The MERSCOPE is an automated platform for the acquisition of high-quality MERFISH data for spatially resolved single-cell transcriptomics. In this video, we will walk you through a general protocol for setting up, running, maintaining, and troubleshooting Vizgen’s MERSCOPE instrument.

Place the MERSCOPE Imaging Cartridge in a 37-degree Celsius water bath at a depth of about 2 centimeters for 45 minutes. Take care not to let the valve contact the water. Record the cartridge barcode number for future reference.

Warm the DAPI and PolyT Staining Reagent for 10 minutes in a 37-degree Celsius water bath.

Keep the Imaging Buffer Activator and RNase Inhibitor in a benchtop cooler until use. Spin down using a benchtop centrifuge before use

Aspirate the Clearing Solution from the sample, ensuring all Clearing Solution is removed from the petri dish. Be careful not to touch the tissue sample. Wash twice with 5 mL of Sample Prep Wash Buffer. Using the lowest setting, gently vortex the tube for around 30 seconds to ensure the reagents are well mixed, and no precipitate is visible before use.
Add 3 milliliters of DAPI and PolyT Staining Reagent. Incubate for 15 minutes on a rocker.

Aspirate the DAPI and PolyT stain. Add 5 milliliters of Formamide Wash Buffer and incubate for 10 minutes. Aspirate the Formamide Wash Buffer. Wash with 5 millilter Sample Wash Buffer. Proceed immediately to MERSCOPE instrument setup.

Click “Start MERFISH” on the display. Allow 1 to 2 minutes for the MERSCOPE Instrument to initialize. Enter your experimental details. Select the applicable panel specific MERSCOPE Codebook.

Click Next to advance through configuration screens to the sample properties screen. Specify the sample thickness. If sample thickness is unknown, 10 micrometers should be selected.

Enter details of any additional auxiliary staining to a maximum of 9, including 3 cell boundary stains that will be imaged in addition to the MERFISH measurement. DAPI and PolyT stains are imaged automatically. At the end of this configuration, a Configure Summary will appear. Click next when ready.

IF the sample was stained with the MERSCOPE Cell Boundary Stain Kit, toggle on Cell boundary stains under Additional Stains.

Toggling on Cell boundary stains automatically enables Aux 1-3.

IF the gene panel contains sequential genes, check the corresponding auxiliary bit and select RNA from the menu to the right of the auxiliary bit under Additional Stains.

Navigate to the panel summary page for a constructed gene panel in the MERSCOPE Gene Panel Design Software. Sequential genes are listed along with the assigned auxiliary bits.

IF the sample was stained with one or more MERSCOPE Protein Stain Kits, check the corresponding auxiliary bit(s) under Additional Stains.

Select the illumination intensity Protein (Bright), Protein (Medium), or Protein (Dim) for each channel from the menu to the right of the auxiliary bit under Additional Stains.

Users should have established the illumination intensity for each channel during verification. Refer to the MERSCOPE Protein Stain Verification Kit User Guide for more information.

Click Next. At the end of configuration, a Configure Summary will appear.

If the Configure Summary is satisfactory, click Next. DAPI and PolyT stains are always imaged automatically. Names of additional stains can be changed after they are enabled by clicking on the box.

We will now demonstrate how to activate and load the MERSCOPE Imaging Cartridge.

Open the imaging cartridge lid and discard any previously used MERSCOPE Imaging Cartridge remaining. Remove the fully thawed MERSCOPE Imaging Cartridge from the water bath and completely dry the outer surfaces. Ensure the barcode is free of any precipitation. Keep a record of the barcode number for reference.

Invert the thawed MERSCOPE Imaging Cartridge 10 times to ensure the cartridge reagents are mixed. Do this before piercing the Cartridge Activation Port. Spray RNaseZap solution onto a Kimwipe and wipe to clean the imaging cartridge. Then repeat with 70% ethanol. Wipe dry.

Prepare the Imaging Activation Mix, which consists of 250 microliters of Imaging Buffer Activator and 100 microliters of RNase Inhibitor.

Using a pipette tip, pierce the foil of the Activation Port, located on the rear left-hand side of the cartridge. Ensure that the foil in the port is completely open.

Using a 1 milliliter pipette, carefully add the Imaging Activation Mix into the Activation Port. Make sure the pipette tip is immersed in the liquid in the Imaging Cartridge before dispensing to avoid introducing air bubbles. Thoroughly mix the solution by carefully pipetting up and down 10 times. Avoid introducing air bubbles.

Carefully layer 15 milliters of mineral oil on top of the liquid in the cartridge via the Cartridge Activation Port.

Insert the activated imaging cartridge into the MERSCOPE Instrument with the valve toward the back and the barcode facing front. Close the MERSCOPE Instrument lid and click Scan Barcode. If the validation is successful, click Prime Fluidics. The instrument will proceed to prime the fluidics.

The MERSCOPE Instrument scans the MERSCOPE Imaging Cartridge barcode for compatibility with the selected MERSCOPE Codebook. If the MERSCOPE Instrument cannot read the MERSCOPE Imaging Cartridge barcode, the barcode number may be entered manually.

We will now demonstrate how to prepare the MERSCOPE Flow Chamber. Open the flow chamber lid and unlock the flow chamber from the stage adapter. Remove the flow chamber and detach the fluidic lines from the instrument.

In general, the MERSCOPE Flow Chamber should be lifted out of, and placed into, the stage adapter by gently holding the fluidic lines to either side of the flow chamber and lifting or placing vertically. For example, do not tilt the Aqueduct.

Click Next to advance through screens as operations are performed.

The MERSCOPE flow chamber is comprised of several parts, including the Top, Aqueduct, Gasket, MERSCOPE Slide, and Base.

Disassemble the flow chamber by rotating the top component counterclockwise. Remove and discard the MERSCOPE slide from previous run. Clean the MERSCOPE Gasket and Aqueduct with RNaseZap solution followed by 70% ethanol. Wipe dry.

Remove and discard the MERSCOPE slide from previous run. Clean the MERSCOPE Gasket and Aqueduct with RNaseZap solution followed by 70% ethanol. Wipe dry.

Gently pick up the MERSCOPE Slide with tweezers and place it into the Flow Chamber Base. The sample gel should be facing up. Place the Flow Chamber Gasket on top of the slide. Place the Aqueduct on top.

Place the Chamber Top next. Ensure that the notch in the Base and the flow direction arrows marked on the Aqueduct connectors are oriented correctly.

Twist the Top clockwise until a loud click is heard to ensure secure assembly. Once assembled, spray the bottom of the MERSCOPE Slide with 100% ethanol and wipe clean. Repeat this twice to ensure the optical imaging surface is clean.

Activate the chamber with imaging buffer using a syringe.

Connect the assembled flow chamber fluidic lines to those in the MERSCOPE Instrument. First connect the output line on the left side. Then connect the input line on the right side. The Aqueduct has flow direction arrows marked on the connectors to help guide into the correct orientation.

Click Wet Flow Chamber to initiate fluidics to the MERSCOPE Flow Chamber. Hold the flow chamber vertically with the output lines upwards as the flow starts to pull the air bubbles. If air bubbles are visible in the MERSCOPE Flow Chamber or input fluid line, click Pull more liquid. When no air bubbles remain, click Next.

Insert the Flow Chamber into the MERSCOPE Instrument with the notch in the base toward the front of the instrument. First insert the back edge of the flow chamber under the overhangs in the stage adapter. Then, lower the flow chamber to lie flat within the stage adapter. Lock the Flow Chamber into place and close the flow chamber lid.

If the locking mechanism will not engage, it is possible that the MERSCOPE Flow Chamber has not been inserted correctly under the overhangs in the stage adapter. Remove the flow chamber from the stage adapter and try the insertion again, ensuring the back edge of the of the flow chamber is first inserted under the overhangs.

We will now select regions of interest for the sample. Click Acquire Mosaic. The MERSCOPE Instrument will now acquire a low-resolution mosaic. Select the regions of interest to be included in the experiment using the touchscreen or mouse. Draw boundaries on the mosaic to define the region of interest for MERFISH imaging. Once a boundary is drawn it is saved and a summary appears on the right-hand side of the screen.

Select an existing region by clicking on it on the right-hand side of the screen. When a region is selected hold and drag a boundary dot to change its location. Click Done to exit out of a selected region. To rename a region, touch the corresponding name on the screen. Up to 10 regions can be selected with a total area up to 1 centimeter squared.

Toggle between PolyT and DAPI and move the Visible Intensity Range slider to adjust the contrast of the image. When ready, click Next.

We will now switch to the high-magnification objective. Open the flow chamber lid and unlock to remove the MERSCOPE Flow Chamber from the stage adapter. Do not detach the fluidic lines. Click Next to advance through screens as operations are performed.

Clean the immersion oil from the high-magnification objective with lens tissue. Slowly Pipette 50 microliters of fresh immersion oil onto the high-magnification objective. Avoid introducing any air bubbles.

Re-insert the Flow Chamber into the MERSCOPE Instrument and lock it into place. First insert the back edge of the flow chamber under the overhangs in the stage adapter. Then, lower the flow chamber to lie flat within the stage adapter. Close the flow chamber lid and click Acquire Focus.

Using the high-magnification objective, the MERSCOPE Instrument will now attempt to find the focal plane. If the focusing is successful, click Next to advance to an Experiment Summary.

When ready, click Start Measurement to initiate the fully automated experiment. The MERSCOPE Instrument will indicate Done when all measurements are complete.

After the experiment is completed, Segmentation Parameters can be selected. In doing so, image processing will run the selected segmentation algorithm to produce single cell outputs. Alternatively, this can be skipped by clicking Skip Selection at the bottom of the screen.

A Boundary Segment may be selected if certain stains were used in the measurement. To preview the segmentation results with a given set of parameters, select the algorithm to preview. The segmentation results will be displayed on the image below.

The image channel displayed may be selected and the Cell Boundary Polygon Opacity and Visible Intensity Range may be adjusted to facilitate evaluation of the segmentation results.

Next, an Image Processing Parameters screen will appear. If this is satisfactory, click Start Image Processing to initiate image processing analysis. The MERSCOPE Instrument will automatically copy the raw image data to the MERSCOPE Analysis Computer. Progress bars will report on the progress of the analysis.

When finished, Click Clean Instrument to proceed to cleaning the MERSCOPE Instrument. The cleaning process and a new experiment can begin on the instrument even while imaging processing of previous experiments is running in the background.