An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

We developed an innovative method for metabolomics. We combined the Belfry spectral imaging of living cells with single-cell mass spectrometry. Our technique can monitor and predict the metabolic changes of single cells against drugs.

And this opens a lot of application for basics research and industry. Our method adds single-cell resolution to current drug evaluation techniques which will greatly aid future drug discovery efforts. Our technique can also help us understand the role played by cellular heterogeneity in cancer resistance.

The single-cell sampling and data analysis are the most challenging aspects in this experiment. We are currently working on automating these two steps, especially the sampling part. We recommend you practice the single-cell sampling well before the experiment, as each micromanipulator setup is slightly different and you should take the time to get familiar with the controls.

After incubating cells to reach 70%confluency, culture cells of interest in an appropriate culture media, add Penicillin-Streptomycin to avoid contamination. After measuring cell density on a hemocytometer, subculture cells into a 35 millimeter glass bottom grid dish or quartz slides, using the same medium at a seeding density of 0.7 times 10 to the sixth. Then incubate at 37 degrees celsius for 24 hours.

The next day, the cells reach a confluency of 50 to 60%Wash the cells two times with pre-warmed PBS buffer at 37 degrees celsius. Divide the cells into drug treated and untreated subgroups in 35 millimeter culture dishes. Mix Tamoxifen dissolved in dimethyl sulfoxide with the culture media to obtain a final volume of two milliliters and Tamoxifen concentration of 10 micromolar.

This is the drug treated group. Mix a corresponding volume of DMSO into the medium as a control group to study the effects of DMSO. Incubate both groups in two milliliters of the spiked media for 24 hours to reach a confluency of 70 to 80%Prior to spectral measurements, verify the pinhole and laser position match using a target.

To calibrate the spectrophotometer prior to each experiment, place ethanol in a glass bottom dish, measure the spectrum at a given laser intensity for one second, and associate the peak to known wavelengths. Then setup the micro-chamber at 5%carbon dioxide and 37 degrees celsius. Once the microscope system is ready, remove cells from the incubator and immediately rinse cells twice with warmed PBS buffer at 37 degrees celsius.

Then add two milliliters of warmed PBS or FluoroBrite DMEM. Add 10 microliters of water onto the water immersion objective lens. And delicately place the glass bottom cell dish onto the microscope stage.

Fix the cell sampling system onto the Raman microscope. Connect the 3D micromanipulator to the glass capillary holder that is attached to an empty syringe for sample sucking. Set the microscope to a high magnification field of 40 times to observe the tip of the glass capillary, and make sure it is not broken.

Control the position of the glass capillary using the micromanipulator. Ensure that the capillary tip is centered in the field of view. Then move the capillary up on the Z-axis to give clearance for the culture dish later.

Place the sample dish on the stage of the microscope, adjust the magnification and focus, select the target cell on the grid dish, and move it to the center of view. Measure each cell by focusing the laser line. A 15 second exposure time per cell is sufficient to obtain a cross section of a cell with a clear Raman signal.

A galvano-mirror allows scanning of one cell or a group of cells within several dozen minutes. Then carefully lower down the glass capillary using micromanipulator until the tip comes into focus. Under microscopic observation, touch the target single cell with the capillary tip.

Then start applying negative pressure using the syringe to trap the cell inside the capillary tip. Record this procedure by taking a video to check the timing and sucked location of the cell precisely. Move the capillary up on the Z-axis.

Then detach the capillary from the capillary holder using forceps in preparation for mass spectrometry analysis. After setting up the mass spectrometry instrument and analyzing the media, add two microliters of the ionization solvent to the capillary containing the cell. Fix the capillary to a nano-electrospray adaptor connected to a suitable mass spectrometer and start the automatic acquisition method.

A comparative analysis of the average spectrum of each condition, with and without drug treatment, is shown here. The average spectrum of the two conditions clearly differ at various peaks. In particular, the peaks at 1, 000 per centimeter, which is a sign to aromatic compounds such as phenylalanine and tyrosine, show strong differences.

Based on the projection on latent structure model, the VIP scores were calculated which represent the importance of wavelengths in discriminating the experimental conditions. Importantly, the highest peaks of the VIP profiles corresponded to Raman peaks for which strong differences were seen between the two treatments. This confirmed the specific molecular differences between treated and untreated cells.

After positive identification, the relative abundance of the drug and its metabolites were measured in each cell and compared to background peaks in untreated cells. Strong variation was observed in Tamoxifen abundance. And this phenomenon was even more pronounced in the case of its metabolite 4-hydroxy-tamoxifen.

Researchers can be interested in exploring the link between spectral images and the metabolic profiles of cells. Our research also has industrial applications because it allows for local screening of cells for pharmaceutical manufacturing. Care must be taken while preparing the ionization solvent for the mass spectrometer measurements.

It should be prepared under a fume hood. Also, be careful not to touch the ion source of the mass spectrometer instrument to avoid being electrocuted. Finally, the sampling capillaries used throughout the measurement are very sharp, so handle them with care to avoid injury.