The innovative design of the ZEISS Airyscan 2 detector enables the expansion of the scanner–detector codependence beyond the traditional 1:1 relationship by fully leveraging the positional information contained at the pinhole plane. MBS 488, main beam splitter used with a 488-nm laser.
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Right, a schematic showing the placement of the Airyscan 2 detector in the pinhole plane, acting as a series of small 0.2–Airy Unit (AU) pinholes to provide improved optical resolution with the collection ability of a 1.25-AU pinhole. Left, a schematic of a traditional laser-scanning confocal microscope with a pinhole–PMT arrangement.
With model systems and organisms getting larger and with lower expression levels (e.g., lower fluorophore concentrations), traditional confocal systems are severely restricted in terms of how quickly desired sample volumes can be scanned with sufficient SNR and resolution, and this ultimately affects the ability to quantify the image data.įigure 1 |Comparison of traditional confocal and Airyscan 2 beampaths.
The interplay and codependence that were once leveraged as experimental flexibility have now become the limiting factors for traditional confocal systems.
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Hence, the scanner–detector relationship directly affects what model systems, experiments, and applications can be used with a laser-scanning system, as researchers have traditionally been forced to compromise between scanning speed (i.e., frame rate), pixel dwell time (i.e., SNR and laser exposure), and pixel size (image resolution). The traditional design yields a 1:1 relationship between the scanner movement and the data readout in which every scan position (i.e., pixel) correlates to one value read from the unitary detector (i.e., pixel intensity). The use of a physical aperture (i.e., pinhole) and unitary detector (standard photomultiplier tube (PMT)) in the design of traditional laser-scanning microscopy systems requires that the final data resolution, SNR, and speed be directly coupled to how finely an image is scanned and how the data are read off the PMT (Fig. The Multiplex mode innovation for Airyscan 2 focuses on the need to capture structural dynamics, cellular signaling, molecular trafficking, and diffusion events with real-time super-resolution and superior signal-to-noise ratio (SNR) in model systems that extend beyond traditional cultured cell preparations and into new 3D model systems of organoids, spheroids or 3D culture, and whole organisms. Through new acquisition strategies for pinhole-plane imaging, the new mode empowers researchers to leverage the unique combination of optically sectioned super-resolution and sensitivity at the highest volume rates. The net result of these innovation steps is not only an improved user experience but the new Multiplex mode. With a focus on usability, utility, and throughput, the Airyscan 2 detector from ZEISS fully leverages pinhole-plane imaging with new data-handling and acquisition strategies. The LSM 9 series with Airyscan 2 represents the next big step in the evolution of confocal microscopy.
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