# Optical Trapping, Sizing, and Probing Acoustic Modes of a Small Virus
## 4 Discussion  
It is well established that optical trapping enables the performance of experiments on single nanometer-scale particles. While the possibility to optically trap large viruses existed for decades, trapping with the aid of DNH apertures of one of the smallest viruses yet reported was demonstrated here. We showed the feasibility of identifying the virus in question, by determining the size of the particle, as well as experimentally measuring the signature acoustic modes. Such identification experiments, along with others such as standard Raman spectroscopy [78; 79; 80; 81], could be useful for diagnostic purposes or for virus particle sorting. Figure 2: Sizing of particles via autocorrelation analysis: (**a**) a typical signal of a trap event; (**b**) autocorrelation of the signal after trapping, with double-exponential fit curve to extract the time constant, \(\tau\)2; (**c**) a linear fit of the values of the time constant obtained from the autocorrelation curve fit, \(\tau\)2 vs. particle diameter for the three particle sizes; (**d**) eight measurements of the extraordinary acoustic Raman (EAR) signal of PhiX174 virus, along with the averaged curve in red, showing a peak at 32 GHz. The sizing technique, which models the optical trap as an overdamped harmonic oscillator, relies on some simplifying assumptions, i.e., that the particle is spherical, and that the particle radius is small compared to the distance between the aperture walls. PhiX174 has an icosahedral geometry, which is nearly spherical, with an established diameter of 25 nm, while the distance between aperture walls is 40 nm; thus, these assumptions are reasonable. Moreover, when the radius of a trapped PhiX174 particle along with those of two polystyrene sphere sizing standards was plotted against the characteristic times extracted from the experimental data, a good linear fit was found, which seems to justify those assumptions. It also helps to give confidence that the trapped particles were the expected ones, instead of some contaminant, which is always a possibility when it comes to trapping nanometer scale particles in liquids. One potential concern could be the effect of temperature on the optical experiments. Temperature could affect the physical attributes of the material being measured, such as acoustic properties or conformation of the structures, as well as affecting the measurement system itself; for example, transmission of the laser through the DNH aperture is likely temperature-dependent. The sample was not actively cooled; however, DNH apertures were in a gold substrate with a large surface area, which should transmit heat away from the sample area inside the DNH aperture, at the focus of the lasers. While it would be difficult to measure the local temperature at the sample, which is held within the focus of the lasers, several steps were taken to minimize the impact of unwanted temperature effects. Firstly, the input lasers were actively temperature- and current-controlled, and the output signal was found to vary in intensity by less than 5%, both when measured before the sample and when passing through the DNH apertures before a trapping event. For the EAR experiments, after varying the wavelength by each wavelength step of \(\sim\)0.001 nm, for each step, a wait time of 5 s was given to allow the system to stabilize before beginning data acquisition. In addition, any short-term fluctuations in signal due to temperature variation would be averaged out over the measurement time of 15 s per step. Moreover, the full frequency range was swept multiple times for each sample type, giving consistent results for each scan as seen in Figure 2d, which is a good indication that any potential long-term temperature drift did not introduce any variation in the measurements. The EAR measurement actually directly measures fluctuations of the laser transmission signal, which are a manifestation of localized heating effects of the interfering lasers coupling with the sample material.