Sample preparation for optical microscopy

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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 preparation

• Fixed cell imaging

• Live cell imaging

General sample preparation

Coverslip thickness

It is important to ensure that the optical components of the microscope and the consumables that are used during sample preparation work together to achieve the best possible image quality. One of the most important consumables that researchers will use is the coverslips, which must be the correct thickness to minimize spherical aberration.

In optical microscopy, spherical aberration is a phenomenon that occurs when parallel rays of incoming light do not converge at the same point after passing through the objective lens, decreasing both contrast and resolution. All lenses suffer from this physical effect, but fortunately most commercially available objective lenses for fluorescence microscopy are corrected for coverslips that are 170 µm thick, as specified by the 0.17 marking on the barrel of the objective. Using coverslips that are 170 µm thick will allow all rays of incoming light to converge at the same point within the sample, maximizing contrast and resolution.

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Coverslips are available in a range of thicknesses from #0.0 (~ 100 µm) to #4.0 (~ 500 µm). The correct coverslips for high- resolution 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.

Type Brand Description DeltaVision Ultra/Elite

DeltaVision OMX

Coverslips, high precision

Marienfeld Precision coverslips, various sizes

Thor Labs Precision coverslips, various sizes

Zeiss Coverslips, high performance, various sizes

35 mm dish

Ibidi™ µm-Dish 35 mm, high glass bottom #1.5H

MatTek™ No. 1.5 Coverslip glass bottom dishes

MatTek High Tolerance 1.5 Coverslip, 14 mm Glass Diameter, P35G-0.170-14-CP35G

WillCo WellsGWST-3512 #1.5 Glass and 0.005 mm Surface Flatness

GWST-3522 #1.5 Glass and 0.005 mm Surface Flatness

Coverslip- based multiwell chambers

Nunc™

Lab-Tek™ II Chambered Coverglass µ2 well (155379 or 155379PK) µ4 well (155382 or 155382PK) µ8 well (155409 or 155409PK)

Ibidi

2 well (80297 or 80287) 4 well (80447 or 80427) 8 well (80827) 6 channel (80607)

96-well microplate

InVitro Scientific P96-1.5H-N

Greiner™ SensoPlate™ 96-Well, F-Bottom, Glass Bottom, Black, Lid, Sterile, Single Packed, Item No: 655892

MatTek SensoPlate 384 Well, F-Bottom, Glass Bottom, Black, Lid, Sterile, Single Packed, Item No: 781892

384-well microplate

Greiner Black 96 well no. 1.5 Coverslip, 5mm Glass Diameter, Uncoated (PBK96G-1.5-5-F)

MatTek Black 384-well Plate, 1.5 Coverslip, Uncoated

Table 1. Recommended coverslips and imaging chambers for DeltaVision Elite, DeltaVision Ultra, and DeltaVision OMX microscopes

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.

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Fig 1. Synaptonemal complex imaged with immersion oils with RI from 1.522 to 1.534 on DeltaVision OMX. In this case, the 1.530 RI oil is optimal.

1.522 Oil RI too low

1.530 Correct oil RI

1.534 Oil RI too high

x

z

x

y

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 especially apparent in the xz view where the signal is concentrated

in the middle of the point spread function (PSF) and the PSF is much more symmetrical than with the other immersion oils.

To learn more about the factors that affect optimal oil RI and how to select the correct RI immersion oil see the Immersion oil optimization application guide 29250659 for DeltaVision OMX SR/Flex, and see the Oil optimization application guide 29308412 for DeltaVision Ultra. In addition, a free immersion oil calculator app is available for Android and iPhone® which estimates the optimal oil RI using information about the sample, environment, and imaging.

Download the apps here.

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Sample placement

Place samples on the coverslip as shown in Figure 2.

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 medium.

• Ensures that the sample is within the working distance of the lens. For high resolution imaging, the 60× 1.42 numerical aperture (NA) objective lens has a working distance of 150 µm. If the sample is on the slide, the mounting medium between the sample and the coverslip can put the sample at the edge of the working distance of the objective.

Sample acquisitionSample-specific preparation protocols can be followed regardless of the fluorescence imaging modality used. However, during sample preparation the thickness of the sample must be tailored to the specific imaging modality. Structured illumination microscopy (SIM) is limited to samples under 30 µm thickness, while confocal microscopy can image thicker samples (< 1 mm), due to the removal of out-of-focus light by optical sectioning. Sample preparation can therefore be further optimized depending on the thickness of the sample.

In this first section, we consider some parameters to be taken into consideration during the preparation of the experimental samples whether the samples will eventually be imaged as fixed or live samples.

Bacteria

It is best to refer to specific literature for varying bacterial species for the best fixation and staining methods for fluorescence microscopy.

For all bacteria, care must be taken during the mounting process to not exert undue force on the cells. Applying too much pressure on the cells between the glass slide and the coverslip may change the morphology or rupture the cells. This is especially important for osmotically fragile species.

For fixed cell imaging, cells may be stained in a tube, applied to a coverslip in a minimal volume of liquid and allowed to adhere for a minimum of 30 min. Coating the coverslip with poly-L-lysine can aid in this process. The coverslip can then be mounted on a slide as described below. Alternatively, if the bacterial cells are sufficiently adherent to the coverslip, staining can proceed after the cells have adhered to the coverslip.

We suggest two methods of immobilizing bacteria for live cell imaging although there are other methods available. In both methods, a semisolid pad is made and bacteria are added to the pad, then coverslipped. This creates a monolayer of cells and immobilizes them. Alternatively, for osmotically fragile bacteria, create a pressure buffer with an adhesive product such as double-sided tape or a Gene Frame that sticks to standard microscope slides to create a well. Fill the created well with a semisolid medium, add bacteria and coverslip. These immobilization methods can also be used with fixed cells if the cells are not sufficiently adhered to the coverslip and excessive Brownian motion is experienced, increasing the possibility of motion artifacts in the acquired images.

Yeast

Yeast-specific protocols in the literature should be followed for sample preparation. Conventional fixation in 4% paraformaldehyde in sucrose is compatible with fluorescent microscopy (see references below).

Mammalian cells

Maintaining good tissue culture technique is important for the acquisition of physiologically relevant microscopy data. To reproduce phenotypic observations, cells must be healthy and growing normally at the start of any experiment.

For adherent cells:

• For live or fixed cell imaging, consider the imaging substrate. As imaging vessels have a glass bottom, test cell growth and viability on glass. It is important to confirm that the vessel will allow the cells to remain viable and undergo the anticipated phenotypic behavior for the experiment. If glass affects normal cell growth, viability, or behavior, consider coating the coverslips or glass-bottomed dishes with collagen, fibronectin, or poly-L-lysine for proper adhesion of the cells to the glass. For live or fixed cell imaging, cells can be grown in a multiwell imaging chamber or in a 35 mm dish fitted with a #1.5 coverslip. Alternatively, for imaging fixed cells, #1.5 coverslips can be placed into the bottom of a plastic multiwell tissue culture plate, and cells can be grown, fixed, and stained in the same well, followed by mounting on a slide for imaging. Imaging a well-spread, thin layer of cells always yields better images.

• Use freshly thawed cells and avoid prolonged culture or multiple passages. Validate the cell-seeding density as well as the frequency of the labeled protein or structure to ensure that there are sufficient target cells available for imaging. Preliminary experiments will help to define the culture duration and seeding density required to have healthy cells growing for imaging.

Fig 2. Place samples on the coverslip, not the slide.

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For non-adherent cells:

• Consider using a shallow flow cell or microfluidic chamber where cells are loaded to keep them within a restricted height for imaging.

• Consider using a gel matrix substrate to restrict cell motility during observation.

Tissue

For fixing tissue samples, preliminary tests should be performed to determine the optimal fixative and duration to ensure proper preservation of all antigens throughout the sample. This is particularly important to allow penetration of fixative into thicker samples. Following fixation, samples can be paraffin-embedded or cryopreserved for sectioning directly onto the coverslip followed by staining, or thick samples can be directly stained for confocal imaging.

When preparing tissue samples, consider whether the samples will be imaged using SIM and/or confocal microscopy, as the methods for sample preparation are dependent on the imaging modality.

For SIM imaging, live samples or fixed tissue sections should be mounted directly onto the coverslip. The main challenge for SIM is background fluorescence from the sample beyond the imaging range. The solution is to limit tissue sample thickness to the minimum needed to answer the biological question, but within a 30 µm imaging limit. Though imaging the first 50 µm from the coverslip is possible, out-of-focus light from the thick sample can affect resolution. To improve resolution in thicker samples, consider the use of clearing agents.

For confocal imaging, the same process for SIM tissue sample preparation can be followed. Unlike SIM, confocal microscopy removes out of focus information from the scan, which allows serial sectioning analysis of thick fixed or living samples. If imaging of thicker samples is desired (tissue sections > 20 µm, whole mount tissues, organoids, or spheroids), samples can be differentially mounted to allow added space for the sample. Sample-specific considerations will define the appropriate mounting or imaging agent and the proper vessel for imaging. For example, the sample thickness will define whether samples are mounted on a coverslip and covered with a slide with a spacer, or alternatively, imaged in a 35 mm dish containing an appropriate imaging medium. Consider the addition of an antifade reagent to the sample to prevent photobleaching.

References

For more information on imaging bacteria, see the following references.

1. Bottomley, A. et al. Immobilization techniques of bacteria for live super-resolution imaging using structured illumination microscopy. Methods Mol Biol. 1535, 197–209 (2017). doi: 10.1007/978-1-4939-6673-8_12.

2. Turnbull, L. et al. Super-resolution imaging of the cytokinetic Z ring in bacteria using fast 3D-structured illumination microscopy (f3D-SIM). J. Vis Exp. 91(e51469), 1–13 (2014). doi: 10.3791/51469.

For preparation of yeast samples, see the following references:

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.

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.

3. Burns, S. et al. Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication. eLife 4:e08586 (2015). doi: 10.7554/eLife.08586.

For references of mammalian cell culture and imaging, see the following references.

For more information on cell culture best practices, including information on maintaining healthy cells, seeding density, growth media, sub-culturing, culture vessels, surface coatings and more, see the ATCC Animal Cell Culture Guide “tips and techniques for continuous cell lines.”

For more information on imaging cells in a collagen matrix, see:

1. Artym, V. and Matumoto, K. Imaging cells in three-dimensional collagen matrix. Curr. Protoc. Cell Biol. 48: 10.18.1–10.18.20 (2010). doi: 10.1002/0471143030.cb1018s48.

2. Application note 26: Fabrication of Collagen I Gels. Ibidi Version 2.4 (2014) Retrieved from: https://ibidi.com/img/cms/support/AN/AN26_CollagenI_protocols.pdf

For more information on 3D assays, see:

1. Ibidi. 3D Cell Culture Assays [website section] Retrieved from https://ibidi.com/content/189-3d-assays

For more information on microfluidic systems, see:

1. Lee, P. et al. Microfluidic systems for live cell imaging. Methods Cell Biol. 102, 77–103 (2011). doi: 10.1016/B978-0-12-374912-3.00004-3.

2. Tam, J. et al. A microfluidic platform for correlative live-cell and super-resolution microscopy. PLoS ONE 9(2014), 1–20 (2014), doi: 10.1371/journal.pone.0115512.

For examples of preparation of tissue samples, see the following references:

1. Klingberg, A. et al. Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J. Am. Soc. Nephrol. 28(2), 452–459 (2017). doi: 10.1681/ASN.2016020232.

2. Li, W. et al. Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D). PNAS. 114(35), 7321–7330 (2017). doi: 10.1073/pnas.1708981114.

3. Richardson, D.S. and Lichtman, J. W. Clarifying tissue clearing. Cell, 162(2), 246–257 (2015). doi: 10.1016/j.cell.2015.06.067.

4. Silvestri, L. et al. Clearing of fixed tissue: a review from a microscopist’s perspective. J. Biomed. Opt. 21(8), 1–8 (2016). doi: 10.1117/1. JBO.21.8.081205.

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Fixed or live sample staining and mounting

Selecting imaging reagents

Selecting bright and stable fluorophores, fluorescent proteins (FPs), and stains is a critical part of experimental design. Always verify that the reagents match the illumination profile of the microscope. For DeltaVision Ultra or DeltaVision Elite, see Table 2 for a list of recommended fluorophores, FPs, and stains. For DeltaVision OMX SR and DeltaVision OMX Flex, see Table 3. For other models, see the system-specific documentation for the excitation and emission filter specifications. When using free spectral viewers, confirm that the reagent has an illumination profile matching the system, that it has good signal, and does not bleach rapidly.

When selecting reagents for SIM on DeltaVision OMX, choose the shortest wavelengths possible for the finest structures within the sample, as SIM resolution is wavelength dependent. For example, for two-color immunofluorescence experiments, choose reagents matching the 488 and 568 laser lines.

Table 2. Recommended reagents for imaging with a DeltaVision Elite/Ultra microscope

DeltaVision Elite/Ultra Channels

Common fluorophores, fluorescent proteins, and stains

Blue DAPI, Hoechst™, CF™ 405M, ATTO™-425

Cyan mCerulean, CFP, mTurquoise

Green

GFP, mEmerald, mNeon, Cy™ 2, CFTM 488A, Alexa Fluor™ 488, ATTO-488, Calcein AM, CellTracker™ Green, MitoTracker™ Green FM, ER Tracker™ Green, CellMask™ Green, LysoTracker™ Green, FM1-43

Yellow TOTO™-1, YFP

OrangeCy3, Alexa Fluor 555, Propidium iodide, mOrange, CellTracker Orange, CellTracker CM-DIL, CellMask™ Orange, MitoTracker Orange

Red

mRuby3, mKate2, mApple, Td-Eos, mEOS2, Alexa Fluor 568, Alexa-Phalloidin, Ethidium homodimer, CellTracker Red, MitoTracker Red, ER-Tracker Red, LysoTracker Red, FM4-64

Far RedCy5, Alexa Fluor 647, TO-PRO™-3, SiR, DRAQ5™, CellMask Deep Red, MitoTracker Deep Red FM, LysoTracker Deep Red

Table 3. Recommended reagents for imaging with a DeltaVision OMX SR/Flex microscope

DeltaVision OMX SR/Flex Laser Lines

Common fluorophores, fluorescent proteins, and stains

405 DAPI, Hoechst, CF 405M

488GFP, mNeonGreen, EmGFP, Alexa Fluor 488, ATTO-488, CellMask™ Green, LysoTrackerTM Green, MitoTracker™ Green FM, ER-Tracker™ Green

561/568mRuby3, mApple, tdTomato, TMR, Alexa Fluor 568, MitoTracker Red, ER-Tracker Red, LysoTracker Red

640/642Cy5, Alexa Fluor 647*, TO-PRO-3, SiR, CellMask Deep Red, MitoTracker Deep Red FM, LysoTracker Deep Red

*Must have antifade for SIM imaging.

Optimizing immunofluorescence assays

Optimization of the immunofluorescence protocol for a high signal-to-noise ratio is important for all imaging but absolutely critical for successful SIM imaging to avoid introducing artifacts into SIM reconstructions. Use best practices, reagents, and techniques at every step, from experimental design through sample preparation and sample mounting.

Consider the following when performing assay optimization:

• Test different fixation methods, such as formaldehyde, glutaraldehyde, or alcohols, depending on the proteins to be investigated. This is important to preserve the native structure of the specimen.

x Typically, tissue culture cells are fixed for at least 10 min at room temperature (RT) with 4% formaldehyde in PBS. Always follow this step with 2–3 washes with PBS to remove excess formaldehyde.

x When using formaldehyde, always prepare a fresh 4% solution or use a fresh ampule.

x Add a quenching step following fixation with aldehyde-based fixatives. This quenches any excess aldehydes, minimizes autofluorescence from the aldehydes and stops the fixation reaction. A simple method is a 5-min incubation in 100 mM glycine, followed by 2–3 washes in PBS. Always use freshly prepared glycine solution.

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• 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 pre-existing antibodies. Select primary antibodies that strongly localize to structure of interest and have a low level of background signal.

• Always centrifuge antibody dilutions before using to avoid precipitates.

• Do not shorten or reduce the number of washes. Proper washing is essential to remove non-specific staining and ensure low background signal.

• To stabilize the label following primary and secondary labeling, consider adding a second short fixation step along with additional washes to remove the fixative.

Choosing a secondary antibody

When choosing a fluorophore, select based on the illumination profile of DeltaVision Elite/Ultra or the laser lines on the DeltaVision OMX. Choose the host species for the primary antibody, the target species of the secondary antibody, and then the conjugate (fluorophore) to identify an antibody appropriate for the experiment. Highly cross-adsorbed antibodies are recommended.

Mounting samples

A common mistake in preparing samples for high- and super-resolution microscopy is carelessness with mounting techniques and selection of mounting medium. Select the mounting medium carefully and use good technique in mounting the specimen on the coverslip (if it isn’t already fixed there).

Select a mounting medium that does not contain DAPI or any other dye. Dye in the mounting medium increases background signal and reduces the signal to noise ratio required for quality images.

Use soft or non-setting mounting media. In hardening mounting media, the samples compress as they cure and shrink in the axial

(Z) direction. In contrast, when using a soft mountant, compression does not occur, and sample morphology is preserved. Additionally, to minimize changes in the RI, a glycerol-based soft mounting medium is recommended. Glycerol-based media are hydrophilic and do not cause shrinkage or expansion of the sample. Finally, whenever possible, select a mounting medium containing an antifade compound to reduce sample photobleaching.

Table 4. Recommended commercially available (ready–to–use) mounting media for high- and super-resolution microscopy

Reagent Soft Mount

Hard Mount RI Note

SlowFade™ Antifade Reagents 1.42

Useful for photosensitive dyes and FPs, see website to select which formulation for the dye or FP

Ibidi Mounting Medium

1.42–1.44

Convenient dropper bottle

Vectashield™ H1000 1.45

Great all-purpose choice, also works for most bacterial samples, recommended for SIM

Phenylene-diamine (PPD) 1.46

Works for most bacterial samples, but oxidation must be monitored, not suitable for CyDye™ fluorophores

ProLong™ Gold Antifade Mountant 1.47

Only recommended for SIM when not cured

ProLong Diamond Antifade Mountant 1.47

Only recommended for SIM when not cured

Pro tips for working with mounting media

• When working with soft mounts such as PPD or Vectashield H1000, completely seal the coverslip with nail polish to prevent sample oxidation.

• Increased viscosity of glycerol in mountants may affect bacterial morphology. To avoid these effects, the glycerol in the mounting medium can be diluted to 50% with PBS.

• When working with hardening mountants like ProLong Gold, or ProLong Diamond, seal the sample with nail polish immediately after mounting. Sealing the sample significantly delays the curing and hardening of the ProLong solution. The RI will be maintained across the sample and the 3D structures will be preserved. Imaging the sample within 7 days is recommended as the ProLong will eventually harden and shrink. This can be a good alternative to using Vectashield.

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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.

• Do not image broken coverslips as they will be tilted.

• Thoroughly remove PBS and pre-incubate the sample in a small amount of mounting medium prior to mounting to improve consistency of RI throughout the sample.

• To mount the slide on the coverslip: place the coverslip on a lab bench, add mounting medium, angle the slide above the coverslip, and lower slowly until the coverslip mounts. This will minimize bubbles that refract light and cause artifacts.

• Seal the coverslips to the slide with two coats of nail polish (with 10 min between coats to allow for curing) to ensure proper sealing. This is important to hold the sample in place and to prevent oxidation.

• Do not use clear nail polish for sealing coverslips as it does not set and bond strongly. It is easy to accidentally dislodge the coverslip when using clear nail polish.

• After sealing, perform a final wash of the outer coverslip surface with double distilled water (ddH2O) and dry thoroughly. Compressed air can be used to ensure the slide has dried completely.

• Always store the slides in the dark at 4ºC to minimize photobleaching.

References

For a discussion of immunofluorescence:

1. Donaldson, J. Immunofluorescence staining. Current Protoc. Cell Biol. 69(4.3) 1–7 (2015). doi: 10.1002/0471143030.cb0403s69.

For a discussion of fluorescence, see:

1. Murphy, D, and Davidson, M. Fluorescence microscopy, in Fundamentals of Light Microscopy and Electronic Imaging, 2nd ed. Wiley-Blackwell, New Jersey, pp. 1–538 (2013).

For a discussion of immunohistochemistry, see:

1. IHC WORLD (www.ihcworld.com/)

2. Taylor, C.R. and Rudbeck, L. eds. Immunohistochemical Staining Methods, 6th ed., Dako Denmark A/S (2013)

3. O’Hurley, G. et al. Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol. Oncology 8, 783–798 (2014). doi: 10.1016/j.molonc.2014.03.008.

4. Ramos-Vara, J. Principles and methods of immunohistochemistry. Methods Mol. Biol. 691 83–96 (2010). doi: 10.1007/978-1-60761-849-2_5.

For SIM sample preparation, see:

1. Kraus, F. et al. Quantitative 3D structured illumination microscopy of nuclear structures. Nat. Protoc. 12(5), 1011–1028 (2017). doi: 10.1038/nprot.2017.020.

For a discussion of fluorophores and imaging controls for SIM experiments, see:

1. Demmerle, J. et al. Strategic and practical guidelines for successful structured illumination microscopy. Nat. Protoc. 12(5), 988–1010 (2017). doi: 10.1038/nprot.2017.019.

2. Pronobis, M. et al. A novel GSK-3 regulated APC: Axin interaction regulates Wnt signaling by driving a catalytic cycle of efficient β-catenin destruction. eLIFE 4(e08022) (2015). doi: 10.7554/eLife.08022.

3. Van de Mark, D. et al. MDM1 is a microtubule-binding protein that negatively regulates centriole duplication. MCB 26(21), 3788–3802 (2015). doi: 10.1091/mbc.E15-04-0235.

For more information on mounting media, including recipes, see:

1. Biomicroscopy. Retrieved from http://biomicroscopy.ucsf.edu/mediawiki/index.php?title=BioMicroscopy.

2. Collins, T. Mounting Media and Antifade reagents. Wright Cell Imaging Facility.

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Additional considerations for live cell sample preparation Live cell imaging has been instrumental in scientific discoveries that characterize dynamic cellular processes. From whole organism development to intracellular processes, live cell imaging has led to breakthroughs in many areas of biology. Increasingly, researchers also turn to live cell imaging to validate results obtained from imaging fixed cells. Whether validating fixed cell imaging results or examining dynamic processes, it is imperative to properly reproduce an appropriate cellular environment for the type of organism being imaged.

Here we discuss the most important considerations of live cell imaging with respect to sample preparation. See the "Live cell instructions" for the DeltaVision Elite, Ultra, or OMX microscope for operating instructions. For more information, see the Additional GE Healthcare cell analysis publications section at the end of this document.

Cell culture media

Cells must be maintained in media that will support the physiological conditions of study. After selecting an appropriate medium, confirm that the cell culture media is non-fluorescent by imaging an area of the sample dish where there are no cells. Mammalian cell culture media commonly contain a fluorescent compound called Phenol Red. If the medium contains Phenol Red, transfer cells to an equivalent Phenol Red-free medium. Usually, at least two passages are needed to eliminate the Phenol Red.

It is imperative to start the experiment with healthy cells in a sufficient volume of medium in the imaging chamber. This is important to maintain normal physiology and buffer against pH and temperature changes over the course of the imaging experiment.

Environmental controls

DeltaVision microscopes have optional environmental control modules that regulate humidity, gas, and temperature during imaging. Contact a Sales representative to learn more about available options for the DeltaVision system.

Temperature

Cells must be maintained at a suitable physiological temperature to maintain a healthy state. Depending on the organism, this may range from room temperature up to 45°C. Allow several hours for the imaging environment to fully equilibrate before commencing an experiment. This allows for the stable maintenance of focus during the experiment. See the user instructions for the live cell module for more details.

Humidity

Most cells will need to be maintained in a culture medium with a constant concentration of salts and other nutrients. For these reasons, providing a humid environment with minimal evaporation is very important. Large increases in the solute concentration due to evaporation of the cell culture medium may induce physiological changes in the cells unrelated to the experimental aims.

To maintain the sample in a humid environment, use the cell cover provided with the live cell module for the DeltaVision microscope. If unavailable, consider using a breathable membrane or overlay mineral oil to decrease evaporation and maintain gas exchange.

Gas and pH balance

Most cells have a requirement for a certain level of oxygen to be supplied for normal growth and development. For some cells, this may be very low and for others, normal ambient air is sufficient. The use of CO2 may also be necessary to maintain the correct pH of the medium. In the absence of a DeltaVision live cell module to regulate CO2 and/or O2 levels, or with short-term live cell imaging (less than 4 h), use an alternative buffer such as ~ 20 mM HEPES. Live cell imaging media containing HEPES are commercially available. As always, be certain that phenotypic observations are reproducible following the culture media change.

Labeling

In designing a live cell imaging experiment, consider the use of fluorescent proteins (GFP, YFP, mCherry, etc.), self-labeling enzymes attached to a fluorescent ligand (HaloTag™, SNAP-tag™, CLIP-tag™, Click-iT™, etc.), cell permeable biarsenical dyes (FlAsH, ReAsH), or dyes compatible with live cells (Di family, FM143, SiR dyes, CellTracker™ dyes, Lysotracker, MitoTracker, DRAQ5, etc.). See Table 2 for a list of common compatible fluorophores, fluorescent proteins, and stains. In selecting a fluorescent protein, consider photostability, maturation time, and quantum efficiency. Replacing an old fluorescent protein with a more modern one may improve signal stability over time.

Transfection and transduction

For live cell imaging of fluorescent proteins in cell lines, consider using stable transfection or transduction where the fluorescent protein is incorporated into the chromosomal DNA. Transduced cells tend to be healthier, and cell lines can be maintained for consistent results. It is also possible to increase the proportion of fluorescently labeled cells in the population by selective sorting. Lentiviral-induced stable cell lines are an excellent choice.

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Place into

Place into

Secure with

Universal Lid*

*Beveled side facing UP

OMX SR and OMX FLEX

35mm dish

LabTekTMII Chamber

µ-Slide Adapterµ-Slide Assembly

µ-Slide

Slide Adapter

Place into

35 mm Dish

Chambered

CoverglassChambered Coverglass

Sample Holder

Universal Lid*Place into

Place into

Secure with

Slide Adapter

Final Assembly35 mm Dish

Sample Holder

Place into

*Beveled side facing UP

Fig 3. DeltaVision OMX SR and OMX Flex sample holder assembly.

Fig 4. DeltaVision Elite and DeltaVision Ultra sample holder assembly. See DeltaVision Elite Quick Reference #6 on the environmental control system (29175413) or the DeltaVision Ultra Live Cell Module Application Guide (29308413) for more information.

Place into

Imaging dishes

Before beginning an imaging experiment, test cell growth in the selected imaging chamber and examine cell health and viability. Depending on the cell type, coating the dish with fibronectin, poly-L-lysine, or collagen can improve viability and cell morphology. For many imaging chambers, coated products are commercially available. See Table 1 for a list of recommended products.

Sample holder assembly

The DeltaVision line of microscopes has sample holder kits optimized for recommended live cell imaging chambers (Table 1). The kits for the DeltaVision Elite and DeltaVision Ultra microscopes include adapters for 35 mm dishes, LabTek II Chambered coverglass, and LabTek II Chambered slides. The kits for DeltaVision OMX SR/Flex include adapters for the 35 mm dishes and the LabTek II Chambered coverglass. Contact your local Sales representative to order the system appropriate sample holder assembly. Assembly diagrams for these kits are provided in Figures 3 and 4.

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Imaging conditions

For acquisition of physiological data, it is essential to expose cells to the least amount of light possible over the course of the imaging experiment. Here are some of the most important factors to consider:

• Use much less excitation light than for fixed cell imaging. As a rule of thumb, using a cell fluorescence signal that is 5× the surrounding background fluorescent signal is often sufficient. Remember that it is far more important to image healthy cells than to acquire a movie of dying cells.

• Use the minimum number of wavelengths that will answer the biological question.

• Where possible, use non-fluorescent techniques such as bright-field, differential interference contrast (DIC), or phase contrast imaging.

• Avoid short wavelengths that cause the most cellular damage.

• Acquire as few z slices as possible to obtain the information needed.

• Minimize the number of timepoints acquired. Consider the dynamics of the visualized event and determine sampling frequency accordingly.

• Consider whether multiple points or positions can be acquired during one experiment to increase sample size.

• Acquire fields of view that are sufficiently far apart that they are not exposed to light from a neighboring field of view.

Tips for beginners

• Think carefully about the design of the imaging experiment ahead of time.

• Maintain samples, oils, and imaging chamber at the optimal imaging temperature.

• Turn on the environmental chamber early: doing this the night prior to imaging is a good idea.

• Start small with only one sample and a minimally complex experiment.

• Do not expose cells to unnecessary fluorescent light.

• Do the minimum possible to answer the biological question.

References

For more information on live cell imaging best practices and techniques, see:

1. Frigault, M. et al. Live-cell microscopy—tips and tools. J. Cell Sci. 122, 753–767 (2009). doi: 10.1242/jcs.033837.

2. Goldman, R. et al. Live cell imaging, a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, Woodbury. NY (2010).

3. Science/AAA Custom Publishing Office (Producer) Live Cell Imaging, the future for discoveries.

4. Labroots (Producer) Live cell imaging: observing changes as they happen Retrieved from https://www.labroots.com/webinar/live-cell-imaging-observing-changes-happen (2017).

For more information on live cell imaging tools, see:

1. Lukinavičius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11(7), 731–737 (2014). doi: 10.1038/nmeth.2972.

2. Specht, E, et al. A critical and comparative review of fluorescent tools for live-cell imaging. Annu Rev Physiol 79, 93–117 (2017). doi: 10.1146/annurev-physiol-022516-034055.

3. Xue, L. et al. Imaging and manipulating proteins in live cells through covalent labelling. Nat Chem Biol. 11(12), 917–923. doi: 10.1038/nchembio.1959.

For more information on transfection and transduction, see:

1. Viral transfection. [website section] Retrieved from https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/viral-delivery.html

gelifesciences.com/DeltaVisionGE, the GE monogram, Cy, CyDye, and DeltaVision are trademarks of General Electric Company. ATTO is a trademark of ATTO-TE GmbH. CF is a registered trademark of Biotium. DRAQ5 is a registered trademark of Biostatus Ltd. HaloTag is a registered trademark of Promega Corporation. Greiner is a registered trademark of Greiner Diagnostic AG. Sensoplate is a registered trademark of Greiner Bio-One. Hoechst is a registered trademark of Hoechst GMBH. Ibidi is a registered trademark of ibidi GmbH. iPhone is a registered trademark of Apple Inc. MatTek is a registered trademark of MatTek Corp. SNAP-tag, CLIP-tag are registered trademarks of New England Biolabs Inc. TOTO-1, Nunc, CellTracker, Image iT, ProLong Gold, Lab-Tek, Click-iT, Alexa Fluor, TO-PRO-3, ER-Tracker, MitoTracker, CellTracker, CellMask, LysoTracker, ProLong, and SlowFade are registered trademarks of Thermo Fisher Scientific. Vectashield is a registered trademark of Vector Laboratories. All other third-party trademarks are the property of their respective owners. Cy and CyDye are trademarks of General Electric Company or one of its subsidiaries. The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes. A license to use the Cy and CyDye trademarks for commercial purposes is subject to a separate license agreement with GE Healthcare. Commercial use shall include: 1. Sale, lease, license or other transfer of the material or any material derived or produced from it. 2. Sale, lease, license or other grant of rights to use this material or any material derived or produced from it. 3. Use of this material to perform services for a fee for third parties, including contract research and drug screening. If you require a commercial license to use the Cy and CyDye trademarks please contact LSlicensing@ge.com. © 2019 General Electric Company. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. DeltaVision OMX and DeltaVision Ultra microscopes are for research use only - not for use in diagnostic procedures. DeltaVision OMX is a Class 1 laser product. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden GE Healthcare Bio-Sciences Corp., 100 Results Way, Marlborough, MA 01752, USA GE Healthcare Europe GmbH, Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare Japan Corp., Sanken Bldg., 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan GE Healthcare UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK For local office contact information, visit gelifesciences.com/contact

KA2388280619AN

Summary checklist ; Use a #1.5 (170 µm) thickness coverslip, 35 mm dish, multiwell

plate, chambered coverglass, or chambered slide.

; Mount the sample ON the coverslip, or as close to the coverslip as possible.

; Select fluorescent proteins, fluorophores, and dyes that match the illumination profile of the microscope chosen, have good signal, and do not bleach rapidly.

; Optimize the fixation and immunofluorescence method to maximize signal-to-noise in the sample.

; Use mounting media that does NOT contain DAPI or any other dye.

; Use immersion oil optimized for the sample preparation.

; Use the appropriate experimental controls.

; Do not image broken coverslips.

Additional GE Healthcare Cell Analysis publicationsSee the references below for more information on using the live cell module for the DeltaVision microscope.

DeltaVision Elite

1. Quick Reference #6 on the Environmental Control System (29175413)

2. Customer Instructions on CO2 Control System—Installation and Operation (29094268)

DeltaVision Ultra

1. Live Cell Module—Operation and Use (29282123)

2. Live Cell Module Application Guide (29308413)

3. Oil Optimization Application Guide (29308412)

DeltaVision OMX SR and Flex

1. Quick Reference #4 on SIM Sample Preparation (29193027)

2. Immersion Oil Optimization Application Guide (29250659)

3. Localization Microscopy Sample Preparation (29196185)

4. Live Cell Module (29144496)