We wish to thank members of the Ugarova laboratory and investigators in the Center for Metabolic and Vascular Biology for helpful discussion of this work. James Faust wishes to express his gratitude to the instructors at the European Molecular Biology Laboratory Super Resolution Microscopy course in 2015. We wish to thank Satya Khuon at Janelia for help with sample preparation for LLSM. During the review and filming portions of this work James Faust was supported by a T32 Fellowship (5T32DK007569-28). The Lattice Light Sheet component of this work was supported by HHMI and the Betty and Gordon Moore Foundation. T.U. is funded by NIH grant HL63199.

Procedures that utilize animals were approved by the Animal Care and Use Committees at Mayo Clinic, Janelia Research Campus, and Arizona State University.
1. Preparing Acid-cleaned Cover Glass
NOTE: Cover glass purchased from many manufacturers may not be as clean as expected. Consider routinely cleaning batches of cover glass before any procedure where microscopy is involved.
<ol>
	<li>Purchase high stringency cover glass. Take special care to choose the correct thickness (0.15 or 0.17 mm). 
	NOTE: The choice of cover glass thickness is dependent on the microscope objective and is listed directly on the objective barrel.</li>
	<li>Incubate the cover glass in 12 M hydrochloric acid in a well-ventilated chemical fume hood for 1 h with sonication (42 kHz, 70 W). Repeat this step for two additional times with fresh 12 M HCl.</li>
	<li>Fill a separate beaker with ultrapure water and add the cover glass. Sonicate the cover glass for 5 - 10 min in a well-ventilated chemical hood. Repeat this step ten times.</li>
	<li>Incubate the cover glass in pure acetone in a well-ventilated chemical hood for 30 min with sonication. Repeat this step for two additional times.</li>
	<li>Fill a separate beaker with sterile ultrapure water and add the cover glass. Sonicate the cover glass for 5 - 10 min in a well-ventilated chemical hood. Repeat this step ten times.</li>
	<li>Incubate the cover glass in 100% ethyl alcohol in a chemical hood for 30 min with sonication. Repeat this step two additional times. 
	NOTE: The purity of the ethyl alcohol is important. Contaminants in the form of dissolved solids will dry on the glass and can promote macrophage fusion.</li>
	<li>For long-term storage, place the acid-cleaned cover glass in a container filled with pure ethyl alcohol. Alternatively, dry the cover glass with nitrogen gas and store in a vacuum desiccator.</li>
</ol>
2. Preparation of Fusogenic Optical-quality Surfaces
<ol>
	<li>Dissolve DMSO-free high-melting point paraffin wax in ultrapure toluene. 
	NOTE: Stock concentrations are made at 10 mg/mL, and are diluted 1:9 in ultrapure toluene to make a 1 mg/mL working solution. 
	Caution: Toluene should be handled with care as a teratogen. Dispose of toluene according to the institutional chemical hygiene plan.</li>
	<li>Apply the paraffin working solution to a dry acid-cleaned cover glass at a volume that evenly covers the glass surface. Decant excess solution and dry the cover glass with nitrogen gas or air. For bulk preparation, use a Coplin jar designed to accommodate the cover glass.</li>
	<li>Polish the cover glass by 3 strokes in the x axis and next by 3 strokes in the y axis with a lint-free wipe (see Table of Materials).</li>
	<li>Immediately before the experiment is conducted, wash the cover glass with sterile ultrapure water, and subsequently sterilize for 15 - 30 min with ultraviolet light in a biological safety hood. Alternatively, sterilize the cover glass by ethylene oxide or gamma irradiation.</li>
</ol>
3. Micropatterning Hydrocarbon-containing Compounds to Confine Fusion to Predetermined Regions
<ol>
	<li>Dry the acid-cleaned cover glass and immobilize the glass on a flat surface.</li>
	<li>Using forceps, carefully immerse a gold transmission electron microscopy finder grid in a working solution of the hydrocarbon compound(s). Choose a hydrocarbon compound that meets the end goal of the experiment (see Table 1). Wick away excess solution by gently tapping the grid on filter paper, and immediately place the grid in the center of the cover glass. Allow the toluene to dry for 2 min before proceeding to the next step.</li>
	<li>Make sure the grid is bonded to the glass by gently inverting the glass. If the grid detaches repeat step 3.2 using a new cover glass. 
	NOTE: If a plasma cleaner is not available, omit step 3.4.</li>
	<li>Plasma clean the cover glass by treatment with vacuum gas plasma. 
	NOTE: The finder grid acts as a mask to protect the underlying surface adsorbed with long-chain hydrocarbons from the plasma. The regions that are unprotected by the grid are rendered non-fusogenic by exposure to plasma. The amount of time the cover glass is expose to plasma should be determined empirically.</li>
	<li>Using fine-tip forceps, carefully remove the grid from the glass surface to expose the micropattern.</li>
</ol>
4. Fabricating Glass Bottom Dishes
<ol>
	<li>Drill a 6 - 10 mm circular hole in the bottom of a 35-mm plastic Petri dish using a step drill bit. 
	NOTE: It is critical to use a step drill bit to create smooth edges. If edges are too rough, the cover glass will not bond flat to the bottom of the dish. Imperfectly flat surfaces make microscopy difficult.</li>
	<li>Carefully mix and degas silicone elastomer according to the manufacturer&#39;s instructions (i.e., polydimethylsiloxane; PDMS).</li>
	<li>Apply a thin coating of elastomer just proximate to the edge of (i.e., circumscribing) the hole. 
	NOTE: The elastomer should appear as a continuous albeit thin band surrounding the hole.</li>
	<li>Gently apply either the fusogenic cover glass (described in section 2), or the dry acid-cleaned cover glass (described in section 1) to the dish in order to cover the hole surrounded by elastomer. Ensure that the cover glass appears flush with the bottom of the dish and extends beyond the diameter of the hole so that a substantial portion of the glass is in contact with the plastic. Cure the elastomer by baking at 50 &#176;C for 2 - 3 h.</li>
	<li>If fusogenic glass is preferred, UV sterilize the dish and culture cells according to standard protocols. However, if a micropattern is preferred, proceed to step 3.2 for micropattern preparation (the gas plasma used during micropatterning sterilizes the dish and strongly couples PDMS to glass).</li>
</ol>
5. Collecting Thioglycollate-elicited Macrophages
<ol>
	<li>Inject 8-week-old C57BL/6 mice with 0.5 mL of a sterile solution of 4% Brewer&#39;s thioglycollate as previously described3,4,6.</li>
	<li>Seventy-two hours later, euthanize the animal according to approved animal care and use guidelines, and collect macrophages by peritoneal lavage with ice-cold phosphate-buffered saline supplemented with 5 mM ethylenediaminetetraacetate.</li>
	<li>Centrifuge the macrophages at 300 x g for 3 min and resuspend in 1 mL of DMEM:F12 supplemented with 15 mM HEPES, 10% FBS, and 1% antibiotics (culture medium). 
	NOTE: The cells are subsequently counted with a Neubauer hemocytometer. Macrophages are diluted to the appropriate concentration and applied to the surfaces. The number of cells to apply to a given surface should be carefully considered by the investigator for the purpose of the experimental question. Consult known standards in the primary literature.</li>
	<li>After 30 min wash the surfaces with HBSS supplemented with 0.1% BSA to remove non-adherent cells, and replace with fresh culture medium. Return the cultures to the incubator (5% CO2 in air at 37 &#176;C).</li>
	<li>2 h later, aspirate the medium and replace with culture medium supplemented with 10 ng/mL of IL-4. Image the cells as described elsewhere4.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Integrins+&#945;+M+&#946;+2+(Mac-1,+CD11b/CD18)+and+&#945;+D+&#946;+2+(CD11d/CD18)+in+Macrophage+Fusion.">The Role of Integrins &#945; M &#946; 2 (Mac-1, CD11b/CD18) and &#945; D &#946; 2 (CD11d/CD18) in Macrophage Fusion.</a> Am J Pathol. 186 (8), 2105-2116 (2016).</li><li>Faust, J. J., Christenson, W., Doudrick, K., Ros, R., Ugarova, T. 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The authors declare that they have no competing financial interests.

The need to identify and subsequently develop optical-quality glass surfaces that promote macrophage fusion stemmed from the fact that until recently no published study directly visualized macrophage fusion in the context of living specimens3,4. This is due to the fact that fusogenic plastic surfaces that are commonly used require LWD objectives and are largely limited to phase contrast optics. These barriers were overcome by engineering an optical-quality glass surface that not only promoted extraordinary rates of macrophage fusion, but imparted a surprising degree of spatial control over the formation of MGCs.
Although this technology enables the use of advanced imaging techniques to monitor macrophage fusion, it does not come without limitation. Although the polished surfaces adsorbed with long-chain hydrocarbons (e.g., oleamide, paraffin wax, etc.) produce extraordinary rates of macrophage fusion compared to fusogenic plastic surfaces, fusion remains a spatially stochastic process (Figure 2). This limitation precludes the use of high magnification objectives to visualize fusion with living specimens since it is impossible to predict where fusion will occur. To overcome this limitation, we micropatterned paraffin to confine the fusion process to predetermined regions (Figure 4; Supplemental Video 1). Spatially confining macrophage fusion to the micropattern enabled us to predict where fusion would occur and therefore utilize higher magnification objectives to study this highly animate event.
What is the significance of using an optical-quality surface that imparts spatial control of macrophage fusion? First, since these surfaces promote high rates of fusion, it is possible to monitor fusion from living specimens within a reasonable timeframe. This is essential in the case of fluorescence microscopy since increased exposure of cells to light leads to photobleaching and phototoxicity. Second, these surfaces enable advanced imaging techniques based on the fluorescence or polarization to be employed (Figure 4; Table 1; Supplemental Video 1; Supplemental Video 2). Finally, the spatiotemporal control afforded by the micropatterned surfaces described in this protocol should expedite studies designed to identify the cellular and subcellular mechanisms that govern macrophage fusion.

The formation of MGCs accompanies a number of pathological states in the human body distinguished by chronic inflammation1. Despite agreement that mononucleated macrophages fuse to form MGCs2, surprisingly few studies have shown fusion in context with living specimens3,4. This is because clean glass surfaces that are required for most imaging techniques do not promote macrophage fusion when induced by inflammatory cytokines5. Indeed, if clean glass is used as a substrate for macrophage fusion, then low to intermediate magnification objectives (i.e., 10 - 20X) and more than 15 h of continuous imaging are often required to observe a single fusion event.
On the other hand, fusogenic plastic surfaces (e.g., permanox) or bacteriological grade plastic promote fusion2. However, imaging through plastic is problematic because the substrate is thick and scatters light. This complicates imaging since long working distance (LWD) objectives are required. However, LWD objectives usually have low light gathering capacity compared to their coverslip-corrected counterpart. Further, techniques that exploit changes in the polarity of light passing through the specimen such as differential interference contrast are impossible since plastic is birefringent. The barriers associated with the use of plastic are further underscored by the fact that it is impossible to predict where macrophage fusion/MGC formation will occur on the surface. Together, these limitations restrict the visualization of macrophage fusion to phase contrast optics, extended total imaging durations (&#62;15 continuous hours), and low resolution.
We recently identified a highly fusogenic glass surface while conducting single-molecule super resolution microscopy with fixed macrophages/MGCs4. This observation was surprising because clean glass surfaces promote fusion at the very low rate of ~ 5% after 24 h in the presence of interleukin-4 (IL-4) as determined by the fusion index4. We found that the capacity to promote fusion was due to oleamide contamination. Adsorption of oleamide or other compounds that similarly contained long-chain hydrocarbons made the glass fusogenic. The most fusogenic compound (paraffin wax) was micropatterned, and it imparted a high degree of spatiotemporal control over macrophage fusion and a 2-fold increase in the number of fusion events observed within the same amount of time compared to permanox. These optical-quality surfaces provided the first glimpse into the morphological features and kinetics that govern the formation of MGCs in living specimens.
In this protocol we describe the fabrication of a variety of glass surfaces that can be used to monitor the formation of MGCs from living specimens. In addition, we show that these surfaces are amenable to far-field super-resolution techniques. Surface fabrication is dependent on the goal of the experiment, and each surface is described with related examples in the proceeding text.

<table><tbody><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PCD-32G</td><td></td></tr><tr><td>Finder grid</td><td>Electron microscopy sciences</td><td>G400F1-Au</td><td>any gold TEM grid will work</td></tr><tr><td>Cover glass (22x22 mm)</td><td>Thor Labs</td><td>CG15CH</td><td>use only high stringency cover glass</td></tr><tr><td>Paraffin wax</td><td>Sigma Aldrich</td><td>17310</td><td></td></tr><tr><td>Petrolatum</td><td>Sigma Aldrich</td><td>16415</td><td>must be &amp;alpha;-tocopherol-free if substituted</td></tr><tr><td>Oleamide</td><td>Sigma Aldrich</td><td>O2136</td><td>prepare fresh</td></tr><tr><td>Isopropanol</td><td>Sigma Aldrich</td><td>278475</td><td></td></tr><tr><td>Toluene</td><td>Sigma Aldrich</td><td>244511</td><td></td></tr><tr><td>Acetone</td><td>VWR International</td><td>BDH1101</td><td></td></tr><tr><td>Ethanol</td><td>Electron microscopy sciences</td><td>15050</td><td>use low dissolved solids ethanol</td></tr><tr><td>Hydrochloric acid</td><td>Fischer Scientific</td><td>A144C-212</td><td>use to acid wash cover glass</td></tr><tr><td>Slyguard 184</td><td>VWR International</td><td>102092-312</td><td>mix in a 1:10 ratio and cure at 50 &amp;deg;C for 4 h</td></tr><tr><td>35 mm petri dish</td><td>Santa Cruz Biotech</td><td>sc-351864</td><td></td></tr><tr><td>Dumont no. 5 forceps</td><td>Electron microscopy sciences</td><td>72705</td><td>ideal for removing TEM grid in section 3.5</td></tr><tr><td>FBS</td><td>Atlanta Biological</td><td>S11550</td><td></td></tr><tr><td>DMEM:F12</td><td>Corning</td><td>10-092</td><td>contains 15 mM HEPES</td></tr><tr><td>Pen/Strept</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>HBSS</td><td>Corning</td><td>21-023</td><td></td></tr><tr><td>BSA solution</td><td>Sigma Aldrich</td><td>A9576</td><td>use at 0.1% in HBSS to wash non-adherent macrophages</td></tr><tr><td>IL-4</td><td>Genscript</td><td>Z02996</td><td>aliquot at 10 &amp;mu;g/mL and store at -20 &amp;deg;C</td></tr><tr><td>C57BL/6J</td><td>Jackson Laboratory</td><td>000664</td><td>use for fixed samples or techniques that do not require contrast agents</td></tr><tr><td>eGFP-LifeAct mice</td><td>n/a</td><td>n/a</td><td>use for live fluorescence imaging</td></tr><tr><td>Kimwipe</td><td>Kimberly Clark&amp;nbsp;</td><td>34155</td><td>use to polish hydrocarbon adsorbed surfaces</td></tr></tbody></table>

fabricating optical-quality glass surfaces to study macrophage fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

Physicochemical parameters of materials have dramatic effects on the extent of macrophage fusion7,8,9,10. Moreover, surface contaminants are known to promote macrophage fusion11. Therefore, it is important to start with clean cover glass as a negative control for macrophage fusion. When cleaned as described in protocol 1, the cover glass is exceptionally flat with no obvious surface features, whereas adsorption of long-chain hydrocarbons followed by surface polishing creates a degree of nanotopography (Figure 1).
On clean cover glass surfaces, macrophages fuse at a very low rate (Figure 2). In contrast, after 24 h in the presence of IL-4, the macrophages applied to surfaces adsorbed with compounds containing long-chain hydrocarbons undergo robust fusion (Figure 2). Further, adsorption of solvent alone (e.g., toluene, isopropanol) does not make glass a fusogenic surface (Figure 2).
Although it is unlikely that a 10-nm layer of adsorbed hydrocarbons dramatically affects resolution, it was nevertheless important to quantitatively assess the potential difference in resolution among the various surfaces. Resolution is typically defined by the Rayleigh criterion. An alternative metric which is often used to approximate resolution is full-width at half maximum (FWHM) of structures smaller than the diffraction limit of light. Importantly, there is no apparent difference in FWHM of 100 nm fluorescent beads among the various surfaces (Figure 3A), suggesting that the characteristics of glass required for fluorescent imaging were sufficiently preserved. Furthermore, we were able to generate an evanescent field and perform 3D direct stochastic optical reconstruction microscopy of the macrophages applied to surfaces containing long-chain hydrocarbons (Figure 3B). A variety of live-imaging techniques are feasible when macrophages are applied to the surfaces (Figure 4, Supplemental Video 1, Supplemental Video 2).
The advantage of each surface is described in Table 1. Note that the majority of advanced imaging techniques that require high NA oil immersion objectives are incompatible with most fusogenic plastic surfaces since they are thick, and have a degree of autofluorescence. It should be noted that surfaces adsorbed with long-chain hydrocarbons enable a large number of imaging techniques including12 PALM/dSTORM/GSDIM13,14,15,16,17, DIC, and others18,19,20,21 (Table 1). Furthermore, not only are a large number of imaging techniques possible (Table 1), but the use of a micropattern enables a high degree of spatiotemporal control over MGC formation (Figure 4B; Supplemental Video 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56866/56866fig1.jpg" /> 
Figure 1: Paraffin adsorbed surfaces have a degree of nanoscale surface topography. AFM scans (5 x 5 &#181;m) of glass surfaces show no obvious topographical features whereas paraffin-adsorbed surfaces contain arrays of material decorating the surface. The intensity scale on the right side of each micrograph shows height along the z-axis. The scale bars are 500 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56866/56866fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56866/56866fig2.jpg" /> 
Figure 2: Surfaces adsorbed with long-chain hydrocarbons promote macrophage fusion on cover glass. (A) In the absence of IL-4, there appears to be very few multinucleated cells on clean glass surfaces as determined by Wright&#39;s stain. (B) After 24 h in the presence of IL-4, surfaces adsorbed with solvent used to solubilize long chain hydrocarbons (e.g., toluene or isopropanol) have few multinucleated cells. (C-E) In contrast, surfaces adsorbed with long-chain hydrocarbons promote fusion and MGC formation. Paraffin adsorption results in the highest degree of IL-4 induced fusion. MGCs are outlined in black. (F) The degree of MGC formation on permanox plastic is shown for comparison. In each image, light blue shows nuclei and the cytoplasm appears purple. The scale bars are 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56866/56866fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56866/56866fig3.jpg" /> 
Figure 3: Surfaces adsorbed with long-chain hydrocarbons do not dramatically affect resolution. (A) Comparison of 100 nm fluorescent beads illuminated with Total Internal Reflection Fluorescence (TIRF) microscopy. The inset in the upper left panel is a magnified view a single fluorescent bead. The red line was superimposed to show an example line profile used to calculate FWHM. No apparent difference in FWHM of 100 nm beads was observed. The scale bars are 5 &#181;m. (B) Comparison of the distribution of Mac-1 integrin as assessed by TIRF and 3D direct stochastic optical reconstruction microscopy. Macrophages were applied to an oleamide-containing surface and incubated with IL-4 for 24 h. The white inset in B highlights the region imaged in subsequent micrographs. The scale bars are 5 &#181;m for the TIRF images and 2 &#181;m for 3D STORM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56866/56866fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56866/56866fig4.jpg" /> 
Figure 4: Surfaces adsorbed with long-chain hydrocarbons enable time-resolved views of macrophage fusion and spreading. (A) The diagram shows the procedure used to generate a micropattern. (B) Micropatterned surfaces impart spatiotemporal control of macrophage fusion. Several hours after the application of IL-4, the macrophages were imaged with phase contrast. Note the migration of cells from the interior of the grid onto the micropattern. Fusion was observed only on the micropattern. The white dashes outline the expansion of a MGC. Time is shown in the upper left corner of each micrograph as hh:mm:ss. The scale bars are 50 &#181;m. (C) Lattice Light Sheet microscopy20 reveals high spatiotemporal dynamics of a macrophage spreading on a paraffin-adsorbed surface. Color encodes the intensity distribution (yellow is most intense) of eGFP-LifeAct. Time is shown in the upper right corner of each micrograph as mm:ss. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56866/56866fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Surface:</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Technique</td>
			<td>Paraffin</td>
			<td>Oleamide</td>
			<td>Petrolatum</td>
			<td>MP paraffin</td>
			<td>Permanox</td>
			<td>Clean glass</td>
		</tr>
		<tr>
			<td>phase contrast</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;*</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>brightfield</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;*</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>DIC</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>N/T</td>
			<td>&#8730;</td>
			<td>x</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>epifluorescence</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;*</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>confocal</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;*</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>TIRF</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>N/T</td>
			<td>x</td>
			<td>x</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>PALM/dSTORM/GSDIM</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>N/T</td>
			<td>x</td>
			<td>x</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>SIM</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>N/T</td>
		</tr>
		<tr>
			<td>STED</td>
			<td>N/T</td>
			<td>&#8730;</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>LLSM</td>
			<td>&#8730;</td>
			<td>N/T</td>
			<td>N/T</td>
			<td>&#8730;</td>
			<td>N/T</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>Fusion Index</td>
			<td>intermediate</td>
			<td>intermediate</td>
			<td>intermediate</td>
			<td>high</td>
			<td>intermediate</td>
			<td>low</td>
		</tr>
		<tr>
			<td>Fusion location</td>
			<td>random</td>
			<td>random</td>
			<td>random</td>
			<td>defined</td>
			<td>random</td>
			<td>random</td>
		</tr>
		<tr>
			<td colspan="7">MP-micropattern</td>
		</tr>
		<tr>
			<td colspan="7">DIC - differential interference contrast</td>
		</tr>
		<tr>
			<td colspan="7">TIRF - total internal reflection fluorescence</td>
		</tr>
		<tr>
			<td colspan="7">PALM - photoactivated localization microscopy</td>
		</tr>
		<tr>
			<td colspan="7">dSTORM - direct stochastic optical reconstruction microscopy</td>
		</tr>
		<tr>
			<td colspan="7">GSDIM - ground state depletion followed by individual molecule return</td>
		</tr>
		<tr>
			<td colspan="7">SIM - structured illumination</td>
		</tr>
		<tr>
			<td colspan="7">STED - stimulated emission depletion</td>
		</tr>
		<tr>
			<td colspan="7">LLSM - lattice light sheet microscopy</td>
		</tr>
		<tr>
			<td colspan="7">&#8730; - compatible</td>
		</tr>
		<tr>
			<td colspan="7">x - incompatible</td>
		</tr>
		<tr>
			<td colspan="7">&#8730;* - possible only with LWD, low magnification, or immersion objectives</td>
		</tr>
		<tr>
			<td colspan="7">N/T - not tested</td>
		</tr>
	</tbody>
</table>
Table 1: Potential uses of each surface. Note that the plastic surfaces preclude the use of most imaging techniques.
<img alt="Movie 1" src="/files/ftp_upload/56866/56866mov1.jpg" /> 
Supplemental Video 1: Time-resolved phase contrast view of MGC formation. Note the apparent synchronicity of the MGC formation. Time is shown in hh:mm:ss in the upper left corner. The scale bar is 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56866/Supplemental_Video1.mp4" target="_blank">Please click here to download this video.</a>
<img alt="Movie 2" src="/files/ftp_upload/56866/56866mov2.jpg" /> 
Supplemental Video 2: Lattice Light Sheet microscopy20 of a thioglycollate-elicited eGFP-LifeAct macrophage spreading on a paraffin adsorbed surface. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56866/Supplemental_Video2.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion at JoVE.com

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

fabricating-optical-quality-glass-surfaces-to-study-macrophage-fusion

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.2K Views.  Mayo Clinic. Dit protocol beschrijft de fabricage van optische kwaliteit glazen oppervlakken geadsorbeerde met verbindingen met lange-keten-koolwaterstoffen die kunnen worden gebruikt voor het controleren van macrofaag fusie van levende specimens en super resolutie microscopie van vaste specimens in staat stelt .

Video: Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biology

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Bioengineering

Microfluidic Droplet Assay for Single-Cell Cytokine Secretion Analysis

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

Author Spotlight: Unveiling the Polyfunctionality and Heterogeneity in Immune Responses

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...

Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.

This protocol describes the fabrication of an implantable integrated imaging window using 3D laser printing. The window consists of a system of microlenses coupled with micro-scaffolds. The method involves two-photon polymerization (2PP) of the biocompatible photoresist SZ2080 in a continuous sequence, optimizing manufacturing efficiency and alignment between the different components.

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

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