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- Page 1 White Paper ZEISS ELYRA Sample Preparation for Superresolution Microscopy – a Quick Guide...
- Page 2 White Paper ZEISS ELYRA Sample Preparation for Superresolution Microscopy – a Quick Guide Authors: Dr. Sylvia Münter Dr. Yilmaz Niyaz Carl Zeiss Microscopy GmbH, Germany Date: November 2013 General Guidelines for Superresolution Structured Illumination Microscopy (SR-SIM) Sample support In general, we recommend using high-precision coverslips (always no. 1.5 thickness) to mount the samples.
- Page 3 Please make sure that the coverslip is centered on the glass slide in order to fit into the sample holder. See below pictures of the ZEISS level adjustable piezo stage insert. Figure 1 ZEISS level adjustable piezo stage insert Fix your cells according to your standard protocol. For SR-SIM imaging, a thoroughly clean glass surface plays a crucial role. Therefore it is beneficial to seal or attach the coverslip in a way that facilitates cleaning with ethanol, without...
- Page 4 White Paper Embedding Ideally, the sample should be embedded in a medium that matches the refractive index (RI) of the immersion oil (n=1,518). The following media perform well, despite having a lower refractive index. • Fluoromout-G (SouthernBiotech) with RI of 1,40. • 86% Glycerol with 2.5g/l DABCO (1,4-deazabicyclo[2.2.2] octane, Sigma Cat. D2522) in Tris-HCl. 1M pH 8.0. For further reading: http://www.nanoimaging.de/ homepages Sample requirements • Non-hardening VECTASHIELD Mounting Medium with RI of 1,44. • ProLong Gold (Life Technologies). Be aware this embedding medium needs 2 day to harden (in order to reach a constant RI). In addition your sample may shrink during this process. Refractive index (RI) of the cured product: 1,46.
- Page 5 • Nunc Lab-Tek chambers (order no.: P35G-0.17-10-C or P35G-0.17-14-C) All these formats will fit into the adjustable piezo stage insert. Please be aware, in order to obtain ideal TIRF illumination you need a sufficient mismatch in refractive indices. In addition you may want to change imaging buffer concentration therefore we recommend no embedding after fixation of PALM and dSTORM samples. Figure 2 ZEISS level adjustable piezo stage insert Note Nunc Lab-Tek chambers are well suited to transport samples between labs. They fit perfectly into a 50 ml Falcon tube, that can be filled with PBS and sealed with parafilm.
- Page 6 White Paper Fluorescent Proteins for PALM The advantage of photoswitchable fluorescent proteins (PS-FPs) lies in their outstanding specificity and their small size, which is around 2 nm. The latter feature potentially allows for high labeling densities. Among photo-switchable proteins photoconvertable ones are the easiest to use as they - and the structure they stain – can be visualized at a different spec- tral range before conversion. Also one can easily check transfection efficiencies and expression levels. E.g. tdEOS or mEOS can be checked in the green spectral range, while the PALM experiment will be carried out detecting photo- switched EOS molecules in a more red shifted spectral band. Therefore tdEOS or its monomeric variant mEOS have been in extensive use as they also yield reasonable photon numbers. Figure 3 Examples of fluorescent proteins that can be used for PALM imaging Most of these proteins are available through e.g. Addgene, Cambridge, MA 02139, USA Recommended FP-Pairs for dual-color PALM...
- Page 7 White Paper Recommended Dyes for dSTORM dSTORM uses organic dyes, which can be switched by employing reducing agents in the buffer. Dyes with a high photon yield per switching event, low on-off duty cycles, high survival fraction and a large number of switching cycles are preferential. Many Xanthene, Coumarin and Cyanine derivatives fulfill these criteria. Companies often trade these under special group names like Bodipy, Alexa Fluor (both from Invitrogen), Atto (from Sigma Aldrich), DyLight fluor (from Thermo Scientific) and FluoProbes (from Interchim). In the literature, Alexa 647 has been mostly used as it has proved to be a dye that matches all imaging criteria very well and can be switched easily between the dark and bright state. The influence of the selected fluorophore has been nicely shown by G.T. Dempsey et al. (Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging, Nature Methods 2011). Please see below an extract of the data from this publication.
- Page 8 White Paper Post-fixation Following Antibody Labeling A post-fixation step can be valuable. Herein, you fix cells a second time after staining in order to improve the stability of the label. This can prevent the label from detaching and floating the imaging medium. Imaging Buffer dSTORM (please see: G.T. Dempsey, Nature Meth 2011 and S. van Linde, Nature Meth 2011)It is recommended to freshly prepare the imaging buffer every day. The oxygen scavenger system will only last for a few hours and is mainly needed for Cyanine dyes like Alexa 647 and Cy5 (Rhodamines and Oxazines do not require it.) Imaging Buffer 100µl PBS 10x (Phosphate buffered saline ; e.g. from Sigma: D1408) 100µl MEA (Cysteamine Hydrochloride) stock concentration 1M (e.g. from Sigma M6500-25G) (toxic!!) Optional: Oxygen scavenger system (for cyanine dyes) 500µl Glucose 20% (e.g. in solution from Sigma (49163-100ML)) 25µl Glucose Oxidase (e.g. 24 mg/ml GluOx from Aspergillus niger; Sigma G0543-50KU) 5µl Catalase (e.g.: 12.6 mg/ml Catalase from Bovine liver; Sigma C3155-50 MG)
- Page 9 White Paper Fiducial Markers for Localization Microscopy Fiducial Markers (FM) are used to: 1) correct for small drifts (tens of nanometer range) during the course of an experiment 2) to align channels in multicolor experiments 3) to correct for chromatic aberrations in multicolor experiments 4) serve as a reference for the software autofocus For PALM imaging we recommend fluorescent beads (e.g. Tetraspek beads from Invitrogen) or gold nanoparticles, dependant on the laser power, as fiducial markers. Choose the beads according to the fluorophores used in your experiment. For dSTORM imaging we recommend nanoparticles (gold colloids) of which the photoluminescence persists through the entire measurement and which should be immobilized on the coverslip.
- Page 10 Prof. Rainer Heintzmann (http://www.nanoimaging.de) Prof. Markus Sauer Lab (http://www.super-resolution.biozentrum.uni-wuerzburg.de/) ZEISS Online Campus edited by Mike Davidson (http://www.zeiss.com/campus) This collection of protocols and information on superresolution sample preparation is just a general guideline. While all attempts are made to provide accurate, current and reliable information we cannot guarantee that the protocols will work or that the...
- Page 11 Carl Zeiss Microscopy GmbH 07745 Jena, Germany BioSciences microscopy@zeiss.com www.zeiss.com/elyra...