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Protocols

On this page I will publish protocols for microscopy in pdf format.

SP8 gSTED microscope 

The gatedSTED technology is able to resolve much finer structures than traditional confocal laser scanning (such as the SP5).

It is therefore generally of great interest to resolve fine details such as dentritic spines, vesicles, cytoskeleton, nuclear pores, golgi, mitochondria etc.

Please contact the manager for further details and see below for some guidelines on sample preparation and data analysis.

Many but not all of the protocols listed under "SP5 confocal microscope" are also applicable for the SP8, accordingly.

NEW Leica guide for sample preparation (07/2019)

Deconvolution of gSTED images with Huygens

SP5, SP8 confocal/STED microscopes

General Info on HyDs

Large dynamic range (BrightR) imaging

Number&Brightness Analysis (recommended for advanced users only): how to extract data on  molecule dimerization from confocal imaging.

Objective_correction : Summary how to optimize the objectives using the correction collars; please not that on the SP8 there are also motorized correction collars.

SP5/SP8 image data: How to extraxt image infos from lif files

SP5/SP8_errorLog: How to generate an error report on the SP5 microscopy.

Dye separation: How to seperate dyes with a strong fluorescence overlap-linear unmixing on the SP5 and SP8.

FRAP: How to perform FRAP and photoactivation experiments.

FRET:FRET acceptor bleaching and FRET sensitized emission wizards.

Glycerol objective:information on the extra-wide working distance glycerol objective-recommended for thick samples such as brain sections.

Huygens Deconvolution

SP5_deconvolution: Instructions how to aquire confocal images for deconvolution using the Huygens software.