Keywords: Single cell photoacoustic microrheology PA Viscoelastic Imaging System PA Elasticity Measurement Method Lock-in Amplifier
Note: This article uses the Sine Scientific Instruments OE2031 lock-in amplifier to make measurements.
[Overview]
In 2019, Professor Xing Da's team from South China Normal University published an article titled "Single-Cell Photoacoustic Microrhezhology" in IEEE Transactions on Medical Imaging.Professor Xing Da's team proposed an innovative photoacoustic microrheology (PAMR) that uses the time and phase characteristics of photoacoustic (PA) response to extract elastic modulus and viscosity.PAMR achieves single-cell elasticity and viscosity mapping of adipocytes and myocytes at the micrometer scale, expands the scope of traditional PA imaging, and opens up new possibilities for the development of new microrheological technologies.
Rheological properties such as elasticity and viscosity are related to the physiological functions and pathological states of cells and have been considered as indicators for testing many diseases.There are many technologies currently used to evaluate the viscoelasticity of tissues and cells, but the measurement of propagation velocity in traditional technologies reduces the accuracy of elasticity and viscosity estimation.Photoacoustic (PA) imaging, as a rapidly developing non-invasive technology, has been applied to elastic property characterization imaging.However, traditional PA imaging has problems such as overestimation of elastic modulus due to friction, inability to distinguish PA intensity contrast, lack of prior knowledge of absorption spectra, and time-consuming measurement.
In 2017, the PA viscoelastic imaging technology developed by Professor Xing Da's team can extract the viscosity-elasticity ratio parameters through PA phase delay, which can non-invasively analyze viscoelastic properties and be used for cancer detection and plaque assessment.
Fig. 1. Schematic diagram of the experimental setup of the PA viscoelastic imaging system
[Measurement methods and some experimental results]
Prof Xingda's team proposed a PAMR method based on thermoelastic expansion and damping effects. Firstly, the elasticity and viscosity were determined by developing a biomechanical model and exploring the time and phase characteristics of the PA response. Secondly, the team performed numerical simulations of the displacements using various rheological parameters. And based on the numerical simulations, a PAMR system was established.
1) Estimation of the modulus of elasticity:
The estimation of the elastic modulus is based on the thermal expansion mechanism, whereby irradiating the absorbing region with a laser pulse will induce oscillations in the sample, which will then generate a PA pressure wave, and using Navier's equation the displacements of the sample can be calculated for each laser excitation point:
（1）
By using the Hankel transform, the displacement can be solved analytically at the focus as
（2）
The modulus of elasticity E can be estimated by the method (3)
（3)
2) Viscosity estimation:
Viscoelastic media exhibit complex rheological behaviour with time-dependent mechanical response. The viscosity η can be calculated by equation (4)：
（4）
Therefore, according to Eqs. (3) and (4), the elastic modulus and viscosity can be extracted simultaneously by PA rise time and phase delay measurements.
Fig.2 illustrates the experimental setup of the PAMR system. A laser with an operating wavelength of 532 nm and a pulse width of 10 ns was used as the excitation source. The laser beam was expanded and collimated by a spatial filtering system and then scanned by a 2D galvanometer scanner. The laser beam was passed through a telescope system consisting of a scanning lens and a cylinder mirror before being focused onto the sample surface by an objective lens. The laser energy density was captured by a photodiode and an ultrasonic transducer with a flat spectrum between 200KHz and 15MHz was used to detect the PA signals.The PA signals were first amplified by a low noise amplifier, and then transmitted to a digital oscilloscope and a lock-in amplifier, OE2031, in order to acquire the rise time and phase delay of the PA signals, respectively. A function generator was used to simultaneously generate the trigger signals T1 (10KHz), T2 (10KHz) and T3 (1MHz).T1 was used to trigger the pulsed laser; T2 and T3 were used to trigger the oscilloscope and the lock-in amplifier OE2031, respectively.A CCD camera was used to acquire an optical image of the sample.
Fig.2 Principle device of photoacoustic microrheology (PAMR). bs: beam splitter; GM: galvanometer; SL: scanning lens; TL: tube lens; MO: microscope objective; UT: ultrasonic transducer; WT: water tank; Osc: oscilloscope; FG: function generator. t1: pulsed laser triggering signal; t2: oscilloscope triggering signal; t3: phase-locked amplifier triggering signal.
The research team conducted validation experiments on a homogeneous agar-gelatin model by comparing the elasticity measured by the PA method with that measured by a conventional mechanorheometer, and the results are shown in Fig. 3(e): there is a high correlation between the results measured by the two methods (R2 > 0.96). Figure 3(f) illustrates the conventional PA amplitude image (PAI) and PA elastography (PAE) images of the homogeneous agar-gelatin model with different concentrations. Due to the same light absorption distribution, there is no contrast in the PAI amplitude image. In contrast, PAE elastography was able to show significant differences. The results indicate that PAMR has the ability to determine the modulus of elasticity.
Fig.3 Validation of the PA elasticity measurement method. (e) Comparison between the elasticity obtained by the PA method and the elasticity obtained by conventional rheometry. (f) Conventional photoacoustic amplitude image (PAI) and photoacoustic elastography (PAE).
[Summary]
This study presents a photoacoustic microrheology-based method, PAMR, for measuring the mechanical properties of individual cells.PAMR utilises the temporal and phase characteristics of the PA response for biomechanical property studies, enabling single-cell elasticity and viscosity mapping of adipocytes and myoblasts on a micrometre scale. The following conclusions can be drawn from the model construction and experimental results:
①PAMR is able to successfully measure the elastic modulus of samples with high correlation with conventional mechanical rheometer measurements, and photoacoustic elastography (PAE) provides a significant contrast regarding the elasticity of the samples when compared to photoacoustic amplitude imagery (PAI), which suggests that PAE is able to reveal elasticity variations induced by differences in biochemical composition or content.
②The PAMR technique possesses the ability to accurately measure various cellular elasticity and viscosity properties, and it has shown remarkable results in distinguishing adipocytes from myocytes.
③The lock-in amplifier OE2031 was successfully used to detect the phase difference between the PA signal and the reference signal, thus proposing a novel acousto-optic signal processing method.
[Reference]
