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Sample preparation for optical microscopy Multiple processing steps are required to prepare biological samples for fluorescence microscopy. Implementation of best practices at each step during sample preparation is essential to ensure high quality images are acquired. This document covers the key concepts and considerations regarding sample preparation to enable empowered choices in experimental design when preparing samples for imaging on DeltaVision™ Ultra or DeltaVision OMX. Read on for more information regarding: • • • • • General sample preparation Mammalian cell culture Non-mammalian sample...

Coverslips are available in a range of thicknesses from #0.0 (~ 100 μm) to #4.0 (~ 500 μm). The correct coverslips for highresolution microscopy are #1.5. Use only coverslips, 35 mm dishes, multiwell plates, or multiwell chambered coverslips with a thickness of 170 μm. See Table 1 for a list of recommended coverslips and other imaging chambers. Do not use #1.0 coverslips (150 μm) for high-resolution imaging. For super-resolution imaging on DeltaVision OMX, choose high precision #1.5 coverslips that are more tightly toleranced (170 µm +/- 5 µm) than normal coverslips. Table 1. Recommended coverslips...

Immersion oil The sample itself is part of the optical pathway and can introduce spherical aberration as seen in Fig 1. To compensate, it is necessary to use immersion oils of varying refractive index (RI) to introduce an equal and opposite spherical aberration and bring the sample back into optimal focus and contrast. This is in essence very simple adaptive optics. The specific immersion oil required will depend on the sample and temperature used for imaging. With the correct oil RI (1.530), less blur and higher contrast between the object and background is readily observed (Fig 1). This is...

Sample placement Place samples on the coverslip as shown in Figure 2. Fig 2. Place samples on the coverslip, not the slide. Why is it so important to place the sample on the coverslip? • Image quality is best closest to the coverslip. • The distance between the coverslip and sample is an important factor when determining optimal oil RI. With the sample placed on the slide, this distance will vary across the coverslip, making oil optimization much more challenging. • Placement on the coverslip enables direct imaging of the sample, rather than imaging the sample through variable amounts of mounting/imaging...

For preparation of yeast samples, see the following references: • Consider using a shallow flow cell or microfluidic chamber where cells are loaded to keep them within a restricted height for imaging. 1. Atkin, A.L. Preparation of yeast cells for confocal microscopy. Methods Mol. Biol. 122, 131–139 (1999). https://www.ncbi.nlm. nih.gov/pubmed/10231788. • Consider using a gel matrix substrate to restrict cell motility during observation. 2. Pemberton, L. et al. Preparation of yeast cells for live-cell imaging and indirect immunofluorescence. Methods Mol. Biol. 1205, 79–90 (2014). doi: 10.1007/978-1-4939-1363-3_6....

Fixed or live sample staining and mounting Table 3. Recommended reagents for imaging with a DeltaVision OMX SR/Flex microscope Selecting imaging reagents DeltaVision OMX SR/Flex Laser Lines Common fluorophores, fluorescent proteins, and stains GFP, mNeonGreen, EmGFP, Alexa Fluor 488, ATTO-488, CellMask™ Green, LysoTrackerTM Green, MitoTracker™ Green FM, ER-Tracker™ Green mRuby3, mApple, tdTomato, TMR, Alexa Fluor 568, MitoTracker Red, ER-Tracker Red, LysoTracker Red Cy5, Alexa Fluor 647*, TO-PRO-3, SiR, CellMask Deep Red, MitoTracker Deep Red FM, LysoTracker Deep Red Selecting bright and stable...

• Consider which detergents may be best suited for permeabilizing cells. Different detergents have different modes of action. Some may create holes in the plasma and interior membranes. Some detergents will create permanent holes in the cells whilst other detergents will create openings that will reseal when the detergent is withdrawn. Test which reagents will be best suited for the sample. • Test blocking buffers to prevent non-specific binding of antibodies. • Perform antibody titrations to obtain the best staining concentration, and explore the literature for what is known to work well for...