Acoustic and Mechanical Properties of Microbubbles Stabilized by Polymeric Nanoparticles

This thesis examines acoustic and mechanical properties of microbubbles stabilized bypoly(butyl cyanoacrylate) nanoparticles. The microbubbles are developed with the purposeof being applied in combination with ultrasound as a novel drug delivery systemto tumors. Knowledge about the acoustic and mechanical properties is essential to understand the interaction between ultrasound and the microbubbles, their drug deliverypotential and stability in the circulatory system.Microbubbles with different surfactant proteins, average diameter, and nanoparticlebatches are analyzed and compared in this work.Backscattered power and attenuation of ultrasound waves propagating through a suspensionof microbubbles are measured in the frequency range 1-20 MHz. The measurementsare done with microbubbles suspended in a sample chamber in a water tank. Attenuationare measured by using the same ultrasound transducer both for transmit andreceive. The attenuation coefficients are plotted as function of frequency, and theoreticalattenuation spectra calculated from the Church model and the Ho model are ttedto the experimental spectra by appropriate estimation of viscoelastic properties of themicrobubble shell. Using casein as a surfactant protein is found to increase the shearmodulus and viscosity of the shell compared to when Bovine Serum Albumin (BSA) is used. Microbubbles with BSA as surfactant protein and smaller average diameter show higher shear modulus, but lower viscosity compared to when the average diameter is larger.The backscatter is received with a transducer different from the transmit transducer.When a transmit transducer with center frequency 1 MHz is used, the scatter spectradisplay higher harmonics at pressures above 5-15 kPa. Furthermore, destruction takesplace above 170 kPa, but apparently more for the microbubbles with casein rather thanBSA as surfactant protein. Smaller microbubbles seem to exhibit more resistance againstdestruction.Measurements of microbubble shell elasticity is performed using atomic force microscopy(AFM) by applying nanoscale compressions (up to 500 nN) with a at cantileveron isolated nanoparticle-stabilized microbubbles. Values of the Young's modulusare found by fitting obtained force-deformation curves to the simple theoretical Reissner model. In accordance with the attenuation measurements, the results indicate that the casein microbubbles have higher Young's modulus than BSA microbubbles. The shell thickness is assumed to be 150 nm. A linear relation between the Young's modulus and the diameter is found for the microbubbles with casein as surfactant protein

Non-linear optical microscopy of cartilage canals in the distal femur of young pigs may reveal the cause of articular osteochondrosis

publishedVersio

Non-linear optical microscopy of cartilage canals in the distal femur of young pigs may reveal the cause of articular osteochondrosis

Background Articular osteochondrosis is a common cause of leg weakness in pigs and is defined as a focal delay in the endochondral ossification of the epiphysis. The first demonstrated steps in the pathogenesis consist of loss of blood supply and subsequent chondronecrosis in the epiphyseal growth cartilage. Blood vessels in cartilage are located in cartilage canals and become incorporated into the secondary ossification centre during growth. It has been hypothesized that vascular failure occurs during this incorporation process, but it is not known what predisposes a canal to fail. To obtain new information that may reveal the cause of vascular failure, the distal femur of 4 pigs aged 82–140 days was sampled and examined by non-linear optical microscopy. This novel technique was used for its ability to reveal information about collagen by second harmonic generation and cellular morphology by two-photon-excited fluorescence in thick sections without staining. The aims were to identify morphological variations between cartilage canal segments and to examine if failed cartilage canals could be followed back to the location where the blood supply ceased. Results The cartilage canals were shown to vary in their content of collagen fibres (112/412 segments), and the second harmonic and fluorescence signals indicated a variation in the bundling of collagen fibrils (245/412 segments) and in the calcification (30/412 segments) of the adjacent cartilage matrix. Failed cartilage canals associated with chondronecrosis were shown to enter the epiphyseal growth cartilage from not only the secondary ossification centre, but also the attachment site of the caudal cruciate ligament. Conclusion The variations between cartilage canal segments could potentially explain why the blood supply fails at the osteochondral junction in only a subset of the canals. Proteins linked to these variations should be examined in future genomic studies. Although incorporation can still be a major cause, it could not account for all cases of vascular failure. The role of the caudal cruciate ligament in the cause of osteochondrosis should therefore be investigated further

Automated polarization-resolved second harmonic generation imaging

Polarization-resolved second harmonic generation (P-SHG) microscopy has evolved as a promising technique to reveal subresolution information about the structure and orientation of ordered biological macromolecules. To extend the adoption of the technique, it should be easily integrated onto commercial laser scanning microscopes. Furthermore, procedures for easy calibration and assessment of measurement accuracy are essential, and measurements should be fully automated to allow for analysis of large quantities of samples. In this paper we present a setup for P-SHG which is readily incorporated on commercial multiphoton microscopes. The entire system is completely automated which allows for rapid calibration through the freely available software and for automated imaging for different polarization measurements, including linear and circular polarization of the excitation beam. The results show that calibration settings are highly system dependent. We also show that the accuracy of the polarization control is easily quantified and that it varies between systems. The accuracy can be tuned by iterative alignment of optics or a more fine-grained calibration procedure. Images of real samples show that the red accuracy of the results is easily visualized with the automated setup. Through this system we believe that P-SHG could develop a wider adoption in biomedical applications.publishedVersionCopyright: © 2018 Romijn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes - Fig 4

<p>(a, b) An overview of the ellipticity, <i>ρ</i>, between the minimum and maximum electric field component of the polarization for different HWP and QWP combinations for two different microscopes. For linearly polarized light the ellipticity is 0, and for circularly polarized light the ellipticity is 1. The overlaying plots indicate which waveplate combinations generate linearly polarized light (black lines) and circularly polarized light (black and white circles) as determined by fitting a theoretical model. The white crosses in each plot indicates the HWP and QWP settings used when measuring the polarization illustrated in (c) and (e). The white circles correspond similarly to plots (d) and (f). The single measurements had an ellipticity of <i>ρ</i> = 0.29 (c), <i>ρ</i> = 0.86 (d), <i>ρ</i> = 0.19 (e) and <i>ρ</i> = 0.97 (f).</p

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes - Fig 6

<p>Waveplate combinations that provide linear polarization were selected at polarization angle intervals of 10° from 0° to 180° (a, white crosses and plus signs). There are two different combinations that provide the same polarization angle, thereof the two separate sets (crosses vs plus signs). The polarization angle and ellipticity were measured for all waveplate combinations in both sets (b). Tendon was imaged twice using all waveplate combinations (c, average). Two pixels were selected (c, white asterisk and star) to demonstrate the variation of the intensity as a function of polarization angle for the two sets (d). The second order susceptibility tensor ratios and fiber angle were calculated separately for the two sets (plus signs: e, f, g, crosses: h, i, j). The difference between the two was emphasized by taking subtracting the results from one of the sets (crosses) from the other (plus signs) (k, l, m).</p

An illustration of the orientation and rotation of the QWP and HWP compared to the polarization of the incoming laser light.

<p>The motors containing the QWP and HWP are not calibrated according to the <i>x</i>-axis. Therefore, a relative angle <i>ϕ</i> = <i>ϕ</i>′ − <i>ϕ</i>₀ and <i>θ</i> = <i>θ</i>′ − <i>θ</i>₀ have to be implemented for the QWP and HWP, respectively. Here <i>ϕ</i>′ and <i>θ</i>′ are the angles provided by the motors, and <i>ϕ</i>₀ and <i>θ</i>₀ are the angles of the fast axis of the QWP and HWP, respectively, compared to the <i>x</i>-axis when the motors are at 0°.</p

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes - Fig 5

<p>(a, b) A detailed and coarser map of the ellipticity, <i>ρ</i>, with overlaying waveplate combinations that provide linearly (mean: black lines, ±3std: dashed lines) and circularly (mean: black circles, ±3std: intervals) polarized light. To illustrate the accuracy smaller regions of interest (a, white rectangles) were measured in more detail (c, d, e). The overlapping plots are based on the fitting of the original map (a), and have an ellipticity of <i>ρ</i> = 0.29 (c), <i>ρ</i> = 0.12 (d) and <i>ρ</i> = 0.86 (e).</p