The Guide to STED Sample Preparation

Download free guide describing the main points for successful STED imaging

Multicolor TauSTED Xtend 775 for Cell Biology applications that require nanoscopy resolution for multiple cellular components. Cells showing vimentin fibrils (AF 594), actin network (ATTO 647N), and nuclear pore basket (CF 680R). Sample courtesy of Brigitte Bergner, Mariano Gonzales Pisfil, Steffen Dietzel, Core Facility Bioimaging, Biomedical Center, Ludwig-Maximilians-University, Munich, Germany. Triple_color_fixed_sample_TauSTED_Xtend_2_3.jpg

This guide is intended to help users optimize sample preparation for stimulated emission depletion (STED) nanoscopy, specifically when using the STED microscope from Leica Microsystems. It gives an overview of fluorescent labels used for single color STED imaging and a ranking of their performance.

Key Learnings

  • Fluorescent label combinations for dual and triple color STED imaging that minimize cross-talk during detection are recommended. 
  • Discussion of considerations for immunofluorescence labeling and a detailed protocol to obtain high quality images, with a high signal/noise (S/N) ratio, of interesting structures in a specimen.
  • Important details for sample mounting and substrates that enable optimal imaging, minimizing aberrations and autofluorescence due to the mounting medium, are reviewed.
  • Finally, for STED imaging of live-cells, the most appropriate fluorescent labels are mentioned, both fluorescent proteins (FPs) and organic fluorophores which give the best performance.
Multicolor TauSTED Xtend 775 for Cell Biology applications that require nanoscopy resolution for multiple cellular components. Cells showing vimentin fibrils (AF 594), actin network (ATTO 647N), and nuclear pore basket (CF 680R). Sample courtesy of Brigitte Bergner, Mariano Gonzales Pisfil, Steffen Dietzel, Core Facility Bioimaging, Biomedical Center, Ludwig-Maximilians-University, Munich, Germany.
Multicolor TauSTED Xtend 775 for Cell Biology applications that require nanoscopy resolution for multiple cellular components. Cells showing vimentin fibrils (AF 594), actin network (ATTO 647N), and nuclear pore basket (CF 680R). Sample courtesy of Brigitte Bergner, Mariano Gonzales Pisfil, Steffen Dietzel, Core Facility Bioimaging, Biomedical Center, Ludwig-Maximilians-University, Munich, Germany.

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