To best reveal the structure of EVs we recommend using a gentle purification method for example size exclusion chromatography, tangential flow filtration or density gradient centrifugation. Ultracentrifugation and precipitation based purification are thought to result in aggregation and disruption of EVs.

The lens chief ray is the ray path from an off-axis point that passes through the center of the aperture stop of the optical system. The lens chief ray angle or CRA is the angle of incidence of the lens chief ray with the image plane.

Matching the lens CRA to the sensor CRA is critical to digital imaging. In modern camera systems, matching the image sensor pixel angle of acceptance to the lens CRA must be accomplished to achieve the highest quality image. In many miniature-sized CMOS image sensors, the location of the microlens that is applied to the pixel to focus light to the active area of the photo sensor varies from the center of the image sensor to the edge. This effect is called microlens shift and can be visualized in the image above. It is done so that the lens can be designed to be very thin, which is required for mobile phone applications, or so that lenses can be fabricated using a wafer-level stack. When the lens CRA is mismatched with respect to the image sensor specification, artifacts such as non-uniformity in the image and color errors can occur.

Chief rayangle

Sample check The system facilitates a rapid assessment of key features within minutes including the density of EVs in the sample, their distribution and the efficacy of biomarker staining. This step ensures the quality and the reliability of the sample before the full imaging process. If necessary, users have the option to modify the autofocus settings on a per-lane basis, based on the insights they gained from the images. This adaptive approach allows for fine-tuning and optimization in real-time, ultimately contributing to the accuracy and the precision of the imaging results.

Focalray

Summary page & EV mosaic Upon completion of the 4-lane acquisition, a mosaic summary view offers a visual representation of the biomarker heterogeneity at the single EV level. This feature enables users to swiftly navigate through different EVs of the same positivity class by clicking on next/previous options. Positioned at the bottom of each lane, this mosaic view facilitates a convenient visual comparison of EVs across various conditions. The immediate insights gained from this overview aid in determining the success of the acquisition process.

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In summary, the lens chief ray angle or CRA is the angle of incidence of the lens chief ray with the image plane. Matching the lens CRA to the image sensor CRA specification is important in achieving the best final image quality. When the lens CRA does not match the image sensor CRA specification, artifacts may occur in the image.

Automatic 4-lane acquisition Users have to flexibility to choose their preferred fields of view (FOV) for imaging or opt for the FOVs recommended by our expert development scientists. The process is streamlined to efficiently and automatically image a 4-lane chip within 90 minutes. Users can access information about the EV count within minutes, thanks to the system’s real-time analysis capabilities. This features provides users with immediate insights, enhancing the overall efficiency and and responsiveness of the imaging workflow.

Chief raylocations

Visualize and size your EVs accurately, quickly and easily Quick and unbiased visualization and sizing of EVs through the pan-EV stain, reveals both membrane and shape details for a comprehensive analysis of their dimensions and characteristics.

The new kit introduces our Pan-EV stain, internal cargo detection as well as major improvements to reproducibility and EV capture efficiency. Capture stunning super-resolution images of EVs labeled with Pan-EV and Tetraspanin Trio for high confidence EV identification and sizing (with accuracy comparable to TEM), while using the only tool on the market that can characterize the spatial distribution and colocalise up to three biomarkers at the individual EV level.

One-click system calibration The system is designed for automatic calibration processes, aimed to enhance data consistency and reproducibility across lanes and chips. These processes include autofocus, automatic-Z-lock, and auto-TIRF, all of which contribute to optimized system performance. The system considers the specific properties of the analyzed sample and automatically determines the appropriate settings. The user simply selects a specific lane and imaging channel where they expect a robust signal for the system calibration, streamlining the overall imaging experience, however, the system also allows for manual settings if the user prefers.

Chief rayOptics

Be confident your EVs are really EVs EV Profiler 2 includes ONI’s Pan-EV Detection and pooled Tetraspanin Trio reagents. This combination ensures the exclusion of contaminating particles or antibody aggregates from the analysis.

*EV Profiler 2 has a shelf life of two months. **AutoEV requires a CODI account and an active internet connection. Access to AutoEV without an internet connection will come later in 2024. EV Profiler Kit 2 and AutoEV are only compatible with Nanoimagers manufactured from 2021 on.

The antibodies for capture and detection are specific to human and have not been tested for cross-reactivity with other species.

Chief rayangle vs field of view

Chief rayand marginalray

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The graphic below depicts the focusing rays from an imaging lens, converging onto an image sensor pixel. The three diagrams show the rays of light coming from different parts of the object. A microlens is drawn on the top of the pixel that helps condense light onto the sensor's active area.

EV Profiler 2* is our second generation reagent kit for visualization and phenotyping of extracellular vesicles using dSTORM microscopy on the Nanoimager**.

AutoEV is built to automatically acquire field of view in the lanes of the EV Profiler 4-lane assay chip with a single click, and is not intended to be used with any other slides or coverslips.

Experiment setup The software has default settings tailored for each variant of the kit. All you need to do is inform the software about your specific reagents, and it will automatically adjust the acquisition settings accordingly. Additionally, users have the flexibility to select and apply specific analysis settings after the imaging process, providing a flexible and efficient experience.

Chief raytracing

Chief raylenses

When I first heard of a CRA requirement for an image sensor, I struggled to understand why it was important. A CRA spec wasn't something that was taught when I went to school! Digging deeper, I learned about the amazing microlens technology developed for the mobile phone camera market. What I learned is shown in the graphic below.

Detect and visualize biomarkers inside or on the surface of EVs Detect and visualize biomarkers inside and on the surface of EVs, allowing for efficient phenotyping and insight into biomarker clustering.

When combined with our newest software offering, AutoEV, your system will be calibrated and optimized to acquire and analyze a 4-lane chip automatically and deliver a comprehensive report of EV size and positivity for each individual lane in 90 mins.

AutoEV introduces great new algorithms like AutoFocus, AutoTIRF, and automated acquisition across lanes. You can manually set the illumination (TIRF) angle if desired, and you can adjust the focus on a per-lane basis prior to starting the automated acquisition, so you can be sure to have the imaging results you want, all while being able to step away from the microscope for imaging.

Custom 1-page EV report By selecting “Generate Report”, the user can quickly generate a concise 1-page report, featuring three essential, easily comparable metrics. This report provides valuable insight on the quality of your sample, including EV count, biomarker heterogeneity count, and EV size. The metrics are presented at both the population level and according to each biomarker positivity, offering a comprehensive overview of the sample characteristics for efficient analysis and decision-making.

In applications such as surgical imaging where the endoscope diameter must be kept very small, image sensors that have been developed for mobile phones are commonly used. Here, the lens diameter must be small, but typically the length of the lens is not as important. When an image sensor with a high CRA is used, the lens design must take this specification into account. The lens CRA must match the CRA specification of the image sensor to prevent masking or shadowing within the image. When the lens and image sensor CRA are mismatched it can cause artifacts in the image such as image shading, or color-mismatch effects. Lens and sensor CRA mismatch errors are a common challenge in developing new miniature camera systems.

Co-localize up to three biomarkers EV Profiler 2 detects biomarkers in up to three channels, enabling measurement of single, double, and triple positivity on individual EVs with a 10% SD between assay chips.