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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

Abstract

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

Introduction

Over 40 million people worldwide are currently affected by neurodegenerative disorders, such as Alzheimer’s (AD)1 and Parkinson’s (PD)2 diseases. More generally, more than fifty pathologies are associated at the molecular level with protein misfolding and aggregation, a process that leads to the proliferation of insoluble fibrillar protein aggregates, known as amyloid deposits3,4. The molecular origins of neurodegeneration and its links with protein conformational changes of proteins leading to amyloid formation, however, remain unclear, in large part because of the high level of heterogeneity, transient nature and nanoscale dimensions of the pathological aggregates4,5.

Highly successful investigations of protein structures in the last several decades have been based widely on the use of bulk methods, including X-ray crystallography, cryo-electron microscopy and nuclear magnetic resonance spectroscopy5,6,7,8,9. Within this class of techniques, infrared (IR) spectroscopy has emerged as a sensitive analytical tool to unravel the chemical properties of biological systems such as proteins8. IR methods allow the quantification of protein secondary and quaternary structural changes during their misfolding and aggregation. In addition, in order to further decipher at the microscopic level the mechanistic details involved in the complex free energy landscapes of protein during their aggregation, a major advance has been the development of chemical kinetics tools to extend to complex self-assembly pathways including amyloid fibrils formation5,6,7,10,11,12. However, bulk spectroscopic methods provide only average information on the heterogeneous ensemble of species present in solution or involved in specific microscopic steps, thus rendering the investigation of the biophysical properties of individual aggregated species challenging at the nanoscale level13,14.

Several microscopy techniques with the capability of operating on scales smaller than the diffraction limit of light have emerged in the last decades. This class of methods includes electron microscopy (EM) and atomic force microscopy (AFM). While scanning electron microscopy (SEM) and transmission electron microscopy (TEM) provide two-dimensional (2D) images of a specimen, AFM has emerged in the last decades as a powerful and versatile technique to study three-dimensional (3D) morphologies, as well as the nanomechanical properties of a sample with sub-nanometer resolution13,14,15,16,17,18,19,20,21,22,23,24,25,26,27. The rationale behind studying protein aggregation via AFM is that this approach enables the investigation of the morphology of individual species present in solution13,14,16,17,19,20,21,25,27,28,29,30,31,32,33,34,35,36,37. In particular, by monitoring the sample as a function of time, AFM allows the investigation of the evolution of the morphology of the species within the sample, which makes it possible to follow and visualize the pathways of amyloid formation23,25,38,39,40,41,42. Furthermore, AFM enables the quantification of structural parameters such as cross-sectional heights and lengths of the individual species present in solution13,19,30,31,32,33,34,35,36,37,40,43,44,45,46,47,48. However, the study of a single biophysical property, such as morphology, is often not sufficient when studying heterogeneous and complex biological systems. AFM, SEM or TEM imaging methods alone do not readily reveal the chemical properties of heterogeneous species of amyloid aggregates at the nanoscale.

A major advance for the analysis of heterogeneous biological samples at this scale has been made recently with the development and application to the field of protein aggregation of infrared nanospectroscopy (AFM-IR)24,26,38,42,49,50,51,52. This innovative method exploits the combination of the spatial resolution of AFM (~1−10 nm) with the chemical analysis power of IR. The AFM-IR technique is based on the measurement of the photothermal induced resonance effect driven by an IR laser, and on the measurement of the thermal expansion of the sample under investigation by the AFM tip. The sample can be illuminated by the IR laser directly from the top or from the bottom in total internal reflection, similarly as in conventional infrared spectroscopy24,42,52,53. The IR laser can be pulsed with typical frequencies in the order of hundreds of kilohertz (1−1000 kHz) and tuned over a wide spectral range, typically between 1000−3300 cm-1. Although the laser source covers an area of ~30 µm diameter, the spatial resolution of the AFM-IR technique is determined nominally by the AFM tip diameter, which detects the local thermal expansion of the system. AFM-IR is well suited to study biological samples because the IR signal is proportional to their thickness up to 1−1.5 µm, and the resulting IR spectra are generally in agreement with the corresponding FTIR transmission spectra13,54,55. For this reason, established methods of analysis in spectroscopy can be readily applied, such as the study of chemical shifts, band shape change and de-convolution by second derivatives analysis52. Overall, combining the spatial resolution of AFM with the chemical recognition power of IR spectroscopy, AFM-IR enables the simultaneous acquisition of a wide range of morphological, mechanical and chemical properties of a sample at the nanoscale.

Here, we illustrate a protocol for the characterization of the process of protein aggregation that exploits the combination of in vitro fluorescence assays, high-resolution AFM imaging and nanoscale AFM-IR. This combined approach has already excelled in providing detailed results in studying the chemical and structural properties of individual micro-droplets formed by protein aggregates, in the study of liquid-liquid protein phase separation, and in investigating the heterogeneity and biophysical properties of individual aggregated species at the nanoscale23,26,38,45,50,53,56,57.

Protocol

1. Aggregation assays on fluorescence plate readers

NOTE: The protocol described here is an example of how to study the aggregation of any protein or peptide by chemical kinetics. In particular, it describes an optimized protocol to study the aggregation of the Aβ42 peptide, which is involved in the onset and progression of Alzheimer’s disease58,59. A similar protocol can be adjusted and adopted towards studying the aggregation of any protein or peptide.

  1. Obtain a highly pure monomeric solution of Aβ42 through ion-exchange and size-exclusion chromatography techniques to distinguish protein fractions containing Aβ42, and to isolate the monomeric fraction from other aggregated forms of Aβ42 obtained58,59.
  2. Dilute the Aβ42 peptide in 20 mM sodium phosphate buffer, 200 µM EDTA at pH 8.058 to a desired final concentration ranging between 1−5 µM, in 1.5 mL low-bind tubes. Samples collected for AFM measurements should not contain the fluorescent dye used in the aggregation assay (thioflavin T, ThT), as it might introduce artefacts during sample deposition for AFM analysis. Thus, prepare triplicates of two identical solutions: i) the first containing monomeric Aβ42 to follow the process of aggregation by AFM; ii) the second containing the Aβ42 monomeric with addition of 20 µM ThT as a tracer to monitor the kinetics of aggregation.
    NOTE: It is important to perform steps 1.1 and 1.2 on ice. This procedure is to ensure that the monomeric Aβ42 solution does not aggregate until initiated in the plate reader. When handling samples, careful pipetting is essential to avoid the introduction of any air bubbles that may affect the protein sample.
  3. Pipette 80 µL of each sample in each well of a 96-well, half-area plate of black polystyrene with clear bottom and nonbinding surfaces.
  4. Seal the plate with a foil to minimize the evaporation of the sample over the course of the aggregation.
  5. Equilibrate the temperature of the plate reader to 37 °C.
  6. Set up the aggregation protocol to read fluorescence measurements at fixed time-point intervals, at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. The aggregation should be initiated in quiescent conditions.
  7. Insert the plate into the plate reader.
    NOTE: It is important to be careful while handling the plate, to ensure that the aggregation is not triggered prior to starting the plate reader. Use the gain adjustment settings to ensure the optimal readout of the fluorescence of each sample.
  8. Start the measurement.
    NOTE: The aggregation experiment is concluded when the ThT fluorescence sigmoidal curve reaches a plateau58.

2. Sample preparation for AFM and nano-IR measurements

  1. Glue highest quality grade V1 mica disc to high quality magnetic stainless-steel disc using adhesive tabs or double-sided tape.
  2. Etch mica by placing a piece of adhesive tape onto the mica surface and pulling off the top layer of the mica. This procedure will produce clean, atomically flat surface, suitable for sample deposition.
    NOTE: Etched surface must be uniform and smooth.
    CAUTION: Do not touch or breathe directly above freshly etched mica, as this will introduce artefacts.
  3. Pause/stop the plate reader measurement to collect the time point of interest during the aggregation process of the protein.
  4. Remove the sealing foil and collect 10 µL aliquot of the samples without ThT from the well into a 1.5 mL low-bind tubes.
  5. Deposit the 10 µL of the sample on the freshly etched mica. For AFM-IR measurements samples must be deposited on freshly stripped gold substrate.
  6. Incubate the solution on the mica for 1 min to allow physisorbtion.
    NOTE: Longer incubation time would allow better absorption on the surface but may induce artificial self-organization and self-assembly25. To avoid these effects, spray deposition may be exploited25.
  7. Rinse three times with 1 mL of ultrapure water.
  8. Dry under a gentle flow of nitrogen to measure the sample in air environment.

3. AFM imaging of the morphology of protein aggregates

NOTE: Morphology measurements can be performed both in contact and dynamic mode, in the following steps the latter is described since it reduces lateral forces to measure the 3D morphology of the sample with high resolution. AFM-IR measurements will be performed in contact mode to enhance AFM-IR signal-to-noise ratio.

  1. Turn on the AFM system at least 30 min before the measurements in order to enable the system to reach thermal stability. Click on Setup, then on Probe. In Probe Change window click on Next to prepare Z, Focus Stage.
  2. Turn off the AFM beam switch (if it is on). Unlock the dovetail locks, disconnect the head from the system and in Probe Change window click on Next.
  3. Mount the AFM cantilever on the probe holder. Connect the head to the system and lock the dovetail locks.
    NOTE: Optimal cantilevers to study biological samples in dynamic mode AFM have spring constant ranging between 2−40 N m-1 and an apex radii ranging between 2−8 nm14.
  4. Turn on the AFM laser beam switch and in Probe Change window click on Next.
  5. In the pop-up window click on No if the cantilever type did not change. Adjust optical alignment knobs to find the cantilever. Adjust focus onto cantilever and click on Next.
  6. In the pop-up window click on Yes if the focus is on the cantilever.
  7. Position the laser beam at the end of the cantilever using the knobs controlling position of the detection laser. Maximize the total signal measured by the four-quadrant photodiode to at least >1 V using the knobs controlling the position of the deflected laser beam on the position sensitive photodiode (PSPD). In Probe Change window click on Next and then on Close.
  8. Wait 15 min for the cantilever to reach thermal stability. Readjust the position of the deflected laser beam on the PSPD if necessary.
  9. Click on NCM SWEEP, choose desired amplitude of oscillation, click on Use Phase, click on auto and tune the cantilever close to the maximum of its first free resonance of oscillation, which is of approximately 300 kHz for a cantilever with a spring constant of 40 N m-1.
    NOTE: Tuning the cantilever out of the maximum of its free resonance assures higher stability of the measurements14.
  10. Place the sample on the sample holder. Choose suitable imaging parameters. Typical scanning rate is 0.3−1.0 Hz for a scan area of 1 x 1 µm2 to 5 x 5 µm2. Typical resolution needed is between 256 x 256 and 1024 x 1024 pixels. Click on Scan Area to choose the scan size and the pixel number.
  11. Focus the optical view on the sample. Click on Approach to approach the sample surface. Once approach is completed click on Lift 100 µm to rise the AFM tip 100 µm above the surface of the sample. Click Expand on the optical image of the sample. Click on the Focus Stage bar to focus the view on the surface of the sample.
  12. Use the arrows to move in the region of the sample of interest. Engage the surface by pressing Approach button. Click on Line Scan button, and check if the tip is following the surface well, if necessary, adjust the Set Point.
  13. Start imaging the sample surface by pressing Scan button. During imaging, to avoid large imaging force and keep consistency of within independent samples, maintain a constant regime of phase change not exceeding Δ20°42.
  14. Enter file name and choose base directory where the acquired image will be saved.

4. Infrared nanospectroscopy measurements of protein aggregates

  1. Turn on the AFM-IR system and the infrared (IR) laser60 30−60 min before measurements for thermal stabilization. Typical lasers for AFM-IR instruments are optical parameter oscillators (OPO) and quantum cascade lasers (QCL).
  2. Open the built-in software to control the instrument. Click on file and then on new to open a new nanoIR file. Press the button initialise to start the AFM-IR system.
  3. Open the instrument cover and mount on the AFM-IR system a silicon gold coated probe with a nominal radius of 30 nm and a spring constant of 0.2 N/m to measure the sample in contact mode.
  4. Click on Load in the section AFM probe and then next. In focus on probe section click on the arrows to focus the camera on the cantilever. In the section sample XY movement, use the arrows to place the cross-air at the end of the cantilever.
  5. Rotate the knobs controlling the position of the detection laser to position laser at the end of the cantilever. Rotate the laser knobs to detect and maximize the sum measured by the four-quadrant photodiode to a value >3 V. Rotate the deflection knob to adjust the cantilever deflection to -1 V and then click next. Close the cover of the instrument.
  6. In the section focus on sample use the arrows to focus the camera on the sample. Then, in the section sample XY movement use the arrows to move in the region of interest of the sample and click on approach and engage.
  7. In the microscope window select as inputs the channels of interest to map the biophysical properties of the sample. In particular, choose height for morphology, Amplitude 2 for IR absorption and PLL frequency to map tip-sample contact resonance.
  8. In the AFM scan section of the controls window, use similar parameters as in section 3 (i.e., scan rate 0.1−1.0 Hz, between 256 x 256 and 1024 x 1024 pixels). Choose as value of the gains according to sample roughness: integral I = 1−10 and proportional P = 10−30. Then, click on scan to acquire a morphology map.
    NOTE: The morphology can be similarly measured in the dynamic tapping mode, but AFM-IR measurements are performed here in the contact mode to have higher signal to noise ratios.
  9. After the mapping of morphology is finished, in the microscope window, click on the height map to position the probe on the top of one aggregate. Then in the nanoIR section of the controls window, click on start IR to illuminate the sample with the IR laser.
  10. To focus the infrared laser on the cantilever, click on optimisation. In this window, write a wavenumber where the sample is going to have high absorbance and click add. For protein, a typical value is 1655 cm-1. Click on scan to find the IR laser position and click on update to align its position with the cantilever. Close the window.
  11. In the section general of the nanoIR setting, write a wavenumber where high absorbance in the relative field is expected. Then, deactivate the Band Pass Filter option and look at the meter reading and at the fast Fourier transform (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 300 kHz. Write this resonance frequency value in the general section in the freq. centre field and use a freq. window of 50 kHz.
    NOTE: Select a laser power that is low enough to not saturate the meter reading and to have distortion in the cantilever response and to not overheat the sample.
  12. Click on laser pulse tune window to use the resonance enhanced mode. Choose a frequency centre of 300 kHz, a tune range of 50 kHz and a duty cycle of the laser of 5%. Click on acquire to sweep the pulse rate of the laser. By using the cursor in the graph, tune the laser pulse to the frequency of the mechanical response of the thermal expansion of the sample absorbing the IR light. Then, select the option PLL to monitor the contact resonance between the sample and the tip. Press the zero button in the PLL window and tick enable to track the sample-tip contact resonance. Choose an integral gain I = 0.5 and proportional gain P = 10. Close the window.
  13. Open again the optimisation window and find the position of the IR laser for at least 3 wavenumbers corresponding to major absorbance bands of the sample (amide band I-II-III) and for at least one wavenumber for each chip of the laser.
  14. Click on Tools | IR Background Calibration | New. In the window select the spectroscopic region of interest (1800−1200 cm-1 to study protein samples); choose the same pulse rate as determined in step 4.12 and a duty cycle of 5%; click on fast acquisition and select the laser speed, typical range for a quantum cascade laser is between 20−500 cm-1. Click on acquire to measure the IR laser background. This background spectrum will be used for normalization of measured nanoscale localized spectra. Close the window.
    NOTE: If a fast laser and resonance enhanced mode is not available, skip step 4.12 and select stepped spectra instead of fast in both the background and IR spectra acquisition windows. However, single aggregate sensitivity will not be reached.
  15. In the IR spectra settings, choose an IR spectrum resolution between 1−4 cm-1 and a number of co-averages of at least 64x. Click on acquire to measure a nanoscale localized IR spectrum in the protein range (1800−1200 cm-1).
  16. To acquire a nanoscale resolved chemical map, select IR imaging option, choose a wavenumber of interest (e.g., 1655 cm-1 for amide band I) and click on scan in the AFM scan window.
  17. Once the mapping is completed, go to file and save the measurements. To analyze the acquired maps of morphology, contact-resonance and chemistry, as well as the nanoscale localized spectra, use the built-in AFM image processing software. Spectra and Images can be saved as .csv or .axz files for further detailed analysis with commercial software.

5. Image processing and analysis of cross-sectional dimensions

  1. Flatten raw images14,17,19,41,42,59 using built-in AFM image processing software or commercial software.
    NOTE: The aggregates should be masked from the calculation to avoid artefacts of analysis and underestimation of their measured height.
  2. Flatten the image by 0 order plane fit subtraction.
  3. Flatten the image by plane and then line by line at a 1st regression order, the second step is repeated until the flat baseline in line profile of the image is reached.
  4. If the sample is very crowded, contains exceptionally high aggregates or scanner bow artefact is present, flatten image using 2nd regression order fit.
  5. Measure aggregate height and width from a line profile taken perpendicular to the fibril axis14,17,19,41,42,59.
  6. Measure fibril length parallel to the fibril axis14,17,19,41,42,59.

Results

A representative time course of Aβ42 aggregation, as measured by the ThT fluorescence assay, is shown in Figure 1. The aggregation process is commonly characterized by a sigmoidal curve, where a lag phase is initially observed, and is followed by a steep growth phase, before the curve reaches a plateau when an equilibrium steady state is reached6,7,58. It is essential to ensure that an optimize...

Discussion

The first critical step in this protocol is the preparation of monomeric proteins, such as in the case of Aβ42 solution described in steps 1.1 and 1.2. It is essential to initiate the aggregation process from a highly pure, monomeric solution, as the presence of oligomeric or aggregated species may result in poor reproducibility of the aggregation kinetics58, and induce artefacts in the AFM measurements (e.g., fibrillar species will be evident at the initial stages of the aggregation), which ...

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Swiss National Foundation for Science (SNF) for the financial support (grant number P2ELP2_162116 and P300P2_171219), the Darwin College, Erasmus+ program for the financial support (grant number 2018-1-LT01-KA103-046719-15400-P3) and the research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) through the ERC grant PhysProt (agreement number 337969), the Newman Foundation (T.P.J.K.) and The Cambridge Centre for Misfolding Diseases (C.G., M.V., and T.P.J.K.).

Materials

NameCompanyCatalog NumberComments
AFM-IR systemAnasys InstrumentsnanoIR 2 or 3Systems to measure thermal expansion in contact and resonance mode
Corning 96-well Half Area Black/Clear Bottom Polystyrene NBS MicroplateCorning3881
Corning Microplate Aluminium Sealing TapeCorning6570
Double Sided Adhesive DiscsAGAR ScientificAGG3347N
FLUOstar OmegaBMG Labtech415-101Platereader
Mica Disc 10mm V1AGAR ScientificAGF7013
Park NX10 AFM systemPark SystemsN/AAtomic Force Microscope
Platypus Ultra-Flat Gold ChipsPlatypus TechnologiesAU.1000.SWTSG
PPP-NCHR-10 cantileversPark SystemsPPP-NCHR-10
Protein LowBind Tubes, 2.0mLEppendorf30108132
Silicon gold coated cantileversAnasys InstrumentsPR-EX-nIR2
SPM Specimen Discs 12mmAGAR ScientificAGF7001

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