Introduction
Fluorescence microscopy provides biologists with a powerful tool to selectively label and visualize specific cellular components with exceptional sensitivity in both fixed and live samples. It remains one of the most influential technologies in advancing discoveries in the life sciences. However, conventional light microscopy is inherently limited by the diffraction of light, which prevents the resolution of structures smaller than around 200 nm (Vangindertael et al., 2018). To overcome this limitation, super-resolution microscopy (SRM) techniques have emerged since the early 2000s, enabling the visualization of biological structures down to just a few tens of nanometers (Sahl et al., 2017; Schermelleh et al., 2019; Sigal et al., 2018). At the ZMB, a diverse array of SRM methods is available, each offering varying degrees of resolution, sample compatibility, versatility, and user-friendliness. In this section, we introduce the SRM techniques available to our users at the microscopy core facility, providing a brief overview of each method's principles. Our evaluations are based on a scoring system detailed in The Journal of Cell Science, helping you choose the best technique for your research needs.
| Experimental design |
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The same challenges present in conventional microscopy also apply to SRM, and it is crucial for researchers to be aware of these to prevent bias during image acquisition, analysis, and data validation. (Jost and Waters, 2019).
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For multicolor imaging, it's advisable to use online tools, such as FPbase, to predict the compatibility between various fluorophores or fluorescent proteins and the selected microscope.
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When selecting an SRM technique, the speed of acquisition is an important factor to consider. Slow acquisition methods can cause motion blur or artifacts during live imaging, compromising image quality.
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Consistency is key for preparation of fluorescent samples for SRM, but this does not eliminate the need for biological and technical replicates.
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| Sample labeling |
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Fixation methods need to be carefully optimized as each fixative has specific advantages, drawbacks and dye compatibility. Live-cell imaging can be used to validate that no fixation artifacts have been introduced (Pereira et al., 2019).
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Reagents should be carefully validated, including antibodies and dyes.
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Aldehyde-based fixatives may be quenched using inert amine-containing molecules (glycine or sodium borohydride).
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To minimize nonspecific adsorption of fluorescent compounds, adding detergent and serum to the washing buffer is recommended. Ultimately, the ratio between the signal of interest and the background is more crucial than the overall brightness of the staining.
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Thick samples can be cleared to remove light-scattering and light-absorbing components of a tissue (Kolesova et al., 2016; Qi et al., 2019), allowing access to deeper structures.
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| Choice of vessel |
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Glass coverslips should be used rather than plastic, as plastics introduce optical aberrations and are not compatible with immersion oils for long-term imaging.
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The thickness of the glass must match the expected value for the microscope used; 170 µm-thick cover glass (#1.5H) is often recommended. To ensure reliable performance, high quality, high tolerance cover glass should be chosen.
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Positioning of the sample must be designed to reduce the distance between the objective and the region of interest. This will help improve the resolution by reducing light dampening and distortion.
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Most techniques can accommodate a slide–cover glass sandwich format, but the flatness after cover glass mounting can vary significantly. In such cases, using glass-bottom imaging dishes and multiwell slides is recommended for optimal focus.
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| Choice of the imaging medium |
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For fixed samples, multiple mounting media are available, all designed to bring the refractive index (RI) of your sample close to the RI of the cover glass (RI=1.52).
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Curing mounting media allow for longer conservation and have an RI that is better for image quality, but lead to a slight shrinkage of cellular structures.
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Non-curing mounting media may be preferred due to convenience of use, and preservation of sample structure, despite the compromise in RI.
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For live-cell imaging, the priority of the medium is to ensure the survival of the biological sample.
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Imaging media need to be nutrient rich and provide a pH-controlled environment. For instance, adding 25 mM HEPES in imaging media may help to ensure cell survival when CO2 access is non-optimal during imaging (Frigault et al., 2009).
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Consider removing any non-essential compounds from the medium that are autofluorescent (e.g. Phenol Red, flavins, nicotinamide adenine dinucleotide and lipofuscin) (Surre et al., 2018).
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