MmSTORM: Multimodal localization based super-resolution microscopy

Tamás Gajdos,József Mihály,Bálint Barna H Kovács,Zsófia Cserteg,Miklós Erdélyi,Szilárd Szikora,Tibor Novák,Gábor Szabó

doi:10.1038/s41598-018-37341-9

Abstract

Super-resolution localization microscopy provides a powerful tool to study biochemical mechanisms at single molecule level. Although the lateral position of the fluorescent dye molecules can be determined routinely with high precision, measurement of other modalities such as 3D and multicolor without the degradation of the original super-resolved image is still in the focus. In this paper a dual-objective multimodal single molecule localization microscopy (SMLM) technique has been developed, optimized and tested. The proposed optical arrangement can be implemented onto a conventional inverted microscope without serious system modification. The performance of the method was tested using fluorescence beads, F-actin filaments and sarcomere structures. It was shown that the proposed imaging method does not degrade the image quality of the original SMLM 2D image but could provide information on the axial position or emission spectra of the dye molecules.

Highlights

Single molecule localization based super-resolution microscopy (SMLM) methods such as PALM1,2, FPALM3, STORM4, dSTORM5 and GSDIM6 provide the highest spatial resolution images captured via optical microscopy techniques
Different point spread function (PSF) engineering methods have been proposed to determine the axial position of single fluorescent molecules
In this paper we report the development and application of an optical setup for multimodal single molecule localization microscopy (SMLM) that can be potentially used for 3D, polarization and spectral sensitive measurements

Summary

Optical Setup Evaluation Based on Simulated and Measured PSF

The proposed experimental setup was built on a standard inverted fluorescent microscope (Fig. 1(a)). Fluorescence beads fixed to the coverslip were used to determine the z position as a function of ellipticity of the measured PSFs. Figure 4(b) shows the normal, unfiltered 2D dSTORM image, while Fig. 4(c,d) are two ~46 nm thin sections of the reconstructed volumetric image at different z positions (915 nm and 685 nm). During this particular measurement the full FOV (512 × 512 pixels2) was excited, which resulted in a spatial overlap of the two images. Switching between different dyes simultaneously requires an optimum photochemical environment and it only works for specific dye pairs[51,52]

Discussion and Conclusion

Author Contributions

Findings

Additional Information