STED microscopy—towards broadened use and scope of applications

Abstract

PostprintThis is the accepted version of a paper published in Current opinion in chemical biology. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the original published paper (version of record):Blom, H., Widengren, J. (2014) STED microscopy -towards broadened use and scope of applications. Current opinion in chemical biology AbstractStimulated Emission Depletion (STED) microscopy has been convincingly demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turnkey laser technology and by implementing specifically designed phase plates and birefringent segmented polarization elements. With these simpler and cheaper realizations of STED now becoming more broadly available, and with a parallel development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes. Highlights-STED imaging of cells, living tissue and organisms has been convincingly demonstrated -The STED technology has in the last 6-7 years become far easier to realize -This largely relies on new laser technology and customized optical elements -New fluorescent markers and sample preparation procedures support the development -By increased accessibility, a strong increase in the use of STED can be expected

Resolution, target density and labeling effects in colocalization studies – suppression of false positives by nanoscopy and modified algorithms

“…The probability of having no label at all on the target molecules will then also increase, which will evidently lead to an underestimation of the colocalization. Nonetheless, in the course of the continuous technical development of optical super‐resolution imaging , our study indicates that there will be good reasons to replace primary and secondary antibody fluorescence staining with smaller sized tag molecules for colocalization analyses.…”

Gated STED microscopy with time-gated single-photon avalanche diode

“…Theoretically proposed in 1994 [2], and experimentally validated in 1999 [3], stimulated emission depletion (STED) microscopy was the first effective super-resolved microscopy technique [4]. In a STED microscope the diffraction limit is overcome by reducing the size of the effective fluorescence volume of a scanning microscope, which, in a conventional microscope, corresponds to the diffraction limited focus of the excitation laser beam.…”

Sensing Inside the Living Cells

“…The application of such approach improves the image but requires defi nite assumptions, and therefore cannot provide the general solution of the problem. The recently developed STED microscopy (Hell 2007 ) being an experimental technique shows such possibility (Donnert et al 2006 ;Blom and Widengren 2014 ).…”