シングルプローブの応用：周囲条件下での質量分析イメージングと単一細胞解析

The overall goal of this method is to create a microscale sampling device, the Single-probe, to analyze live single cells using mass spectrometry under ambient conditions. And to perform ambient mass spectrometry imaging of biological tissues with high spatial resolution. This method can help answer key questions in biological and pharmaceutical studies.

Such as what are the molecules present in live single cells, and how are they distributed in tissues. The main advantage of this technique is that the single-probe is a multifunctional device that can be used for both live single cell analysis and the high-resolution ambient mass spectrometry imaging experiments. Though this method can analyze single cells and tissues, it can also be applied to other systems where microscale sampling is desirable, such as spar analysis of chemicals and live microcolonies and biofilms.

Demonstrating the procedure with me will be Wei Rao, a postdoc from my lab. Place a whole mouse organ of interest into the center of a small plastic well. Submerge the organ in tissue embedding compound up to a height of about 10mm.

Immediately place the tissues into liquid nitrogen for flash freezing. To prepare the mouse tissue section, take the frozen mouse organ and thaw to 15 degrees Celsius in a temperature-controlled cryo-microtome. Secure the tissue onto a steel base with approximately 500 microliters of tissue embedding compound, and place onto a cryo-microtome sectioning mount so that the desired sectioning orientation is presented to the knife.

Section the tissue to a 12-micron thickness. Then, place the sectioned tissue slices onto polycarbonate microscope slides and leave them to dry for 30 minutes at room temperature. Obtain a cell sample, such as 1 milliliter of HeLa Cell Suspension, and add it into 9mL of complete cell culture medium in a standard 10cm cell culture plate.

Keep the cells in culture at 37 degrees Celsius, with 5%CO2 for two to three days, until the growing surface is covered at 70-80%on the cell culture plate. Perform cell passaging in the cell culture plate. To do so, aspirate the growth medium and rinse the cells with 5mL of 1x phosphate buffered saline.

Remove the PBS using a sterile aspiration tip and incubate the cells with 2.5mL of trypsin for approximately 5 minutes at 37 degrees Celsius to detach the cells from the culture plate. Stop trypsin activity by adding 7.5mL of complete cell culture medium and then, uniformly resuspend the cells. To prepare the cell samples for a single-cell mass spectrometry, or SCMS experiment, first place individual micro-cover slides into the wells of a 12-well plate.

Then, add 1.8mL of cell culture medium and 0.2mL of cell suspension into the well. Gently mix the cells with mild agitation of the plate before incubating in a 5%CO2 environment at 37 degrees Celsius, for approximately 24 hours. To perform drug treatment of the cultured cells, add a drug compound solution into the 12-well cell culture plate.

Place the dual-bore quartz tubing into a laser micro-pipette puller, and pull a dual-bore quartz needle. As starting points, set the heat to 400, the filament to three, the velocity to 80, the delay to 150, and the pull to 250. Cut the pull tip so that there is approximately 5mm of unpulled dual-bore quartz capillary left at the other end.

Cut an 80mm section of fused silica capillary as a solvent-providing capillary. Then insert the capillary into one bore at the flat end of the dual-bore quartz needle. Using the flame to pull a fused silica capillary as a nanoESI emitter, cut to 7-10mm long and insert it into the other bore at the flat end of the dual-bore quartz needle.

Apply a minimal amount of UV curing resin onto the flat end of the dual-bore quartz needle. Solidify the resin using an LED UV lamp for approximately 20 seconds, to secure the solvent-providing capillary and the nanoESI emitter. Next, cut a standard microscope glass slide in half lengthways.

Then, place the single-probe onto one end of the glass slide, so that the nanoESI emitter is pointed outwards. Apply the regular epoxy to the body of the single-probe so that it becomes secured onto the glass slide before leaving overnight for hardening. This system includes a customized ion source interface flange of the mass spectrometer, and an aluminum optical board that is attached with USB digital microscope, miniature manual XYZ translation stage, and the motorized XYZ translation stage system.

Modify the ion source interface flange of the mass spectrometer and fabricate the stand of the digital stereomicroscope as described in the text protocol. Attach the modified digital stereomicroscope, a USB digital microscope, a miniature manual XYZ translation stage with a flexible clamp holder, and the motorized XYZ translation stage system to the aluminum optical board. Use the flexible clamp holder to fix the glass slide attached with a single-probe.

Then, attach the single-probe setup to the mass spectrometer. 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. 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. Use a syringe to pump the sampling solvent at an appropriate rate. And then, apply the ionization voltage.

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 to monitor the distance change between the single-probe tip and tissue surface. Set the parameters for rastering across the section of interest within the sample.

For the mouse kidney sections presented here, use a 10 micrometer per second rastering speed and a 20 micrometer distance between lines. Set the parameters for rastering across the section of interest within the sample. For the mouse kidney sections presented here, use a 10.0 micrometer per second rastering speed, and a 20 micrometer distance between lines.

Set up a method for the automated acquisition of MS spectra from the mass spectrometer. For high mass resolution MSI on a mouse kidney sample, use a mass resolution of 60, 000, a 5kV positive mode, 1 microscan, 150 milliseconds maximum injection time, and automatic gain control on. Initiate the MSI data acquisition, and then construct MS images from raw MS files as described in the text protocol.

Set up the single-probe system as per instructions for MSI and adjust the solvent flow rate. Wash the cultured cells attached on the microcover glass slides with PBS to remove cultural media and extracellular drug components. Then place the cell-containing glass slide onto the motorized XYZ translation stage system for the experiment.

Focus the digital stereomicroscope 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 nanoESI emitter on the single-probe. Use the motorized XYZ stage control program and digital stereomicroscope 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. Lower down the cell-containing plate to pull the single-probe tip out of the cell. Finally, let the solvent flow for approximately three minutes, to completely flush the single-probe.

The ambient mass spectrometry imaging of mouse kidney sections was performed, and a large number of molecules present on the tissue's surface can be detected. The spatial distribution and relative abundance of multiple molecules on the tissue's surface were plotted. A spatial resolution of 8.5 microns has been achieved through the observation of sharp transitions of features present within the mass spectrometry data.

Fabricating the single-probe with a sharp tip and open channels is critical for single-cell analysis. Microscopes are needed to locate the target cells and monitor the cell penetration process. The signal change from mass spectra also provides additional information to confirm cell penetration.

A large number of molecules have been detected from live single cells. When used for anti-cancer drug studies, control cell experiments are used to show the difference of anti-cancer drug treatment. After watching this video, you should have a good understanding of how the single-probe is fabricated, and how this device is used for mass spectrometry analysis of single cell and ambient mass spectrometry imaging of biological tissues.

Generally, individuals new to this method might struggle because detailed instructions are necessary to fabricate the single-probe device, set up the whole system, and operate the instruments. Once mastered, the single-cell analysis can be continuously performed for many cells. And each analysis takes less than three minutes.

Finishing an ambient MS imaging experiment may take a few hours, depending on sample size, spatial resolution, and the mass spectrometer used. While attempting this procedure, it's important to remember that fabricating a high-quality single-probe is the key step. In addition, the tuning and operation of the instrument are critical to successfully conduct an experiment.

Other methods can be developed following similar procedures to the ones presented here. For example, reagents can be added into the sample solvent and reactive single-cell analysis and ambient MSI can be performed to improve the detection of molecules from samples. After its development, this technique paved the way for researchers in both single-cell analysis and mass spectrometry imaging studies under ambient conditions.

This technique can be potentially used for cell biology research, pharmacology studies, and clinical tests. Don't forget that working with sharp quartz capillaries and UV curing epoxy can be hazardous, and precautions such as wearing gloves and UV safety goggles should always be taken while performing this procedure.