Extracellular vesicles (EVs) are nanoparticles secreted from cells into bodily fluids. EVs are potential biomarkers for diseases such as thrombosis or cancer. However, the small size and low refractive index of EVs complicates their detection. A flow cytometer is suited for EV characterisation, but typically lacks scatter sensitivity on one or both scatter detectors for derivation of both particle size and refractive index. Here, we aim to improve the FACSCanto (Becton Dickinson) forward scatter detector for the detection of 100 nm EVs, which requires an improvement in SNR of 10<sup>7</sup>-fold based on Mie theory. This was achieved through replacement of the 20 mW laser by an 200 mW laser, replacement of the photodiode detector with a photomultiplier tube and a confocalized optical geometry. Using a prototype optical setup, we obtained an improvement in SNR which was 1,11·10<sup>4</sup> – fold better than the standard design. However, the optics was suboptimal and far from diffraction-limited. Zemax simulations led to a nearly diffraction limited optical design which is expected to yield another 200-fold improvement. Taken together these changes will improve the SNR 2.2·10<sup>6</sup>-fold and thus improve the detection limit of the FACSCanto to 130 nm EVs.
The speed improvement is a game-changer in optical coherence tomography (OCT) imaging because it opens up for new and very exciting applications. The frame rate of an OCT system is limited by the speed of the camera or the sweep rate of the light source. This problem can be overcome by multiple-beam imaging, in which different locations on the sample are illuminated by an array of light simultaneously. This technique allows parallel imaging from multiple sample locations and therefore improves OCT axial scan rate by a factor equal to the number of beams used simultaneously which can go up to very high frequency ranges (e.g. MHz) easily. In this work, we introduce a compact integrated-optics based multiple-beam illumination design in which several waveguides with certain length differences are combined with wavelength-independent couplers for space-division multiplexing. Electrodes will be placed on each beam path in order to separate desired signal from unwanted reflections at the optical surfaces or tissue. The imaging speed will be improved by the number of the beam paths used. In addition to fast imaging, the proposed design will be very compact which makes it very suitable to be used in endoscopic probes. The proof-of-concept of this idea was experimentally demonstrated using a design which consists of 2 times 4 parallel OCT channels that are realized with a total of 6 Y-couplers. Each individual OCT channel has an optical path length delay with respect to the other channels.
Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles <70 nm in suspension.
Method: Urinary vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope.
Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section.
Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.