Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Animal use and welfare should adhere to the NIH Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Mouse tissue samples were provided by collaborator Dr. Chuanbin Mao.

1. Mouse Tissue Section Preparation

1. Place a whole mouse organ of interest (brain, kidney, liver, etc.) into the center of a small plastic well (e.g., 12-well cell culture plate), and submerge in tissue embedding compound up to about 10 mm height. Make sure there is no bubble formed in the tissue embedding compound and that the organ is placed in the desired orientation (i.e., sagittal, coronal, etc.).
2. Immediately place tissues into liquid nitrogen for flash freezing. For long-term storage, store the frozen samples in a -80 °C freezer.
3. Take the frozen mouse organ and thaw to -15 °C in a temperature controlled cryomicrotome.
4. Secure tissue onto a steel base with approximately 500 µl of tissue embedding compound and place onto a cryomicrotome sectioning mount so that the desired sectioning orientation is presented to the knife.
5. Section the tissue to a 12 µm thickness. Place sectioned tissue slices onto polycarbonate microscope slides and leave to dry for 30 min at room temperature. For long-term storage, store the frozen slide in a -80 °C freezer.

2. Cell Culture

Note: Cell culture was performed in biological safety cabinet (Biosafety Level II) under sterile conditions. HeLa cell line was used as a model system, and cells were cultured in complete culture medium with the following conventional protocols:

1. Warm reagents (i.e., trypsin, phosphate buffered saline (PBS), and cell culture medium) to 37 °C.
   Note: The cell culture medium contains inorganic salts, amino acids, vitamins, and others. For a complete list of constituents, refer to the formulation from the manufacturer.
2. Obtain cell sample (e.g., 1 ml HeLa cell suspension) and add it into 9 ml of complete cell culture medium in a standard 10-cm cell culture plate. The initial cell number is around 0.5 x 10⁶ cells/ml. Keep cells in culture at 37 °C with 5% CO₂ for 2-3 days until the growing surface is covered at 70-80% on the cell culture plate. Record cell passage number for each successive round.
3. Perform cell passaging (i.e., cell splitting) in the cell culture plate.
   1. Aspirate growth medium, and use 5 ml of 1x PBS to rinse the cells. Remove PBS using a sterile aspiration tip, and incubate the cells with 2.5 ml of trypsin (0.25%) for ~5 min at 37 °C to detach the cells from the culture plate.
      Note: The actual trypsin treatment time need to be optimized according to the specific trypsin product purchased from the manufacturer. Inadequate treatment time leaves the cells attached to the plate, whereas excessive treatment leads to cell death.
   2. Stop trypsin activity by adding 7.5 ml complete cell culture medium, and then uniformly resuspend the cells (total volume 10 ml). Use the cell suspension for culture (step 2.2) or preparation of SCMS samples (step 2.4).
4. Prepare the cell samples for SCMS experiments.
   1. Place individual micro cover slides in a 12-well plate, and add 1.8 ml cell culture medium and 0.2 ml of cell suspension into the well.
   2. Gently mix cells with mild agitation of the plate, and incubate in a 5% CO₂ environment at 37 °C for ~24 hr. To perform drug treatment to the cultured cells, add a drug compound solution (e.g., in DMSO (dimethyl sulfoxide)) into 12-well cell culture plate.
      Note: The final drug concentration (e.g., 10 nM, 100 nM, 1 µM, and 10 µM) and treatment time (e.g., 4 hr) are varied according to the specific purpose of studies. Cells are attached to the micro cover slides and ready for the CSMS experiments (step 6).

3. Single-probe Fabrication

1. Place the dual-bore quartz tubing (inner diameter (I.D.) 127 µm, outer diameter (O.D.) 500 µm) into a laser micropipette puller and pull a dual-bore quartz needle. Use the following parameters as starting points: Heat = 400, Fil = 3, Vel = 80, Del =150, and Pul = 250 (all units are manufacturer's units). Ensure the pulled dual-bore quartz needle has a tapered tip for optimal probe properties. Cut the pulled tip so that there is ~5 mm long of unpulled dual-bore quartz capillary left at the other end.
   Note: The actual parameters of the laser puller should be optimized according to the specific conditions of the instrument.
2. Cut a ~80 mm section of fused silica capillary (I.D. 40 µm, O.D. 105 µm) as the solvent providing capillary, and insert it into one bore at the flat end of the dual-bore quartz needle.
3. Cut a ~40 mm section of fused silica capillary (I.D. 40 µm, O.D. 105 µm) and use a razor to shave ~5 mm of the polyimide coating from the mid-point. Use a propane flame to quickly heat and pull the fused capillary into a nano-electrospray ionization (ESI) emitter with a fine taper. Cut a nano-ESI emitter (~7-10 mm long), and insert it into the other bore at the flat end of the dual-bore quartz needle. Alternatively, use the laser puller to produce a fine taper.
4. Apply a minimal amount of (~1-2 µl) UV curing resin onto the flat end of the dual-bore quartz needle, and solidify the resin using a LED UV lamp for ~20 sec to secure the solvent providing capillary and the nano-ESI emitter. The procedures to assemble the individual parts into a single-probe are shown in Figure 1a.
5. Cut a standard microscope glass slide (1" x 3") into half lengthways. Place the single-probe onto one end of the glass slide so that the nano-ESI emitter is pointed outwards. Apply the regular epoxy to the body of the single-probe so that it becomes secured onto the glass slide (Figure 1b). Leave overnight for hardening. Couple the fabricated single-probe with the integrated single-probe setup (Figure 1c), which is attached to a mass spectrometer as illustrated in Figure 1d.

4. Build the Integrated Single-probe MS Setup

1. Modify the ion source interface flange of the mass spectrometer and fabricate the stand (with adjustable position and height) of the digital stereoscope (Figures 1e and 1f).
   1. Drill an ion source interface flange with two holes allowing for the attachment of an aluminum optical board. Make a slide rail device and a height adjustment rod (attached to a XY stage for fine position tuning), such that the digital stereoscope system can be attached to the aluminum optical board (Figure 1e).
2. Attach the modified digital stereo microscope, a USB digital microscope, a miniature manual XYZ translation stage with a flexible clamp holder, the motorized XYZ translation stage system to the aluminum optical board, which is mounted on the customized ion source interface flange of the mass spectrometer (Figures 1c and 1f). Use the flexible clamp holder to fix the glass slide attached with a single-probe.
3. Attach the single-probe setup to the mass spectrometer (Figure 1f). Adjust the flexible clamp holder and the miniature XYZ stage to place the emitter of the single-probe in front of the inlet of the mass spectrometer. Use the USB digital microscope (with adjustable view angle) on the side of single-probe to provide a zoomed-in image of the single-probe tip or the nano-ESI emitter, and the digital stereoscope (with adjustable height) above the single-probe to view the cells and the probe tip.
   Note: Using the corresponding ion source flange, this integrated single-probe system can be coupled to any other types of mass spectrometers equipped with ambient ionization sources.

5. Ambient MSI

1. Thaw the sample section at room temperature and place it onto the motorized XYZ translation stage system underneath the single-probe. Adjust the sample position by changing the coordinates in the control software.
2. Using the syringe to pump the sampling solvent at an appropriate rate (e.g., 0.2 µl/min), and apply the ionization voltage (e.g., 5 kV). The selection of the sampling solvent is flexible, and the common ones include MeOH:water (9:1) and acetonitrile. The dead volume of the nano-ESI emitter was estimated to be ~3 nl, and the time between the probe-surface contact and ion signal observation is usually less than 1 sec ¹⁵.
   Note: The customized ion source interface flange allows the ionization voltage to be delivered from the mass spectrometer to a conductive union through a crocodile clip. The ionization voltage is then transmitted through a conductive union to the solvent inside the capillary and the single-probe channels, and applied onto the nano-ESI emitter to ionize the sampled analytes. Ensure that the ionization voltage is turned off when connecting the crocodile clip with the conductive union.
3. Adjust the height of the single-probe so that it is resting just above the surface of the sample and able to perform surface-extraction of metabolites. Carefully lift the Z-stage, and then use the USB digital microscope (on the side of the single-probe) to monitor the distance change between the single-probe tip and tissue surface. Monitor changes in the mass spectrum during this height adjustment, and stop lifting the Z-stage when a change of the ion signal from solvent background to tissue metabolites is observed.
4. Repeat step 5.3 three times to set three different points within the stage control program for automated surface flattening adjustment. Place the tip of the single-probe at three points on the sample surface at a distance of about 10 mm apart from each other. Perform height adjustment by pressing the up and down icons, and lock the three points into position under the "Plan method".
5. Set other parameters for rastering across the section of interest within the sample using this program. For the mouse kidney sections presented here, use a 10.0 µm/sec rastering speed and 20 µm distance between lines. The motorized stage system has a 0.1 µm minimum increment motion. The distance between the single-probe tip and tissue is obtained from step 5.3.
6. Set up a method for the automated acquisition of MS spectra from the mass spectrometer. For high mass resolution MSI on mouse kidney sample, use the following parameters: mass resolution 60,000 (m/Δm), ~5 kV positive mode, 1 microscan, 150 msec max injection time, and AGC on. All acquired MS spectra representing individual lines of the MS image had the same number of scans with uniform time spacing between each scan, indicating that the pixel sizes for the images produced were uniformly distributed.
7. Initiate the MSI data acquisition. Initiate the MS acquisition sequence for the mass spectrometer, and then initiate the rastering sequence for the XYZ control program.
   1. For example, in the MS data acquisition program utilized here, go to "Sequence setup", select "New sequence", generate a set of files for a new sequence numbered from 01 to X, where X is the number of lines used for the desired MS image to be taken, and then press "Run sequence".
   2. Use a homemade electronic device to allow the software to produce a contact closure signal for the mass spectrometer to collect the data. The circuit diagram is shown in the supplementary figure (Figure S1) as a reference.
8. Construct MS images from raw MS files using appropriate MSI visualization software. For example, when using the software package developed by Laskin's group at PNNL ¹⁷, perform the following steps.
   1. Click "Brows File." Select the first file obtained from the MSI experiment. Specify where the file starts and finishes under "Number of Lines." Select a range of m/z values for the MS image range under "Enter MZ Range".
   2. Press the "Start" button to initiate the image creation process. Once the MS image is made, click "Save Image" under "Toolbar" to store images in the computer.

6. In-situ Live SCMS

1. Setup the single-probe system as per instructions for MSI. Adjust the solvent (e.g., MeOH/H₂O or acetonitrile) flow rate (e.g., ~25 nl/min).
2. Wash the cultured cells, which are attached on the micro cover glass slides, with PBS to remove cultural media and extracellular drug components. Place cell containing glass slide onto the motorized XYZ translation stage system for experiment.
   Note: Alternatively, use the fresh cell culture medium (without containing fetal bovine serum) to rinse the cultured cells. Less ion suppression has been observed. In addition, cell can survive for longer time during the experiment where the ambient temperature (~20 °C) is significantly lower than the culture temperature (37 °C). The drug type, solution concentration, and treatment time vary in different studies.
3. Focus the digital stereo microscope (above the sample) onto the tip of the single-probe to monitor cell penetration during the analysis. Use the USB digital microscope (on the side of the single-probe) to monitor the working conditions of the nano-ESI emitter on the single-probe.
4. Use the motorized XYZ stage control program and digital stereo microscope (above cells) to locate a cell of interest, and precisely position the single-probe tip above the sample. Start MS data acquisition before the single-probe tip is inserted into the cell.
   1. Use the following parameters as references for MS analysis using a high resolution mass spectrometer: mass resolution 100,000 (m/Δm), ~3 kV positive and negative mode, 1 microscan, 150 msec max injection time, AGC mode on. Automated acquisition of MS spectra is done by clicking "Start" in the MS data acquisition program.
   2. Lift the motorized Z-stage by clicking the icon to penetrate the cell membrane and keep recording the MS signal generated from the cell. A time delay of 1-2 sec is usually observed between the probe insertion and the MS signal detection. As the other confirmation of cell penetration, a dramatic change of the MS signals can be observed upon the penetration of the cell membrane. The MS signals of intracellular compounds usually can last for ~15-20 sec before a significantly decrease.
   3. Lower down the cell containing plate to pull the single-probe tip out from the cell. It usually takes <15 sec for the ion signals of the cellular compounds to approach the noise level. Let the solvent flow for ~3 min to completely flush the single-probe. Meanwhile, position the motorized XYZ stage system to locate the next cell to be analyzed. Each cell experiment requires ~3 min to be accomplished.