Potential use of microbubbles (MBs) as contrast material in x-ray dark field (DF) imaging: How does the DF signal change with the characteristic parameters of the MBs?

Abstract

One of the most exciting aspects of the grating based x-ray differential phase contrast (DPC) acquisition method is the concurrent generation of the so-called dark field (DF) signal, along with the classical absorption signal and the novel DPC signal. The DF signal is associated with local distribution of small angle scatterers in an image object, while the absorption signal and DPC signal are often used to characterize the relatively uniform structure of the image object. Besides the endogenous image contrast, exogenous contrast media are often used in x-ray imaging to locally enhance the image signal. This paper proposes a potential contrast medium for DF signal enhancement: microbubbles (MBs). MBs have already been developed for clinical use in ultrasound imaging, and recent experimental studies have shown that MBs may also enhance the DF signal, although it remained unclear how the physical characteristics of the MBs quantitatively impact the DF signal. In this paper, a systematic study was performed to investigate the quantitative relationships between the DF signal and the following properties of MBs: size, concentration, shell thickness, size uniformity, and whether gold nanoparticles were attached. The experimental results demonstrated that, an increased MB size (about 4 microns) may generate a stronger DF signal for our DPC imaging system; additionally, a moderately increased shell thickness and the use of gold nanoparticles on the shell surface also resulted in further enhancement of the DF signal. These findings may provide critical information needed for using MBs as the contrast agent of x-ray DF imaging.

1. INTRODUCTION

Grating-based x-ray differential phase contrast (DPC) imaging has shown promise in potential applications in medical imaging. With recently improved data acquisition techniques, images with three different contrast mechanisms, i.e., conventional absorption contrast, DPC, and dark field contrast (DF), can be simultaneously generated from a single x-ray exposure.¹ The differential phase signal originates from the refraction of the x-ray beam as it propagates through the image object. In contrast, the DF signals originates from the x-ray small-angle scattering events when an x-ray beam interacts with relatively dilute small angle scatterers in image object. Both DPC and DF signals can be appropriately tailored in a specific application to improve material discrimination capability and thus can be used to potentially improve disease detection.^2,3 For instance, DPC imaging may be used in conjunction with absorption imaging to provide a quantitative imaging method to differentiate materials better.⁴ DF signal may have potential applications in imaging microcalcifications for the early diagnosis of breast cancer.^5,6

In x-ray medical imaging, aside from the endogenous contrast generated by interactions between x-rays and tissue of human body, contrast media can also be injected locally into the vasculature or muscles to enhance signal. Iodinated contrast media are most commonly used to enhance x-ray absorption signals. Microbubbles (MBs) have been introduced in ultrasound imaging as an effective molecular probe;^7,8 studies have also demonstrated its potential as a contrast agent for x-ray DF imaging.^8–10 In general, MBs consist of a gas core surrounded by a solid shell of lipid, protein, or polymer. The average diameter of the MBs is on the order of micrometers, and MBs are usually not very water soluble. Several important characteristics can be used to describe MBs, including the size, shell thickness, shell composition, add-on nano-particles, uniformity of the MBs, and so on. However, there have been no systematic investigations on how each of these parameters impacts x-ray DF signal. Without knowing the quantitative relationship between these parameters of MBs and the DF signal, the optimization of the size and composition of the MBs as a potential DF contrast agent will lose the needed scientific guidance. In this paper, we present the first results of an systematic investigation to address the aforementioned question.

2. MATERIALS AND METHODS

2.1. Brief review of dark field (DF) signal generation

In grating-based x-ray multi-contrast imaging acquisitions, a fundamental relationship between a set of detected intensity measurements I^(k) (k = 1, 2, ⋯, M ≥ 3) are used to determine three parameters, I₀, I₁, and ϕ in following signal model^11,12

$$I^{\left(k\right)}=I_0+I_1\text{cos}\left(2\pi \frac{k}{M}+\varphi \right).$$ (1)

The DPC image and absorption image are generated from ϕ and I₀ respectively. DF signal is determined by both I₀ and I₁, defined as follows

$$DF=-\text{ln}\left[\frac{I_1^{obj}}{I_0^{obj}}\times \frac{I_0^{bkg}}{I_1^{bkg}}\right],$$ (2)

where bkg denotes the measured quantity when there is no object presented in the x-ray beam, while obj labels the measured quantities when an image object is present in x-ray beam. The generation of DF contrast is due to the visibility degradation of the intensity modulation on the detector surface during data acquisition when the scanned object is added into the x-ray beam. The visibility degradation is caused by ultra-small angle x-ray scattering which originates from the local inhomogeneities in the sample.^5,13–15

2.2. Microbubble samples

Microbubbles used in this paper were produced at the Center for Ultrasound Molecular Imaging and Therapeutics at the University of Pittsburgh Medical Center (UPMC). Figure 1(a) shows a schematic diagram of the microbubble. It is a N2 gas encapsulated hollow microcapsule with a polymer and human serum albumin double layer shell. Figure 1(b) is the scanning electron microscope (SEM) image of a broken microbubble.

Figure 1.

(a) Schematic diagram of a microbubble. (b) A scanning electron microscope (SEM) image of the microbubbles.

Specifications of the used MB samples were presented in Table 1. All of the eight samples used in experimental studies had been reconstituted, washed to remove the flooded MBs and Coulter counted. These operations provide very concentrated MBs with the exception of the #2 sample which was modified with gold nano-particles. On average, there are about 800 gold nano-particles attached to each MB in sample #2.

Table 1.

List of eight batches of MB samples prepared at UPMC.

Sample #	Batch number	Concentration (MB/mL)	Microbubble size μm)
			mean	median	mode
1	V010515-2μm	2.00E9	2.661	2.539	2.577
2	Au NP-v010515-2μm	6.40E8	2.698	2.565	2.327
3	V010515-4μm	1.80E9	3.794	3.792	4.056
4	V091014-spg	1.40E9	4.372	4.314	5.088
5	V09	3.00E9	2.690	2.483	2.577
6	V061215	3.95E9	3.616	3.536	4.341
7	V062415	2.55E9	4.138	4.065	4.700
8	V062515	2.54E9	4.565	4.560	4.595

In sample #4, an SPG model was used to refine the distribution of MB size to generate a more uniform distribution of MBs in sample. Sample #5 was made with a different polymer than the others. Moreover, sample #6 has doubled shell thickness, sample #7 has four times thicker shell thickness, and sample #8 has eight times shell thickness compared with the normal shell thickness. The sample solutions were stored at 4° Celsius. Prior to imaging, each solution was shaken gently to mix the MBs.

2.3. Experimental imaging system

MB samples were imaged using an experimental multi-contrast imaging benchtop system at the University of Wisconsin-Madison. The benchtop system consists of a diagnostic x-ray tube (Varian G1582, Palo Alto, CA), which was operated at 40 kVp and 20 mA. The mean beam energy is 28 keV which is also the design energy for the grating interferometer. The x-ray sensor was a CMOS flat panel detector with 48 μm isotropic pixel size (Rad-icon Shad-o-Box 2048, Sunnyvale, CA). The x-ray exposure time was 5 seconds per projection.

Three gratings were employed in the interferometry. The source grating G0 was placed in proximity to the x-ray tube housing to produce spatially coherent x-ray beams. Its period is 22.7 μm and has a duty cycle of 40%. The silicon G1 phase grating has a period of 8.0 μm. The G2 absorption grating has a period of 4.8 μm, and the deposited gold is 60 μm high. The G1 and G2 gratings both feature a 50% duty cycle.

3. RESULTS

3.1. Visual comparison of absorption image and DF image of the eight samples

Shown in Figure 2 are images of the eight samples for absorption contrast (top row) and the corresponding DF images (bottom row). As one can observe from the results, despite the difference in MB parameters in all eight samples, absorption images show very similar visual perception. However, the eight samples show significantly different DF contrasts. The visual observation demonstrates that DF signals are sensitive to the characteristic parameters of the MBs, while absorption images are insensitive to these characteristic parameters of the MBs.

Figure 2.

Initial experimental results of the eight batches of MB samples. Images in the top row show the absorption results, displayed at [0.0, 0.4]; images in the bottom are the DF results, displayed at [0.0, 3.0].

3.2. Quantitative comparison of DF signals on each characteristic parameter

Quantitative measurements show that there are no significant differences in the absorption images of the eight samples as shown in Figure 3(a). These results are not surprising, since the density of MBs is very low in all eight samples compared with the density of water solution. In addition, the volume fraction of MBs in each vial is also small. These two facts may be used to explained similar absorption signal in all eight samples. In contrast, dramatic variations are observed in DF images as shown in the bottom image row in Figure 2 and the quantitative measurements in Figure 3(b). Therefore, for a given imaging system, the parameters of the MBs should be carefully selected to maximum the DF signal.

Figure 3.

Measured absorption and DF signal intensities for the eight samples without concentration normalization.

3.3. Impact of parameters of MBs on DF signals

The above different behaviors between the absorption and DF signals were due to their particular signal formation mechanisms. Namely, the eight MB solutions have similar absorption properties, but with different abilities to generate x-ray small-angle scattering signals. These interesting results inspired us to perform a more detailed study on the DF signals.

Impact of concentration on DF signal

The DF signal intensity is affected by the total small-angle scatterers in the sample. Therefore, the concentration of the MB solution should have an impact on the DF signal. In order to study the impact of concentration on the DF signal, two samples were selected (sample #3 and #4) and their concentrations were changed by gradually adding water to the vials. Figure 4 shows the experimental results for both samples. It is clear from figure 4 that DF signal increases linearly with concentration. In a real application, the concentration will always have a upper limit depending on the tolerance of the animals or humans. Therefore, each measured DF signal in Figure 3(b) was normalized by its own concentration. The normalized results can be found in Figures 5(a) and 5(b). This normalized signal was used to study the impact of each individual MB parameter.

Figure 4.

DF signals as a function of concentration for two different samples.

Figure 5.

The normalized DF signals for standard shell thickness samples #1 to #5 (a) and samples #6 to #8 with varied shell thicknesses (b). Note that the x-axis on both plots was reordered to show increasing mean MB size.

Impact of size of MBs on DF signal

We investigated the impact of MB size on the DF signal. One can see in Figure 5(a) that with our setup, the #3 MB sample with 4 μm diameter improves the DF signal by over 50% compared with the #1 MB sample with 2 μm diameter.

Impact of shell thickness on DF signal

The shell thickness of MB is another important factor that needs to be considered. As demonstrated by sample #6 to #8 in either Figure 3(b) or Figure 5(b), MBs with doubled shell thickness may be helpful to generate stronger DF signals. However, further increasing the shell thickness may reduce the DF signals significantly.

Impact of shell composition on DF signal

Since it’s not easy to change the shell material, we only tested one different MB shell composition. Results show that no improvement was gained for this particular polymer, see results for sample #1 and #5 in Figure 5(a).

Impact of added nano-particle to MBs on DF signal

If the bubble size is fixed, as the case of #1 and #2, we found that the addition of gold nano-particles in sample #2 enabled strong DF signals, with about 40% improvement as shown in Figure 5(a).

Impact of uniformity of MBs on DF signal

The refined #4 sample, see Figure 5(a), provides the highest DF signal. This may be due to the narrow distribution of MB sizes resulting from discarding very large and very small MBs.

4. DISCUSSION AND SUMMARY

In summary, a comparison of DF signals of eight MB samples with different parameters has been performed to study the potential impact of these parameters on the final DF signal in grating-based x-ray multi-contrast imaging system. After preliminary investigations of the impact of different parameters of MB on DF signals, the DF signal has been found to have a strong dependence on the MB size, shell thickness, add-on nanoparticles, and the refinement procedure. We found that the strongest DF signal was generated when the 4 μm diameter MBs were used. Additionally, doubling the shell thickness also generated stronger signals. However, one should keep in mind that the shell thickness can not be made too thick. Moreover, attaching the gold nanoparticles on the shell surface while refining the MB samples before usage can also significantly benefit the DF imaging. In the future, we would like to explore a potential theoretical signal model for MBs in DF imaging and further explore whether MB is a clinically viable contrast agent. Experimental findings obtained in this work hopefully will add value towards contrast-enhanced x-ray DF imaging.