T. G. van Leeuwen

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing

Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25‐fold in size, 300 000‐fold in concentration, 20 000‐fold in volume, and 10 000 000‐fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 104 to 1012 vesicles mL–1.

Optical and non-optical methods for detection and characterization of microparticles and exosomes

Summary.  Microparticles and exosomes are cell‐derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport. Despite increasing scientific and clinical interest, no standard procedures are available for the isolation, detection and characterization of microparticles and exosomes, because their size is below the reach of conventional detection methods. Our objective is to give an overview of currently available and potentially applicable methods for optical and non‐optical determination of the size, concentration, morphology, biochemical composition and cellular origin of microparticles and exosomes. The working principle of all methods is briefly discussed, as well as their applications and limitations based on the underlying physical parameters of the technique. For most methods, the expected size distribution for a given microvesicle population is determined. The explanations of the physical background and the outcomes of our calculations provide insights into the capabilities of each method and make a comparison possible between the discussed methods. In conclusion, several (combinations of) methods can detect clinically relevant properties of microparticles and exosomes. These methods should be further explored and validated by comparing measurement results so that accurate, reliable and fast solutions come within reach.

Single vs. swarm detection of microparticles and exosomes by flow cytometry.

See also Harrison P, Gardiner C. Invisible vesicles swarm within the iceberg. This issue, pp 916‐8.

Velocity-estimation accuracy and frame-rate limitations in color Doppler optical coherence tomography

Color Doppler optical coherence tomography (CDOCT) is a recent innovation that allows spatially localized flow-velocity mapping simultaneously with microstructural imaging. We present a theoretical model for velocity-image formation in CDOCT. The proportionality between the heterodyne detector current Doppler power spectrum in CDOCT and the optical source power spectrum is established. We show that stochastic modifications of the Doppler spectrum by fluctuating scatterer distributions in the flow field give rise to unavoidable velocity-estimation inaccuracies as well as to a fundamental trade-off between image-acquisition rate and velocity precision. Novel algorithms that permit high-fidelity depth-resolved measurements of velocities in turbid media are also reported.

Localized measurement of optical attenuation coefficients of atherosclerotic plaque constituents by quantitative optical coherence tomography

Optical coherence tomography (OCT) is a novel, high-resolution diagnostic tool that is capable of imaging the arterial wall and plaques. The differentiation between different types of atherosclerotic plaque is based on qualitative differences in gray levels and structural appearance. We hypothesize that a quantitative data analysis of the OCT signal allows measurement of light attenuation by the local tissue components, which can facilitate quantitative spatial discrimination between plaque constituents. High-resolution OCT images (at 800 nm) of human atherosclerotic arterial segments obtained at autopsy were histologically validated. Using a new, simple analysis algorithm, which incorporates the confocal properties of the OCT system, the light attenuation coefficients for these constituents were determined: for diffuse intimal thickening (5.5/spl plusmn/1.2 mm/sup -1/) and lipid-rich regions (3.2/spl plusmn/1.1 mm/sup -1/), the attenuation differed significantly from media (9.9/spl plusmn/1.8 mm/sup -1/), calcifications (11.1/spl plusmn/4.9 mm/sup -1/) and thrombi (11.2/spl plusmn/2.3 mm/sup -1/) (p<0.01). These proof of principle studies show that simple quantitative analysis of the OCT signals allows spatial determination of the intrinsic optical attenuation coefficient of atherosclerotic tissue components within regions of interest. Combining morphological imaging by OCT with the observed differences in optical attenuation coefficients of the various regions may enhance discrimination between various plaque types.

Measurement of the axial point spread function in scattering media using single-mode fiber-based optical coherence tomography

The authors studied the axial point spread function of optical coherence tomography for Gaussian intensity profiles emitted from and coupled back into single-mode fibers for signals from a scattering medium. The determined Rayleigh length of the axial point spread function was roughly twice the one measured from the reflection of a mirror. Using the measured point spread function in combination with the single backscatter model allowed determination of the attenuation coefficient of the suspension.

Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae

Abamectin is one of the most important insecticides worldwide. It is used against major agricultural pests and insects of public health importance, as well as against endoparasites in animal health. Abamectin has been used successfully for the control of the spider mite Tetranychus urticae, a major agricultural pest with global distribution, an extremely diverse host range, and a remarkable ability to develop resistance against insecticides including abamectin. Target site resistance mutations may explain a large part of resistance, although genetic evidence and transcriptomic data indicated that additional mechanisms may also be implicated in the abamectin resistant phenotype. To investigate a functional link between cytochrome P450-mediated metabolism and abamectin resistance, we recombinantly expressed three cytochrome P450s (CYP392A16, CYP392D8 and CYP392D10) that have been associated with high levels of abamectin resistance in a resistant T. urticae strain isolated from Greece. CYP392A16 was expressed predominately in its P450 form however, both CYP392D8 and CYP392D10 were expressed predominately as P420, despite optimization efforts on expression conditions. CYP392A16 catalyses the hydroxylation of abamectin (Kcat=0.54 pmol/min/pmol P450; Km=45.9 μM), resulting in a substantially less toxic compound as confirmed by bioassays with the partially purified metabolite. However, CYP392A16 did not metabolize hexythiazox, clofentezine and bifenthrin, active ingredients that also showed reduced toxicity in the abamectin resistant strain. Among a number of fluorescent and luminescent substrates screened, Luciferin-ME EGE was preferentially metabolized by CYP392A16, and it may be a potential diagnostic probe for metabolic resistance detection and monitoring.

Toward Spectral-Domain Optical Coherence Tomography on a Chip

We present experimental results of a spectral-domain optical coherence tomography system based on an integrated optical spectrometer. A 195-channel arrayed-waveguide-grating (AWG) spectrometer with 0.4-nm channel spacing centered at 1300 nm and a 125-channel AWG with 0.16-nm channel spacing centered at 800 nm have been fabricated in silicon oxynitride waveguide technology. Interferometric distance measurements have been performed by launching light from a broadband source into a free-space Michelson interferometer, with its output coupled into the AWG. A maximum imaging depth of 1 mm and axial resolution of 25 and 20 μm in air are demonstrated for the 800- and 1300-nm ranges, respectively.

Imaging Tumor Vascularization for Detection and Diagnosis of Breast Cancer

Breast cancer is one of the major causes of morbidity and mortality in western women. Current screening and diagnostic imaging modalities, like x-ray mammography and ultrasonography, focus on morphological changes of breast tissue. However, these techniques still miss some cancers and often falsely detect cancer. The sensitivity and specificity for detecting the disease can probably be improved by focusing on the consequences of tumor angiogenesis: the increased microvessel density with altered vascular characteristics. In this review, various techniques for imaging breast tumor vasculature are discussed. Dynamic contrast enhanced magnetic resonance imaging is the most-used imaging modality in this field. It has a proven high sensitivity, but a low specificity and cannot be applied in all women. Moreover, it has problems with detecting ductal carcinoma in situ (DCIS). On the contrary, contrast enhanced digital mammography can detect DCIS, but requires the use of ionizing radiation. Contrast enhanced ultrasound provides real-time information about true intravascular blood volume and flow. However, this technique still has difficulties with discriminating benign from malignant tissue. Moreover, these three imaging modalities all require the injection of contrast agents. Two relatively new techniques that do not use external contrast agents are diffuse optical imaging and photoacoustic imaging. Both visualize the increased concentration of hemoglobin in malignant tissue and thereby provide a high intrinsic contrast.

Integrated-optics-based swept-source optical coherence tomography

We designed, fabricated, and characterized an integrated-optics-based swept-source optical coherence tomography (SS-OCT) system in TriPleX technology. An external 1300 nm swept source is coupled to the chip, which contains waveguide structures for interferometric depth ranging and balanced detection. The complete OCT chip has a footprint of 0.4 cm × 1.8 cm. Light from the chip is focused onto the sample using an aspheric lens; the lateral resolution is 21±1 μm. OCT measurements, performed with a moveable mirror, demonstrate a sensitivity of -80 dB and imaging up to the maximum depth of 5.09 mm. Corrected for dispersion, the measured OCT axial resolution of 12.7±0.5 μm is in good agreement with the bandwidth limited resolution. Finally, we demonstrate cross-sectional OCT imaging of a multilayered tissue phantom over the whole depth range with the integrated-optics-based SS-OCT system.