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Sample preparation for optical microscopy
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Sample preparation for optical microscopy - 1

Sample preparation for optical microscopy Intellectual Property Notice: The Biopharma business of GE Healthcare was acquired by Danaher on 31 March 2020 and now operates under the Cytiva™ brand. Certain collateral materials (such as application notes, scientific posters, and white papers) were created prior to the Danaher acquisition and contain various GE owned trademarks and font designs. In order to maintain the familiarity of those materials for long-serving customers and to preserve the integrity of those scientific documents, those GE owned trademarks and font designs remain in place, it being specifically acknowledged by Danaher and the Cytiva business that GE owns such GE trademarks and font designs. cytiva.com GE and the GE Monogram are trademarks of General Electric Company. Other trademarks listed as being owned by General Electric Company contained in materials that pre-date the Danaher acquisition and relate to products within Cytiva’s portfolio are now trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an affiliate. All other third-party trademarks are the property of their respective owners. © 2020 Cytiva All goods and services are sold subject to the terms and conditions of sale of the supplying company operating within the Cytiva business. A copy of those terms and conditions is available on request. Contact your local Cytiva representative for the most current information. For local office contact information, visit cytiva.com/contact

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Sample preparation for optical microscopy - 2

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...

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Sample preparation for optical microscopy - 3

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...

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Sample preparation for optical microscopy - 4

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...

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Sample preparation for optical microscopy - 5

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...

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Sample preparation for optical microscopy - 6

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:...

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Sample preparation for optical microscopy - 7

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...

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Sample preparation for optical microscopy - 8

• 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...

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Sample preparation for optical microscopy - 9

When mounting samples • Use only #1.5 coverslips. We strongly recommend using high precision coverslips for super-resolution imaging. • When using a DeltaVision OMX microscope, do not put any stickers or labels on the coverslip side of the slide as this can cause tilt. It is very important to avoid a tilted coverslip for SIM imaging. • For the DeltaVision OMX SR and Flex microscopes, mount the sample in the middle 50 mm of the slide to image the entire specimen. 1. Donaldson, J. Immunofluorescence staining. Current Protoc. Cell Biol. 69(4.3) 1–7 (2015). doi: 10.1002/0471143030.cb0403s69....

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