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Experimental Procedure
Feasibility of Biopsy Collection
Buffer TestingDefine Piercing Parameters

Once set-up is complete, feasibility experiments can begin. The feasibility analysis requires the following experiments:

  • Buffer testing
  • Define piercing parameters

  

Buffer Testing

In the biopsy experiment, the buffer is used both for preloading the cantilever and for depositing the extract. Preload buffer helps prevent RNA degradation and initiates protein lysis as soon as the extraction occurs. Deposition buffer helps prevent RNA degradation while the biopsy sample is in storage, awaiting library preparation.

Note: Some buffers do not require testing. For example, the cell lysis buffer (CLB) from Lexogen (Lexogen/LUTHOR High-Definition Single-Cell 3’ mRNA-Seq Kit: LUTHOR High-Definition Single-Cell 3' mRNA-Seq Kit | Lexogen), with some modifications is frequently used by Cytosurge and some customers, and is known to work well with FluidFM. Therefore, a buffer test is unnecessary. If using a different buffer, test it first, as not all buffers are compatible with droplet entry. Additionally, if the buffer is not water-based, the evaporation rate should be measured (water-based buffers will likely have an evaporation rate similar to that of H2O Triton 0.02% buffer).

Tip: If trying out a new buffer, droplets should contain a strong surfactant at low concentration (Tx-100, 0.2 %) to ensure droplet entry and smooth aspiration of the buffer into the cantilever. 

Begin buffer testing with the contact angle test:

  

Buffer Contact Angle Test

Materials

  • Microscope or a high-resolution camera
  • Plain backdrop (box, white wall etc.)
  • Laboratory jack
  • FluidFM droplet slide without holder
  • Kim wipe
  • 70% ethanol
  • 1 µL pipette and pipette tips
  • Test buffer

Procedure

  1. Place a microscope or high-resolution camera on its side, facing a plain backdrop (e.g. sheet, cardboard box)
  2. Use a laboratory jack to ensure the sample is at the correct height and is visible in the camera’s field of view
  3. Take a droplet slide and clean it with a Kim wipe and 70% ethanol
  4. Place the droplet slide on top of the laboratory jack in the field of view of the camera. Ensure that the numbered side of the columns (top of the slide) faces the camera and put the slide as close to the edge as possible, as shown in Figure 1. 
    Figure 1. Example of a buffer contact angle test set-up. 
  5. Take an image of the clean droplet slide, focusing on the side of the glass slide to establish the  scale (height of droplet slide = 1 mm)
  6. Pipette 1 µL of buffer on the droplet slide
  7. Take an image sequence of the droplet from the side with the microscope/camera at 1 minute intervals for 10 minutes and save the images
  8. Analyse the results. We recommend ImageJ with the Contact_Angle.jar plugin (ImageJ Plugins/Contact_Angle.jar: Contact Angle). This YouTube video (YouTube/ImageJ Contact Angle Measurement Tutorial: ImageJ Contact Angle Measurement Tutorial) explains how to install and use it. Measure the following:
    1. Contact angle
    2. Evaporation rate (if required)
  10. Ensure that:
    1. The contact angle is < 75°
    2. The buffer droplet does not evaporate too quickly for the biopsy experiment (remains > 5 minutes)

Once the buffer contact angle test is passed, the entry of the probe into a buffer droplet, the aspiration of the buffer and deposition in the buffer should all be tested.


Buffer Entry, Aspiration and Deposition Test

Carry out a biopsy experiment to ensure that the buffer can be entered, aspirated and deposited

Materials

Procedure

Before Beginning:

  1.  The experiment is conducted at room temperature; ensure that the CO2 and the temperature controller of the incubator are turned off.
  2. Place the prepared biopsy plate in the left port.
  3. Set-up the probe (see Probe Filling and Preparation Advanced).
  4. Replace the probe plate in the right port with the wash plate.
  5. Start the Biopsy Workflow in ARYA.

Preload Droplet Entry:

  1. Make sure the cantilever is dry and clean before entering a preload droplet (see Probe Drying below).
  2. Remove the droplet slide holder containing the droplet slide from the OMNIUM.
  3. Pipette 1 µL droplet of buffer into the centre of one of the engraved circles on the droplet slide (see Figure 2).
    1. Note: The droplet should be as round as possible, without bubbles or spills.
      Figure 2: Pipetting a 1 µL droplet onto circle A1 of the biopsy slide
  5. Carefully place the droplet slide with the droplet holder back into the biopsy plate. 
  6. In ARYA, confirm the placement of the droplet after selecting the droplet location in the software (see Figure 3).
     
    Figure 3. Select the location of the pipetted droplet. 
  7. Enter the droplet:
    1. Switch to 4x magnification.  
    2. Focus on the edge of the droplet and place crosshair tool so that the centre of the crosshair is in the centre of the droplet (see Figure 4).  
    3. Adjust the outer edge of the crosshair tool to match the outer borders of the droplet (using the mouse scroll option) or the arrows in the workflow (see Figure 4).

      Figure 4. Adjusting the crosshairs on the droplet.
    4. Droplet approach parameters. We recommend using default parameters:
      • Y Offset from the droplet centre (µm) - The stage moves -300 µm from the centre down
      • Setpoint (mV) - The cantilever approaches until this setpoint is reached
      • Approach Speed (µm/s) - Z-stage movement speed
    6. By pressing “Approach”, the cantilever will start approaching the droplet.
    7. In the case of a successful entry, proceed with “Continue” (see Figure 5). If the cantilever has not yet reached the droplet, restart the approach. If the cantilever has reached the droplet, but cannot enter, lower the probe manually in -5 µm steps. If the cantilever went too far inside the droplet, wash the cantilever in the wash plate using the water wash, dry the cantilever and attempt again.
    8. If droplet entry fails multiple times, consider changing the buffer.


      Figure 5. Image of probe after successfully entering the droplet.

Aspirate Preload Buffer:

  1. Adjust the active and hold pressure:
    • Active pressure – Pulls the preload buffer into the cantilever. When this happens, it forms a buffer/oil interface and typically occurs around -100 mbar. Start with gradual changes of pressure from - 50 mbar with increments of - 10 mbar until you visually see the oil/buffer interface moving.
      Note: If the interface is not straight or both active and hold pressures are in the positive range, it is possible that the coating was poor, and this will hinder the downstream extraction. Make sure to carefully follow the coating protocol.
    • Hold pressure – Minimum pressure needed to stabilise the movement of the buffer/oil interface with preload inside the cantilever. This pressure is normally around -50 mbar.
      Note: Hold pressure is different for the preload aspirated from the well and droplet. Hold pressure can be adjusted in each step of the workflow if it becomes necessary.
  3. If the buffer cannot be aspirated, consider changing the buffer.
  4. After successfully loading the preload, the next couple of steps can be skipped. Go directly to the Wash Probe step in the workflow.

Probe Washing:

  1. The wash tool will appear, from which the washing steps can edited in the washing tool and copied to the wash step.
  2. Configure the wash to run the water wash as described in Wash Plate Preparation. The selection from the wash tool can be copied into the workflow and will be remembered for the following extractions within the same session.

Probe Drying:

  1. The extract deposition droplet needs to be loaded to the system prior to the probe drying, as extract can be lost from the dry probe. Load the collection droplet as described in steps 1. – 5. of Preload Droplet Entry. 
  2. If the consumable has been immersed into liquid, there will be a droplet around the cantilever (see Figure 6).

    Figure 6. Illustrations and corresponding images of a droplet on the end of the probe consumable.

  3. To proceed with the extraction workflow and deposit the sample, the droplet needs to be removed and the probe dried. The wet cantilever approaches the surface of the Whatman paper using the force feedback control. The probe automatically moves left and right for the desired distance. After the user confirms the dry probe, the head retracts, and the workflow can proceed (see Figure 7).

    Figure 7. Side view (top) and bottom view (bottom) of the probe drying process. 
  4. Select the desired drying pad for drying. ARYA indicates by colour if the drying pads have been used. A blue colour indicates an unused pad, yellow means that the pad was used before, and red indicates multiple (>5) usages.
  5. Adjust the position of the cantilever so it is in the middle of the slit.
  6. Select the parameters for approach. We recommend:
    • Setpoint: 100 mV
    • Speed: 500 µm/s
  8. After the initial force-controlled approach, the drying pad will remove most of the water (some will remain around the 2 pillars and at the base of the cantilever). The cantilever “drops” for another 500 µm. This allows the cantilever to move in the same plane as the drying pad. The consumable moves laterally left and right for the paper to completely remove the liquid. We recommend keeping the default parameters (or lowering lateral speed if facing difficulties with drying).
  9. Confirm that the cantilever is dry. If it is not:
  10. Move the probe manually in the x-direction until the liquid is completely gone.
    • If this does not work, manually lower the probe an additional 200 µm and repeat the movement in the x-direction.
    •  If the probe still remains wet, adjust the “Drop after reaching setpoint” and/or “Lateral movement distance” value to -800 µm.
    •  If this still does not help, consider changing the Whatman filter pads.

      Important!       A dry cantilever can cause the extract to escape from the probe due to evaporation on the aperture and oil pressure within the microchannel. The risk of runaway increases the longer the probe is kept dry. Therefore, to reduce the loss, keep the cantilever dry for as short a time as possible and, if possible, increase the volumes of preload buffer.

Extract Deposition:

  1. Approach the droplet as described before in Step 6 of Preload Droplet Entry.
  2. To deposit the extract, apply a positive active pressure. We recommend using pressures between 50 and 400 mbar for deposition.
  3. If deposition fails, consider changing the buffer.

Rinsing:

  1. After the extract is deposited, rinse the cantilever by aspirating and depositing the buffer up to the volume of the total extract volume, at least 3 times, to remove any residual content from the cantilever. Make sure the FluidFM Nanosyringe only contains oil before moving to the next step.
  2. Repeat the all steps (Preload Droplet Entry, Aspirate Preload Buffer, Probe Washing, Probe Drying, Extract Deposition and Rinsing) at least three times. Ensure that:
    • The buffer droplet can be entered with the probe
    • The buffer can be aspirated
  4. The buffer can be deposited.


Define Piercing Parameters

The piercing parameters required for extraction can change for different cell types, so it is also important to optimise these before use. The piercing parameters are:

  • Setpoint – Force applied to the cells by the cantilever, typically use 500 nN for biopsy experiments. Note: Always use the nN units instead of mV (default)for greater accuracy and reproducibility between probes.
  • Approach speed – Speed of the Z-stage movement towards the cell. The speed is usually set to 50 µm/s.
  • Medium entry check pressure – The pressure applied once the cantilever is in contact with the cell to check whether medium enters the probe. It can be adjusted during pressure application and is typically set to -500 mbar.
  • Medium entry check pressure duration - How long the medium entry check pressure is applied before automatically switching back to the hold pressure, usually only a few seconds is necessary. In case of cell culture medium entering the cantilever, release the content slowly back into the dish and thoroughly wash the cantilever with surfactant solution (as outlined under ‘Wash plate preparation’, the sequence for a blocked probe, above). This is crucial, as the cantilever will likely become unusable.
  • Extraction Pressure – The negative pressure that is applied to extract the cellular content. Typically set to -800 mbar.
  • Holding pressure – The pressure needed to stabilise the oil/content interface (preload and biopsy) in the cantilever (typically -80 mbar).
  • Active Pressure Duration – Duration of the applied extraction pressure. The extraction can, however, be stopped at any point or extended if there is not yet enough extract. Use the volume guide tool to estimate the extracted volume.

Not all parameters require adjustment. Normally, the parameter optimisation focuses on setpoint, approach speed, and extract volume.

To optimise the piercing parameters:

Materials

 

Procedure

Before Beginning:

  1. The experiment is conducted at room temperature; ensure that the CO2 and the temperature controller of the incubator are turned off.
  2. Place the prepared biopsy plate in the left port.
  3. Set-up the probe (see Probe Filling and Preparation Advanced).
  4. Replace the probe plate in the right port with the wash plate.
  5. Make sure that the channel height specified in ARYA (Settings>View>Channel Height) corresponds to the channel height that the probes have. This is crucial for volume estimation.
  6. Start the Biopsy Workflow in ARYA.

​Preload Well Entry:

  1. Switch on training mode to aspirate the buffer from the well instead of using the droplet:
    1. Press ctrl + alt + c on the keyboard at the same time, with ARYA open.
    2. Type scriptmanager training on into the window that appears.
    3. Select the option to aspirate the preload from the well in the workflow.
  3. Using the workflow, navigate to one of the wells in the wash plate that contain 0.2% Triton-X

​Aspirate Preload Buffer:

  1. Adjust the active and hold pressure:
    1. Active pressure – Pulls the preload buffer into the cantilever. When this happens, it forms a buffer/oil interface and typically occurs around -100 mbar. Start with gradual changes of pressure from - 50 mbar with increments of - 10 mbar until you visually see the oil/buffer interface moving.
      Note: If the interface is not straight or both active and hold pressures are in the positive range, it is possible that the coating was poor, and this will hinder the downstream extraction. Make sure to carefully follow the coating protocol.
  3. Hold pressure – Minimum pressure needed to stabilise the movement of the buffer/oil interface with preload inside the cantilever. This pressure is normally around -50 mbar.
    Note: Hold pressure is different for the preload aspirated from the well and droplet. Hold pressure can be adjusted in each step of the workflow if it becomes necessary.

Wash the Probe:

  1. Configure the wash to run the water wash as described in Wash Plate Preparation.

Go to Sample:

  1. After successfully loading the preload, move to the cell area. The cell area can be accessed via "Go to Sample" step (see Figure 8). Select a target well and click on "Move to sample area". This will bring you to the selected well.

    Figure 8: Select the location to navigate to to locate the next cell for biopsy. Points that were already used can also be selected
    1. If there are any saved point groups in the target well, they will appear in this step (see Figure 8). 
    2. Select a cell of interest (e.g. #5) and click "Move to point" (Figure 8).

Probe Alignment:

  1. The probe should be aligned to ensure more accurate volume tool alignment and volume calculations. Approach an empty spot on the surface of the dish with the default setpoint.
  2. Align the crosshairs in the centre of the probe aperture.

Extraction:

  1. Select the cell that needs to be extracted and name it, as well as the group it will belong to.
  2. Adjust the extraction parameters:
    1. For the first five extractions, use the default parameters as suggested above. After every subsequent set of five extractions, observe the outcomes from the previous set. If issues occur, adjust the parameters for the next group of cells according to the guidelines below. Once satisfactory results are achieved, use those optimised settings for the remaining cells: 
      1. If cells are visibly compressed on approach: Lower the Setpoint by 100 nN and/or lower the Approach speed by 25 µm/s.
      2. If >60% of cells detach during biopsy: Decrease the target Extract Volume by ~0.2 pL.
      3. If >60% of cells detach or show blebbing after biopsy: Lower the Setpoint by 100 nN, and/or lower the Approach speed by 25 µm/s, and/or decrease the Extract Volume by ~0.2 pL.
  4. Adjust the imaging parameters:
    1. Save an image - To be able to calculate the volume per extracted cell, after the biopsy has been collected, images before and after extraction of the cantilever are taken as well as from the cell. The step will ask you to focus on the cantilever before the extraction to visualise the amount of preload, and after the extraction to visualise the amount of preload and extracted volume.
  6. Use the volume guide tool (see Figure 9) to estimate the extracted volume. It is important to focus on the cantilever before the extraction, refresh the images, and keep the focus once the extraction starts. (Not advised to use the refresh button in the middle of the extraction).

    Figure 9. The volume guide. The probe (left) can be seen in the centre of the screen simultaneously while the probe with the volume tool overlayed can be see next to the workflow tab (right).
  7. Once finished with the extraction, proceed and the probe will move out of the cell. Keep (or adjust) the negative holding pressure to keep the extracted volume in. The image step will ask you to focus on the cantilever after the extraction to visualise the amount of preload with the extracted volume.
  8. After successfully extracting from the cell, the next couple of steps can be skipped. Use the navigation tool to go directly to the second 0.2% Triton-X well to deposit the extract, and skip to the “Extract Deposition” step in the ARYA workflow.

Extract Deposition:

  1. To deposit the extract, apply a positive active pressure. We recommend using pressures between 50 and 400 mbar for deposition.
  2. If deposition fails, consider changing the buffer.

Rinsing:

  1. After the extract is deposited, rinse the cantilever by aspirating and depositing the buffer up to the volume of the total extract volume, at least 3 times, to remove any residual content from the cantilever. Make sure the FluidFM Nanosyringe only contains oil before moving to the next step.
  2. If viability is required, additional steps will need to be carried out (see section Define Percing Parameters in Feasibility of Biopsy Viability)
  3. To prevent an imbalance in the salt concentration that may affect the cell viability, top up the cell media with 0.5 mL of sterile water every three hours.
  4. Repeat the workflow steps from Preload well entry until Rinsing at least twenty times.
  5. Ensure that:
    • The cell piercing area (as close to the nucleus as possible without coming into contact with the nucleus) is clear and reachable with the probe
    • Cells are not killed or visibly damaged after piercing (only if viability is important)
    • Excessive medium aspiration does not occur after piercing
    • Volumes of >0.5 pL can be extracted from the cell without cell detachment


If more than one condition is to be used in the biopsy collection (e.g. normal state and state after exposure to a drug), then the feasibility experiment should be repeated under these additional conditions.

The experiment is ready for the next phase when:

  • Buffer is known or confirmed experimentally to be compatible with the biopsy workflow.
  • The piercing parameters for the cell line have been defined, and cells can be biopsied with minimal issues under all experimental conditions.


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