSF, including astigmatism, biplane detection, double-helix PSF, interferometric detection, and spherical aberration, have been developed in enabling 3D imaging. On the other hand, we recently developed spectroscopic single-molecule localization microscopy (sSMLM), is capable of simultaneously capturing the spatial distributions of photons emitted by individual molecules and their inherent spectroscopic signatures. We have previously implemented 3D imaging capabilities using the astigmatism imaging approach. The use of additional optical elements such as a cylindrical lens, spatial light modulators, deformable mirrors, and second objective lens for 3D imaging not only increase the complexity of system alignment, but also results in undesirable attenuation of the photons especially for adaptive optics-based 3D imaging. Recognizing the sSMLM method already divides the photons into two channels, the 3D imaging can be conveniently implemented using the bi-plane method, without the need for any additional optical element. Using this system, we demonstrated multi-color 3D nanoscopic imaging of Tubulins and TOM20 in fixed COS-7 cells, labeled with Alexa Flour 647 and CF 660C dyes, respectively. We have achieved 40 nm lateral resolution and 70 nm axial resolution over a depth of ~ 1.5 um. We expect that this system will open up new opportunity to explore the complex 3D structures of biological systems and to study the interaction of individual molecules with their local environment at nanoscale precision.

Three-dimensional spectroscopic single-molecule localization microscopy using bi-plane detection

2019 Spring National Meeting

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