TITLE: No Title
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[DESCRIPTION]
<p>The basic imaging process for MERFISH involves several fluid handling steps, e.g. introduction of hybridization buffers and wash and imaging buffers, in conjunction with the collection of smFISH images and the photobleaching of the sample. While it would be possible to perform these fluid exchange and imaging steps manually, we strongly recommend the use of an integrated and automated fluid exchange and imaging approach. Here we provide protocols for how to construct a fluid handling system that should be compatible with a variety of microscopes. We also discuss the basic protocols for hybridizing readout probes to samples on the microscope, imaging the sample, and bleaching residual signal.</p>

[GUIDELINES]
<p><strong>The protocol requires the following reagents:</strong></p>
<p> </p>
<p>1. Readout probe hybridization buffer</p>
<ul>
<li>25 mL 20% w/v dextran sulfate (Section 5.1)</li>
<li>5 mL 20X SSC</li>
<li>5 mL 100% deionized formamide</li>
<li>14.5 mL nuclease-free water</li>
<li>0.5 mL 200 mM VRC</li>
<li>Make fresh each day</li>
</ul>
<p> </p>
<p>2. Imaging buffer master mix</p>
<ul>
<li>In an RNAse-free bottle (such as an empty nuclease-free water bottle) add the following</li>
<li>50 mL 20X SSC</li>
<li>25 mL 1 M TrisHCl pH 8 (Ambion; AM9856)</li>
<li>325 mL nuclease-free water</li>
<li>50 g glucose (Sigma; G8270)</li>
<li>A D/RNaseFree-treated stir bar</li>
<li>Stir until glucose is dissolved</li>
<li>Add nuclease-free water to 500 mL</li>
<li>Store at room temperature for up to a few weeks</li>
</ul>
<p> </p>
<p>3. Trolox solution</p>
<ul>
<li>20 mg (±)-6-hydrozy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox; Sigma; 238813)</li>
<li>200 μL methanol (Sigma; 322415)</li>
<li>Make fresh each day</li>
</ul>
<p> </p>
<p>4. Imaging buffer</p>
<ul>
<li>40 mL imaging buffer master mix</li>
<li>200 μL Trolox solution</li>
<li>200 μL catalase (Sigma; C30)</li>
<li>42 mg glucose oxidase (Sigma; G2133)</li>
<li>Make fresh and use immediately</li>
</ul>
<p> </p>
<p>5. Photobleaching buffer</p>
<ul>
<li>5 mL 20X SSC</li>
<li>44.5 mL nuclease-free water</li>
<li>0.5 mL 200 mM VRC</li>
<li>Make fresh each day</li>
</ul>
<p> </p>
<p>6. Readout probe wash buffer</p>
<ul>
<li>5 mL 2X SSC</li>
<li>10 mL 100% deionized formamide</li>
<li>34.5 mL nuclease-free water</li>
<li>0.5 mL 200 mM VRC</li>
<li>Make fresh each day</li>
</ul>
<p> </p>
<p>7. Light mineral oil (Sigma; M5904)</p>
<p> </p>
<p> </p>
<p><strong>Components for an automated fluidics system:</strong></p>
<p> </p>
<p>1. Flow/imaging chamber (Bioptechs; FCS2)</p>
<p>2. 5”-long 20-gauge needles (Hamilton; 7750-11)</p>
<p>3. Luer to ¼-28 female fitting adaptor (IDEX; P-655)</p>
<p>4. 1/16” barb to .-28 female fitting adapter (IDEX; P-646)</p>
<p>5.¼-28 fitting (IDEX; XP-202)</p>
<p>6. Clear ETFE tubing, 0.02” inner diameter (McMasterCarr; 5583K52)</p>
<p>7. Clear PVC tubing, 1/16” inner diameter (McMasterCarr; 5233K51)</p>
<p>8. 0.38-mm inner-diameter peristaltic tubing (Pulse Instrumentation; 116-0549-04)</p>
<p>9. Peristaltic pump (Gilson; Minipuls 3)</p>
<p>10. Computer-controlled valve (Hamilton; MVP; 36798)</p>
<p>11. 8-way valve (Hamilton; HVXM 8-5; 36766)</p>
<p>12. 5-minute epoxy (VWR; 300050-778)</p>
<p>13. 25-gauge needles (VWR; BD305125)</p>
<p>14. 18-gauge needles (VWR; BD305196)</p>
<p>15. USB to RS-232 converter (Keyspan; USA-19HS)</p>
<p> </p>
<p> </p>
<p><strong>The protocol requires the following equipment:</strong></p>
<p> </p>
<p>1. FCS2 flow system (Bioptechs; FCS2)</p>
<p>2. Objective heater (Bioptechs; 150803)</p>
<p>3. Automated flow system (see Section 6.2)</p>
<p>4. Microscope capable of smFISH imaging (see Section 6.3)</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Assembly of</strong> <strong>and Operation of Flow System</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Each round of MERFISH hybridization and imaging requires the controlled exchange of four different buffers at precise intervals. For this reason, integration of a fully automated fluid handling system with the microscope control software can significantly facilitate the collection of MERFISH data. Here we describe the construction and operation of the automated fluid system that we have used. This system is constructed around a peristaltic pump (Gilson; Minipuls 3) which controls the flow rate of different buffers through the flow system and a series of valves (Hamilton; MVP) that control which buffer is being pulled across the sample at any given time. The components required for the flow system are listed in Before Starting Section, and a schematic diagram of the flow system required for 16-rounds of hybridization is included in Figure 4. Once assembled, the components of this flow system do not need to be replaced between measurements, unless otherwise specified.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rw6fw26.png" width="479" height="471" data-src="https://s3.amazonaws.com/pr-journal/rw6fw26.png" data-ofn="Screen Shot 2017-12-28 at 12.22.30 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Microscope Requirements</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The physical requirements for a microscope for MERFISH measurements are essentially identical to the requirements for smFISH measurements. Even with many probes per RNA, the signal from individual RNAs is often relatively dim; thus, high numerical aperture objectives and sensitive cameras such as electron-multiplying CCD (EMCCD) or scientific CMOS are often needed. MERFISH requires relatively high laser powers to bleach the sample after each round of imaging; thus, the microscope should be able to illuminate the sample with at least 100 mW of light in the wavelength used to image the labeled RNAs, as measured at the back focal plane of the objective. Only a few mW of light is needed for illumination of the fluorescent fiducial beads. If less illumination light is available in the color channel used for smFISH, it should still be possible to perform MERFISH measurements. The user will simply need to devote more time to photobleaching each region of the sample. Finally, to achieve any reasonable level of automation and throughput with MERFISH, a motorized sample stage will be required as well as some form of automatic focus system.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">For published MERFISH measurements, we used a 1.45 NA, 100X oil immersion Olympus objective, and illuminated our sample with ~200 mW of 641-nm light and ~20 mW of 561-nm light, as measured at the back focal plane of the objective, using solid state lasers (MPB communications; VFL-P500-642; and Coherent, 561-200CWCDRH). The 641-nm laser was used to excite our Cy5-labeled readout probes while the 561-nm laser was used to excite the fiducial beads. A custom dichroic (Chroma, zy405/488/561/647/752RP-UF1) and a custom notch filter (Chroma, ZET405/488/561/647-656/752m) were used to couple this light into the objective and filter the emission. These custom optics were used so that our home-built microscope had the capability of imaging at 750-nm. This color band was not used in our published MERFISH work (Chen et al., 2015), and thus stock dichroics and emission filters would also have worked. For example, the Chroma 89016bs dichroic in combination with the Chroma ZET561/10x and ZET642NF notch filters would also work. The fluorescent signal from the sample was imaged onto an EMCDD camera (Andor; iXon-897) through a QuadView (Photometrics), which used several stock dichroics (Chroma, T560lpxr; T650lpxr, and 750dcxxr) and emission filters (Chroma; ET525/50m, WT59550m-2f, ET700/75m, HQ770lp) to image several different color channels onto different quadrants of the camera. The 640-nm channel, corresponding to our RNA signal, and the 561-nm channel, corresponding to our fiducial beads were excited and imaged simultaneously with this setup. The 750-nm channel allows future work to include probes of this color but again was not used in our published MERFISH work (Chen et al., 2015). However, it would also be possible to forego the use of a QuadView and image each color channel one at a time, using the selective excitation of each laser to discriminate the color channels corresponding to Cy5 and the fiducial beads. That being said, it is crucial that no offset be introduced between the image of the smFISH signal and the image of the fiducial beads; thus, the same dichroic filter cube should be used for both color channels. We collected images corresponding to a 40x40 μm field-of-view with a pixel size of 167 nm in the sample plane. Our sample position was controlled with a motorized stage (Marzhauser) and our focus was maintained with a home-built autofocus system consisting of an objective nanopositioner (Mad City Labs, Nano-F) whose position was locked to the position of a IR laser spot (940-nm) reflected off of the sample-coverslip interface and imaged with a CMOS camera (Thorlabs, uc480).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>MERFISH Imaging Protocol</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The general MERFISH imaging protocol involves the hybridization of each readout probe, a brief wash step to remove some of the non-specifically bound probe, imaging of the sample, and then photobleaching of the sample, before hybridization of the next readout probe.</p>

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