Materials and Reagents

FUS sample at a concentration of 1-10 μM (the production and purification of FUS is discussed in previously published protocols (Qamar et al., 2018)
ZnSe window (Thorlabs, catalog number: WG70530)
MilliQ water

Equipment

AFM-IR with thermomechanical detection: nanoIR1, nanoIR2, or nanoIR3 (Anasys Instruments, Bruker, USA)
PR-EX-nIR2 AFM probes: silicon, gold-coated, with a nominal radius of 30 nm and a spring constant of 0.2 N/m (for contact mode measurements; Anasys, Bruker, USA)
P10 Pipette (Starlabs, UK)

Software

In-built nanoIR software (Bruker, USA)
SPIP (Image Metrology, Denmark, https://www.imagemet.com/products/spip/)
OriginPRO (Origin Labs, USA, https://www.originlab.com/origin)

Procedure

Sample preparation for AFM-IR measurements
1. Pipette 10 μl sample onto the hydrophobic ZnSe crystal. Incubate for 1 min to allow for physisorbtion. Longer deposition times are possible, but this may induce artefacts due to artificial self-organisation on the surface.
2. Rinse three times with 1 ml MilliQ water to remove the excess salt.
3. Dry under a gentle flow of nitrogen.

Infrared nanospectroscopy measurements of liquid-liquid phase-separated droplets
1. Turn on the AFM-IR system and the infrared (IR) laser. This should be done 30-60 min before measurement to allow the system to reach thermal equilibrium.
2. Open the in-built software to control the AFM-IR (Figure 3). Select file > new to open a new nanoIR file. Press the initialise button to start the AFM-IR system.
  
  Figure 3. An overview of the nanoIR software. In the controls window, imaging parameters are given as input. In the microscope window, the relevant information is presented as imaging proceeds.
3. Open the instrument cover and mount the probe (Figure 4).
  
  Figure 4. A photo of the AFM head. The cantilever is mounted on the AFM head (red box). Then, the knobs on the AFM head are used to adjust the position of the detection laser.
4. Now, focus the optical camera on the cantilever. Select AFM Probe > Load > Next. Under focus on probe, click on the arrows to change the focus. Under sample XY movement, use the arrows to position the crosshair on the cantilever.
5. Focus the detection laser on the end of the cantilever using the knobs. The value of the sum measured by the four-quadrant photodiode should be at least 3 V. Use the deflection knob in the AFM head and adjust the cantilever deflection to -1 V and then click next.
6. Close the cover of the instrument.
7. Focus the camera on the sample using the arrows under focus on sample (Figure 5).
  
  Figure 5. Illustration of Steps B4-B7. The cantilever is loaded, and the optical camera and laser focused on the end of the cantilever. Then, the camera is focused on the sample surface. Arrows can be used to navigate around the sample.
8. To select a region of interest on the sample, select sample XY movement. Use the arrows to move around the sample surface. When you have identified the region of interest, click on approach and engage.
9. In this context of the study of protein droplets, the information on morphology, IR absorption, and stiffness are of particular interest. To measure these parameters, select the display channels under the microscope window. Choose Height for morphology, Amplitude 2 for IR absorption, and PLL frequency to map tip-sample contact resonance (Figures 6 and 7).
  Note: During morphology acquisition, the tip and the sample in contact can be considered a system of coupled springs. The contact resonance of these springs is related to the intrinsic stiffness of the sample. A stiffer sample causes an increase in contact resonance, which we can term ‘frequency shift.’
  
  Figure 6. Demonstration of Step B9, in which display channels are selected according to the parameters of interest. In this case, they are morphology (Height), tip-sample contact resonance frequency (PLL Frequency), and IR absorption (Amp2).
  
  Figure 7. The contact resonance between the tip and the sample is related monotonically to the intrinsic sample stiffness. A stiffer sample causes a shift to a higher frequency, which can be used to investigate the relative stiffness of heterogeneous samples.
10. Set the imaging parameters for morphology measurements in the AFM scan section of the controls window (Figure 8).
  Note: It is important to consider the sample properties when selecting imaging parameters. For example, choosing a lower scan rate will decrease the lateral force exerted on the sample, which is relevant for biological samples; however, this choice will increase the image acquisition time. A higher number of pixels will increase the image resolution but will similarly increase the image acquisition time. The gain values should be selected considering the sample roughness (i.e., a higher integral gain will allow the system to respond to changes in sample height but can also introduce noise). The following are the recommended range of values: scan rate 0.1-1.0 Hz and 256 × 256-1024 × 1024 pixels. The appropriate range for gain values are as follows: integral I = 1-10 and proportional P = 5-30. The value of the gains should be chosen as the minimal values to have overlapping trace and retrace scan lines.
  
  Figure 8. A demonstration of Step B10, in which the imaging parameters are input for morphology mapping. The parameters that should be considered first are the scan rate, image size, resolution (pixels), setpoint, and gain (integral and proportional).
11. Select scan to acquire a morphology map. Representative morphology maps and contact resonance values of FUS droplets are shown in Figures 9 and 10.
  Note: The morphology can be similarly measured in dynamic tapping mode, but AFM-IR measurements are performed here in contact mode to have higher signal-to-noise ratios during infrared spectra and map acquisition.
12. When the mapping of morphology is finished (Figure 9), set up the instrument to perform the IR and contact resonance (Figure 10) measurements. In the microscope window, click on the height map to position the probe on top of one droplet. Then in the nanoIR section of the controls window, click on start IR to illuminate the sample with the IR laser.
  
  Figure 9. A representative image of the morphology of a FUS droplet. The 3D morphology of a droplet is represented as a 2D colour map, with lighter colours indicating increasing height.
  
  Figure 10. A representative contact resonance map of a FUS droplet. A contact frequency map is simultaneously acquired during morphology acquisition. Tip-sample contact resonance provides information regarding sample stiffness. A stiffer sample would cause an increase in contact resonance, which we can term ‘frequency shift.’
13. To focus the infrared laser on the cantilever, click on optimisation. In this window, write your chosen wavenumber click add.
  Note: The wavenumber should be chosen such that your sample will have significant absorbance. If the sample absorbs IR light at a specific wavelength, chemical bonds will vibrate, causing a thermal expansion of the sample that can be detected by the AFM tip in contact with the droplet. A typical value for protein samples is 1,655 cm^-1, which corresponds to the amide I band. The response of the cantilever is proportional to the absorption of IR light by the sample; thus, differences in IR absorption can be mapped and correlated to the corresponding morphology map.
14. Select scan to find the IR laser position and then update to align its position with the cantilever (Figure 11). Close the window.
  
  Figure 11. A demonstration of Steps B13 and B14, in which the wavenumber of interest, in this case 1,655 cm^-1, is entered. Then select scan to find the IR laser position and update to align its position with the cantilever.
15. Select general in the nanoIR settings. Input a wavenumber where IR absorption is expected. Then, deactivate the Band Pass Filter option and look at the meter reading and fast Fourier transform (FFT) of the cantilever response. In the FFT window, move the green cursor to read the resonance frequency of the cantilever (Figure 12). A typical value for the FFT of the resonance of the cantilevers is around 300 kHz. Write the found resonance frequency value in the general section of the freq. centre field and use a freq. window of 50 kHz. Select a laser power that is low enough to avoid saturating the meter reading, distorting the cantilever response, and overheating the sample.
  
  Figure 12. A demonstration of B15, in which the parameters of the IR laser are set up. On the left-hand side, input the parameters as described above. On the right-hand side, the meter reading displays the thermomechanical response of the cantilever. Use the cursors to centre the green course at the peak of the resonance frequency. Once the value for the resonance frequency has been determined, input it in the Freq. Centre on the left-hand side.
16. Click on the 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% (Duty cycle is the fraction time during which there is laser pulse emission). Click on acquire to sweep the pulse rate of the laser. Use the cursor in the graph to tune the laser pulse to the frequency of the mechanical response of the thermal expansion of the sample absorbing the IR light (Figure 13).
  
  Figure 13. A demonstration of laser tuning (Steps B16 and B17). In the laser pulse tune window, input the parameters as described in Step B19. Click on acquire to display the contact resonance frequency and phase. Under the auto-tune window, select PLL to track the contact resonance frequency of the resonance enhanced mode.
17. To avoid cross-talk between the chemical and mechanical information in the sample, it is important to track the contact resonance frequency in resonance enhanced mode by a phase locked loop (PLL). Select PLL, then click the zero button in the PLL window and tick enable. Choose an integral gain I = 0.5 and proportional gain P = 10.
18. Open the optimisation window again and identify the position of the IR laser for at least 3 wavelengths corresponding to the major absorbance bands of the sample. For a protein sample, this should be amide bands I, II, and III. Moreover, identify the position of at least one wavelength for each chip of the laser.
19. Click on Tools > IR Background Calibration > New (Figure 14). In the window, select the spectroscopic region of interest, which is 1,800-1,200 cm^-1 in the case of a protein sample. Choose the same pulse rate as determined in Step B16 and a duty cycle of 5%.
  
  Figure 14. A demonstration of the steps for background calibration (Steps B19-B21). Input the parameters as described above. Then, select acquire to generate a spectrum of the background (i.e., the substrate).
20. Select fast acquisition and choose the laser speed. For a quantum cascade laser, a typical range is between 20-5,000 cm^-1/s. The background spectrum will be used for normalisation of the measured nanoscale localised spectra. Close the window.
21. If a fast laser and resonance-enhanced mode is not available, skip Step B16 and select stepped spectra instead of fast in both the background and IR spectra acquisition windows. This will still allow for chemical characterisation. While this sensitivity is not sufficient to reach the single -molecule level, it still allows for full characterisation of liquid-liquid phase-separated droplets with a typical size ranging from hundreds of nanometers to micrometers.
22. Now, measure a nanoscale-localized IR spectrum in the protein range (1,800-1,200 cm^-1) (Figure 15). In the IR spectra settings, choose an IR spectrum resolution of 1-4 cm^-1. For the number of co-averages, choose at least 64. Select acquire.
23. Additionally, a nanoscale-resolved chemical map can be acquired (Figures 15 and 16). To do this, select the IR imaging option, choose a wavenumber of interest (e.g., 1,655 cm^-1 for amide band I), and click on scan in the AFM scan window.
  Note: The principles of acquiring localised IR spectra and IR maps are generally the same. When acquiring IR maps, the tip is scanned across the sample while maintaining a fixed laser wavenumber, so that the absorption of the sample and surface is mapped at that wavenumber. When acquiring a localised spectrum, the tip is held in a fixed position on the sample, and the IR laser wavenumber is varied to obtain absorption across a range of values. This provides a full IR spectrum that can then be used to provide specific information regarding the secondary structural contributions (discussed further below).
  
  Figure 15. Representative IR map and localised IR spectra of FUS droplets. An IR absorption map can be measured simultaneously during morphology mapping (together with the contact resonance map). The localised IR spectra can also be acquired at a single point on the surface of a droplet (indicated by the blue cross).
  
  Figure 16. An example of completed raw morphology and IR maps, as well as typical raw localised IR spectra. The blue cross indicates where the localised IR spectra were recorded.
24. Our advice is to acquire multiple localised spectra within one droplet to enable appropriate statistical significance for studying its heterogeneity. Repeat Step B22 multiple times within each droplet.

Data processing and analysis
1. Open an imaging processing software, such as SPIP, to process AFM maps.
2. First-order flatten AFM maps by selecting General > Plane Correction > Plane Correction Set-Up.
3. Under Global Correction, select Average Profile Fit.
4. Select Degree to 0, then Apply.
5. Repeat for Degree = 1.
6. Zero-order flatten IR and stiffness-related maps by repeating the sample flattening procedure (excluding Step C2).
7. To evaluate the relative sample stiffness, consider the distribution of the values for the pixels of the contact resonance frequency to calculate its average and standard deviation inside each region of interest in your sample (Figure 8). Then, plot these averages as a normalised ratio (Figure 17). Due to the complicated nature of determining the absolute stiffness of soft droplets, the stiffness of the droplets should be considered relative to the ZnSe substrate. To prove that the measured mechanical differences are real, the mechanical differences should be evaluated for independent tips and samples. Any software of your choice can be used.
  
  Figure 17. Measuring sample stiffness. A. An example of plotted average frequency shifts for each droplet measured. B. Relative stiffness of the droplets can be determined by representing the data as a ratio of the average frequency shifts.
8. Next, IR spectra can be processed by the OriginPro software to have normalised smoothed spectra and their second derivatives (Figure 18).
  
  Figure 18. The second derivative of the IR spectrum provides information on the secondary structure of the droplets. A. Representative processed IR spectrum of the droplets in the protein range (1,800-1,400 cm^-1). B. A second derivative operation was performed to identify the secondary structure.
9. For each spectrum acquired, subtract the spectrum acquired in a location close to the droplet on the ZnSe substrate to eliminate buffer absorption or tip contamination contributions. Select Analysis > Data Manipulation > Reference Data. Select the Input1 as your droplet spectrum and the reference data input as your ZnSe substrate spectrum. Select Okay.
10. Then for each droplet or for a specific location on its area, calculate an average spectrum by averaging spectra taken at different positions within the droplet. To reduce the level of instrumental noise in the spectra, at least 5 spectra should be averaged. Select Analysis > Mathematics > Average Multiple Curves > Open Dialogue.
11. For Method, select Average; for Averaged X, select Common X Range.
12. Select additional output parameters as desired and select Okay.
13. Smooth the average spectrum by applying a Savitzky-Golay filter (second order, 9 points). Select Analysis > Signal Processing > Smooth > Open Dialogue.
14. From the drop-down list for Method, select Savitzky-Golay.
15. For Points of Window, input 9.
16. For Polynomial Order, select 2. Select Okay.
17. Next, normalise the spectrum. Select Analysis > Mathematics > Normalise Curves > Open Dialogue.
18. For Normalize Methods, select Normalize to [0,1], then Okay.
19. Take the second derivative of the smoothed normalised spectrum. Select Analysis > Mathematics > Differentiate.
20. For Derivative Order, select 2.
21. Under Smooth, check the box for Savitzky-Golay Smooth. For Polynomial Order, select 2; for Points of Window, select 9.
22. Tick the box for Plot Derivative Curve and select Okay.
  Note: The amide I band contains information on the secondary structure, as C=O stretching is influenced by the backbone conformation. Deconvoluting the spectrum by taking the second derivative provides quantitative information on the structural contribution (i.e., provides information on the content of α-helices, β-sheets, and random coils). An example of this is shown in Figure 18.

Acknowledgments

The authors acknowledge funding from Darwin College, University of Cambridge (FSR) and Emmanuel College, University of Cambridge (AMM). This protocol was largely derived from previous work presented in Qamar et al. (2018).

Competing interests

The authors have no competing interests to declare.

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