Optimizing Sample Preparation for High Quality Images
- Super-Resolution Microscopy - Optimizing Sample Preparation for High Quality Images
- Fig. 1: Example showing actin stained with phalloidin488 and Paxillin stained with Alexa 561. The image shows a widefield view on the right side and the SR-SIM processing view on the left side. Scale bar: 5 µm
- Fig. 2: There are three classes of fluorescent proteins useful for PALM imaging. Here we show the mechanism of switching and some common examples for such proteins used in the literature.
- Fig. 3: a) tdEOS Paxillin reconstructed superresolution image and b) same image as widefield reconstruction. The two bright spots are fiducal markers for image alignment purpose. Scale bar: 0,5 µm.
- Fig. 4: Tubulin stained with Alexa647. The image shows a 3D dSTORM acquisition. The z scale is represented as a color code ranging from red to blue as indicated below.
In the year 2008 Nature Methods has selected super-resolution microscopy or nanoscopy as "method of the year" . Since that time super-resolution microscopy has emerged from specialized physics laboratories and become a powerful tool for biologists. The field of application is constantly growing and since recently even expanding in the axial dimension for single molecule localization microscopy. The process to get high quality super-resolution images can be divided in three important steps: sample preparation, image acquisition and image processing. Especially the first step has to be considered with a lot of care, since higher resolving power demands more stringent sample preparation.
Super-Resolution Structured Illumination Microscopy (SR-SIM)
In general, we recommend using high-precision coverslips (no. 1.5) to mount the samples. High-precision coverslips feature an exceptionally accurate thickness of 170 ± 5 µm. Also mind thickness issues when ordering glass-bottom petri dishes or multi-well plates. Grow and fix your cells according to your standard protocol on this glass support. 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 moving the coverslip.
All common types of organic dyes usually conjugated to antibodies or fluorescent proteins are suitable to be used for SR-SIM. Make sure to have a highly specific labeling with low background for a good signal to noise ratio. For multicolor samples the fluorophores should be selected for minimal spectral overlap to avoid crosstalk. Please choose fluorescent labels according to available filter sets and laser lines to obtain an optimal match of excitation and emission for good image contrast (fig. 1). Note that cytosolic or other non-specific fluorescent protein expression (e.g. GFP) will result in staining of extended areas. Since well-defined structures are missing to interfere with the grid pattern, modulation contrast will be low and the final image will lack high resolution information.
Ideally, the sample should be embedded in a medium that matches the refractive index of the immersion oil.
Unfortunately all of the commercially available embedding media have a lower refractive index (around 1.4). For further recommendations see . In order to have stable imaging conditions, especially concerning the refractive index (RI) of the mounting medium prepare the slides at least one week before use, as during curing the refractive index will rise.
Photo Activation Localization Microscopy (PALM) and Direct Stochastic Optical Reconstruction Microscopy (dSTORM)
Samples for PALM and dSTORM are ideally prepared on LabTek chambers or glass bottom dishes with cover glass thickness no 1.5. Please be aware, in order to obtain ideal TIRF-illumination you need a mismatch in refractive index. In addition, you may want to change the imaging buffer concentration. Therefore we recommend no embedding after fixation of PALM and dSTORM samples.
The advantage of photoswitchable fluorescent proteins lies in their outstanding specificity and their small size, which is around 4 nm. The latter feature potentially allows for high labeling densities. Of photoswitchable proteins photoconvertable ones are the easiest to use as they and the structure they mark can be visualized at a different spectral range before conversion (fig. 2). 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 (fig. 3).
If two differently stained molecules in the same sample are subjected to PALM, it is advisable to first image the higher wavelength dye as this does not cross-talk into the shorter wavelength channel. Hence, cross-excitation of the longer wavelength dye by the shorter wavelength and cross-emission of the shorter wavelength dye into the longer wavelength channel are minimized. Under experimental conditions many molecules of the shorter wavelength dye are equally activated with the 405 nm laser line, that is used for conversion/PALM imaging of the longer wavelength dye. Therefore, reversible switchable fluorophores are the preferred choice for the shorter wavelength as they can be recovered and are not irretrievably lost. Recommended pairs for dual-color PALM are: (1) mEOS2 and Dronpa, (2) NeonGreen and PA-mCherry and (3) Padron and Dronpa. A list of suitable fluorescent proteins can be found in .
PALM can also use organic dyes, which can be switched by employing reducing agents in the buffer. In this case the method is also referred to as dSTORM. The performance of these dyes is dependent on at least four criteria: (1) photon yield per switching event (brighter is better), (2) on-off duty cycles (longer off-times are generally better), (3) high survival fraction (low bleaching rate) and (4) a large number of switching cycles (the more the better). In addition to those, the properties of any given dye are dependent on buffer conditions and laser power. It is recommended to freshly prepare the imaging buffer, containing a reducing agent and for cyanine dyes in addition an oxygen scavenger system [2, 4]. In the literature, Alexa 647 has been mostly used as it has proved to be a dye that matched very well all imaging criteria and can be switched most easily between the dark and bright state (fig. 4).
For a multi-color experiment any combination between Alexa 488/Atto 488; Cy3B/Alexa 561 and Alexa 647/DyLight 654 will work. Specifically the combination of Atto 488 with Alexa 647 has proven to be useful . Please consider the size of antibodies. It is preferable to do direct antibody labeling without secondary antibody. Smaller antibodies such as nanobodies (cameloid like antibodies from camels, llamas and sharks) with sizes in the range of 2 nm may be preferred. A post-fixation step can proof to 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.
The stability of the acquisition during a PALM/dSTORM experiment is crucial for a correct localization of the recorded molecules. Therefore it is beneficial to introduce fiducial markers in the assay. Fiducial markers are used to correct for small drifts (tens of nanometer range) during the course of an experiment, to align channels in multicolor experiments, to correct for chromatic aberrations in multicolor experiments and they can serve as a reference for the software autofocus. For PALM imaging we recommend fluorescent beads (e.g. Tetraspek beads) or gold nanoparticles, dependent 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. Ideally one has about 1-3 fiducial markers in the field of view.
 Nature Method 6 no1 (2009)
 Münter S. and Niyaz Y.: Zeiss White Paper: Sample Preparation for Super-Resolution Microscopy - a Quick guide (2013)
 Allen J.R. et al.: Phys. Chem. Chem. Phys. 15, 43 (2013)
 Metcalf D. et al.: Jove 79 e50579 (2013)
 Dempsey G.T. et al.: Nature Methods 8, no12 (2011)
Dr. Sylvia Münter
Carl Zeiss Microscopy GmbH
Zeiss Microscopy Labs Munich, Germany