A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

The overall goal of the following experiment is to measure protein secretion from individual cells with high temporal and spatial resolution. This is achieved by lithographically fabricating gold plasmonic Nanosensors on glass cover slips for the label free imaging of single cell secretions. As a second step, the sensor chip is cleaned and coated with the self-assembled monolayer and then functionalized with ligands that have a high affinity for the secreted analyte proteins.

Next cells are integrated onto the functionalized sensor chips in order to measure their secretions in space and time. The results show that protein secretions from individual cells can be mapped both spatially and temporally without the need for labeling. This method can help answer key questions in the field of intercellular communication, such as how paracrine and autocrine signaling pathways govern cell motility.

To begin this procedure, deposit a 10 nanometer chromium thin film on previously cleaned cover slips by electron beam evaporation to avoid charge effects during the patterning and imaging of nanostructures. Following this spin, the first layer of bilayer resist consisting of ethyl lactate, methylmethacrylate copolymer at 2000 RPM for 45 seconds. Then bake the substrate at 150 degrees Celsius after allowing the substrate to cool to room temperature.

Spin the second layer of polymethylmethacrylate at 3000 RPM for 45 seconds and bake the substrate at 150 degrees Celsius.Pattern. The bilayer resist using electron beam lithography or EBL. Develop an isopropyl alcohol or IPA methyl isobutyl ketone, or MIBK at a two to one ratio and rinse in IPA.

Remove chromium from the developed area of nanostructures via wet etch using CR seven etching for 10 seconds at room temperature and then rinse in deionized, distill distilled water Deposit a titanium gold film on the substrate using the electron beam evaporator following the gold deposition. Lift off the copolymer bilayer. Resist by soaking the substrate in acetone for four hours.

Next, inspect the substrate using a scanning electron microscope to confirm the nanostructure shape and size. The resulting nanostructures are typically separated by a pitch of 300 nanometers. 20 by 20 arrays of nanostructures are spaced by 30 microns center to center.

Leaving ample room for cell imaging, A typical chip will contain 300 arrays. Remove the remaining chromium from the substrate via wet edge using CR seven etching for 60 seconds at room temperature. Then rinse the substrate in deionized distilled water.

Then inspect a subset of arrays using the atomic force microscope for the verification of size and uniformity. This patterned glass cover slip can be used multiple times as long as the proper cleaning procedures are followed for cleaning and regenerating the localized surface plasma and resonance or LSPR chips. Plasma ash at a power of 40 watts in a 300 milli to mixture of 5%hydrogen and 95%argon for 45 seconds.

Functionalize the gold surface and the nano structures immediately after plasma ashing. By immersing the chip in a two component ethanol thiol solution consisting of a three to one ratio of SPO and SPC. After leaving the chip in the thiol solution overnight to form a self-assembled monolayer, rinse it with ethanol and dry with nitrogen gas load the LSPR chip within a custom made microfluidic holder by placing the chip on an aluminum bottom piece.

Then sandwich the chip between this bottom piece and a silicone gasket and a clear plastic top piece using four screws to clamp the assembly. For a typical SPC based thiol application, drop coat 300 microliters of a one to one mixture of 133 millimolar EDC and 33 millimolar NHS in deionized distilled water to activate the carboxyl groups of the SPC thiol component. After waiting for 10 minutes, manually rinse the surface with 10 millimolar PBS following this conjugate the activated carboxyl group with the ligand of interest by drop coating 300 microliters of the ligand solution.

After waiting for one hour and rinsing the surface with 10 millimolar PBS drop code, 300 microliters of 0.1 molar ethanolamine in PBS on the chip to minimize nonspecific binding. After waiting for 10 minutes, wash the ethanolamine with PBS containing 0.005%between 20 or PBST 20. Next, place a quartz piece above the chip to reduce fluctuations in the data related to a changing meniscus.

Keep the chip wet with PBST 20 buffer while mounting on the microscope. Attach the microfluidic tubing to the assembly and flow buffer until a steady state is reached. Then place the LSPR chip assembly firmly into the heated stage sample holder.

The optical setup for this method is shown here. The light from the halogen lamp is passed through a 593 nanometer long pass filter to eliminate wavelengths, which do not contribute to the nano plasmonic response. The light is then linearly polarized and the sample is illuminated via a 40 x 1.4 numerical aperture objective for the excitation of the plasmonic gold nanostructures.

The scattered light is collected by the objective and passed through a crossed polarizer to reduce the background signal from the glass substrate. A 50 50 beam splitter is inserted into the collected light path for simultaneous spectroscopic and imagery analysis. After allowing the assembly and microscope to equilibrate for at least two hours, align the chip using the joystick so that the central array is aligned with the fiber optic for spectroscopy.

After culturing and pelleting hybrid DOMA cells by centrifugation, harvest them, then test for viability before introducing them onto the LSPR chips. Following this, introduce 50 microliters of the cell solution manually onto the LSPR chips with a micro pipette. Finally, wash away the remaining cells in solution with fresh serum free media using a microfluidic profusion system to prepare the LSPR chips for imaging.

An overlay of an LSPR imaging image, which highlights the square arrays and a transmitted light illuminated image, which highlights the cell at lower left is shown here. A cell stained with the fluorescent membrane dye rod Domine, DHPE exhibits filopodia like extensions. If such extensions were to overlap with the arrays, they would give a false positive for protein secretion spectrometry data before and after the introduction of a saturating solution of antis cmic antibodies to the cmic functionalized arrays is displayed here.

This method is used in order to calibrate the arrays. The calibration of the arrays helps in normalizing the response of the sensors and determining the fractional occupancy based on the bio functionalization profile of each run. A commercial SPR instrument is used to determine the dissociation constant as well as to study the resistance to non-specific binding using the same protocol as outlined for the LSPR chip.

Normalized values from two arrays are shown here. One array was within 10 micrometers of the cell while the control was 130 micrometers from the cell. The sudden increase in the normalized response of the array closest to the cell relative to the flat response of the control array is indicative of a localized burst of secreted antibodies.

After watching this video, you should have a good understanding on how to measure protein secretions from individual cells with high spatial and temporal resolution.