Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

Methods based on atomic force microscopy make it possible to characterize biomolecular processes at the nano scale with morphological chemical or structural resolution, finally opening a new front window of observation on biology. Single molecule methods enable probing highly heterogeneous systems, a feature which is particularly relevant for understanding protein aggregation. By combining single molecules with back approaches, we can obtain fundamental information about the behavior of proteins, their links with human disease and design pharmacological interventions which can be successful.

Furthermore, this method can be successfully applied to study the chemical and structural properties of biomaterials for drug delivery, applications and other biotechnological purposes. It may be challenging initially to set the correct AFM parameters for high resolution and it's easy to set up your AFM while measuring your fibrillar aggregates. 30 minutes before the AFM imaging, turn on the AFM system and click set up and probe.

In the probe change window, click next to prepare Z focus stage. Turn off the AFM beam switch if it is on and unlock the dovetail locks. Disconnect the head from the system and in the probe change window, click next.

Mount the AFM cantilever on the probe holder. Connect the head to the system and lock the dovetail locks. Turn on the AFM laser beam switch and in the probe change window, click next.

In the pop up window, click no if the cantilever type did not change and adjust the optical alignment knobs to find the cantilever. Adjust the focus onto the cantilever and click next. In the pop up window, click yes if the focus is on the cantilever.

Use the detection laser beam knobs to position the laser beam at the end of the cantilever. Use the deflected laser beam knobs to maximize the total signal measured by the four quadrant photo diode to at least greater than one volt. In the probe change window, click next and close.

After waiting 15 minutes for the cantilever to reach thermal stability, readjust the position of the deflected laser beam on the position sensitive photo diode if necessary. Click NCM sweep, select the desired amplitude of oscillation, click use phase and click auto. Tune the cantilever close to the maximum of its first free resonance of oscillation to approximately 300 kilohertz for a cantilever with a spring constant of 40 newtons per meter.

Place the sample on the sample holder. Click scan area and set the resolution to between 256 by 256 and 1024 by 1024 pixels and then set the scan size and the pixel number. Set the scanning rate to 0.3 to one hertz for a scan area of one by one to five by five micrometers squared.

Focus the optical view on the sample and click approach to approach the sample surface. Next, click lift 100 micrometers to raise the AFM tip 100 micrometers above the surface of the sample and click expand on the optical image of the sample. Click the focus stage bar to focus the view on the surface of the sample and use the arrows to move in the region of the sample of interest.

Click approach to engage the surface and click line scan to check if the tip is following the surface well. Adjust the set point as necessary. Then click scan to begin imaging the sample surface, maintaining a constant regime of phase change not exceeding delta 20 during the imaging to avoid a large imaging force and to maintain consistency between independent samples.

For IR nanospectroscopy imaging, 30 to 60 minutes before the analysis, turn on the AFM IR system and the IR laser. Open the built in software to control the instrument and click file and new to open a new nano IR file. Click initialize to start the AFM IR system and open the instrument cover.

Mount a silicone gold coated probe with a nominal radius of 30 nanometers and a spring constant of 0.2 newtons per meter onto the AFM IR system to measure the sample in contact mode. Click load in the AFM probe section and next. Use the focus on probe arrows to focus the camera on the cantilever and use the tip to crosshairs to place the crosshair at the end of the cantilever.

Rotate the knobs controlling the position of the detection laser to position the laser at the end of the cantilever. Rotate the laser knobs to detect and maximize the sum measured by the four quadrant photo diode to a value greater than three volts. Rotate the deflection knob to adjust the cantilever deflection to minus one volt and click next.

Then close the cover of the instrument. Use the focus on probe arrows to focus the camera on the sample and tip to crosshairs arrows to move in the region of interest of the sample. Click next and engage.

In the microscope window, select height for morphology, amplitude two for the IR absorption and PLL frequency to map the tip to sample contact resonance. In the AFM scan section, set the parameters as demonstrated for the AFM imaging and click scan to acquire a morphology map. When the morphology mapping is finished, in the microscope window, click the height map to position the probe on the top of one aggregate.

In the nano IR section, click start IR to illuminate the sample with the IR laser. To focus the infrared laser on the cantilever, click optimize and enter 1655 centimeters for the wavenumber. Click add and scan to determine the IR laser position and click update and okay to align its position with the cantilever.

In the general section, enter a wavenumber for which a high absorbance in the relative field is expected and deactivate the band pass filter option. Click start IR then check the meter reading and the FFT of the cantilever response. In the FFT window, move the green cursor to read the resonance frequency of the cantilever.

A typical value of the FFT of the resonance of the cantilevers is around 200 kilohertz. Enter the generated resonance frequency value in the general section in the frequency center field and use a frequency window of 50 kilohertz. Click laser pulse tune window to select the resonance enhanced mode and set a pulse rate of 222 kilohertz, a tune range of 50 kilohertz and a laser duty cycle of 5%Click acquire to sweep the pulse rate of the laser.

Use the cursor to tune the laser pulse to the frequency of the mechanical response of the thermal expansion of the sample absorbing the IR light. Then select phase locked loop to monitor the contact resonance between the sample and the tip and click zero. For other chemical properties at the nano scale, track the contact resonance during the spectra acquisition to ensure that the sample spectrum is not affected by overheating and softening.

Select enable to track the sample to tip contact resonance and select an integral gain of 0.5 and a proportional gain of 10 then click OK.In the optimize window, click the wavelength and scan to locate the IR laser for at least three wavenumbers corresponding to major absorbance bands of the sample and for at least one wavenumber for each chip of the laser. Click tools, IR background calibration and new, and set the wavenumbers to between 1200 and 1800 centimeters. Set the backgrounds to average to one, the duty cycle of 5%and the sweep speed to 100 centimeters squared.

Click acquire to measure the IR laser background for normalization of the measured nano scale localized spectra, save the file and close the window. In the IR spectra settings, select an IR spectrum resolution between one and four centimeters and a number of co-averages of at least 64 times. Then click acquire to measure a nano scale localized IR spectrum in the protein range.

To acquire a nano scale resolved chemical map, select 1655 centimeters and IR imaging and click scan in the AFM scan window. When the mapping is completed, save the measurements. Then use the built in AFM image processing software to analyze the acquired maps of morphology, contact resonance and chemistry and nano scale localized spectra.

It is a critical step to obtain a highly pure monomeric solution as the presence of aggregated species may result in poor reproducibility of kinetics and introduce artifacts in your measurements. A fundamental factor for successfully studying biological samples which have high heterogeneity is the correct the position of solid substrates. Microfluidic spray deposition can be exploited instead of manual one in order to preserve the sample architecture and heterogeneity.

These methods pave the way for unraveling the chemical structure and properties of individual biomolecules and their interactions in physiological and native liquid environment.