The Airyscan Detector: Confocal Microscopy Evolution for the Neurosciences
First introduced in August 2014, the Airyscan detector from ZEISS represents a new detector concept for laser scanning microscopy (LSM) that enables a simultaneous resolution and signal-to-noise ratio (SNR) increase over traditional LSM imaging. The Airyscan detector innovation replaces the conventional LSM detector and pinhole scheme for an array of 32 sensitive GaAsP detector elements placed in the pinhole plane to generate an optical section. The new detection geometry allows for the collection of the spatial distribution of light originating from every point of a microscopic fluorescent object at the pinhole allowing access to higher frequency information and while additionally collecting more light for ultra-efficient imaging. In May 2015, the fast mode innovation for Airyscan combines illumination shaping with pinhole-plane imaging that enhances acquisition speeds by four times while simultaneously increasing SNR and resolution overcoming the traditional compromises of LSM imaging.
Over the last 25 years, the technique of confocal imaging has grown to become the standard choice for most fluorescence microscopy applications. The increase in utilization of confocal imaging systems in basic biomedical research can be in attributed to the technique’s ability to produce optically sectioned images with high contrast while providing acquisition versatility to address many sample and application demands . Hence, during the last decades in order to meet the growing utilization of confocal microscopy across many different fields of application and model systems, most commercially available confocal imaging systems have developed novel approaches and options to increase image contrast and instrument flexibility. While novel approaches of commercially available systems have certainly enabled researchers to answer new questions and address new model systems across, it is within the field of neuroscience that the confocal principal was born and is still driving the evolution of the most fundamental aspect of confocal microscopy: the creation of an optical section.
4.2 Airyscan for Neurosciences
With the aim of understanding structure and function of the brain, confocal imaging was an integral part of extensive analysis and experiments, which would drive the development of the molecular tools as well as the microscopy systems over the decades. The growing number and increased functionality of fluorescent compounds would drive the integration of laser lines, spectral imaging, and photomanipulation options. Advanced analysis tools such as fluorescence-lifetime imaging (FLIM) and fluorescence correlative spectroscopy (FCS) give insights into physiological processes. High-end LSM systems have grown into multimodal imaging and analysis tools. Over this time, the constant hallmark of every commercial laser scanning confocal system has been the use of a physical aperture for a pinhole (as Minsky described in 1955) in combination with a unitary detector (Fig. 4.1). Unfortunately, as current neuroscience research needs advance and new molecular biology capabilities expand, the combination of a traditional pinhole and unitary detector are starting to serve as the limiting factor for utilizing confocal microscopy in the neurosciences. The conventional design choices of modern laser scanning confocal microscopy systems limit the achievable image resolution, signal-to-noise, and speed subsequently limiting the research questions that can be answered. For the neurosciences, these limitations ultimately restrain neuroscience research centered on brain function and structure.
The fundamental limitations of the pinhole and unitary detector arrangement were recognized almost 30 years ago. In 1987 and 1988, Bertero  and Sheppard  both described approaches for pinhole-plane imaging systems where the detector is placed in the pinhole plane allowing researchers to improve spatial resolution and detection efficiency improving on Minsky’s original design. Much more recently, 2013, Sheppard et al. described the extension of so-called pixel-re-assignment technique to fluorescent dyes . Further in 2012, York et al. describe a technique to parallelize pinhole-plane imaging to increase resolution, signal-to-noise, and speed [6, 7]. The first commercial implementation of the pinhole-plane imaging concept was in August 2014 when the Airsycan detector from Carl Zeiss Microscopy was introduced.
4.3 Airyscan Detection for Enhanced Structural Information
Like all fluorescence imaging techniques, image quality in point-scanning confocal microscopy is directly related to ratio of the amount of signal (i.e., photons) and the amount of noise of an image, the so-called SNR. In point-scanning confocal microscopy, the number of detected photons (signal) are generally extremely small due to labeling densities and/or the optical section created by the pinhole. The resulting statistic variation in the number of detected photons, the so-called shot noise or photon noise, will then become a dominant factor for image contrast; i.e., the available gray levels per image pixel. As shot noise follows Poisson statistics, it will be equal to the square root of the signal. Hence, the SNR will also be proportional to the square root of the signal or synonymous to the square root of the number of photons (N): S/N ~ \( \sqrt N \).
Another source of statistical noise is background fluorescence originating from out of focus signals and auto-fluorescence from the specimen or optical components. Both will limit the contrast of the signal in respect to the background and decrease the image SNR such that insufficient contrast exists for sample signals to be distinguished. The signal-to-background ratio (S/B) can be increased by closing the pinhole and would indeed be greatest if the pinhole aperture would be closed to zero. It is evident that this is not practical as the fluorescence signal would also be excluded from reaching the detector. Therefore, a pinhole aperture size must be chosen to maximize image SNR while retaining an adequate S/B to yield a good image contrast. Traditionally, a pinhole size of 1 airy unit (AU) has traditionally proven to be a good compromise to achieve this goal.
4.3.1 Signal-to-Noise and Confocal Microscopy
The gain in SNR achieved by the Airyscan detector does not require any compromise in regards to speed, resolution, or sensitivity. However, to have the same SNR at its disposal a traditional confocal has to compromise on speed, resolution, or sensitivity (or combinations of those), which is an immediate consequence from the theory of the eternal triangle. If acquisition speed and sensitivity should match that of the Airyscan, the pinhole has to be opened to 1.25 AU, which will result in lower resolution (Fig. 4.3). Likewise, if resolution and speed should equal Airyscan at otherwise identical settings, the pinhole would have to be set to 0.2 AU reducing light level and hence sensitivity (Fig. 4.3). Finally, when resolution and sensitivity should be kept equal, again the pinhole has to be set to 0.2 AU to keep resolution and the sensitivity has to be increased by slower scanning or averaging (Figs. 4.4 and 4.5).
4.3.2 Photon Detection Considerations for Confocal Super-Resolution: Airyscan Versus LSM + DCV
Hence, the downside to utilizing a pinhole below the 1 AU limit on a traditional confocal system is the dramatic reduction in signal reaching the detector (95% loss at 0.2 AU). Subsequently, if signal reaching the detector is decreased, the resulting image quality will also decrease. This fact negatively impacts the utilization of smaller pinhole diameters in most neuroscience imaging applications as most neuroscience samples and fluorophores cannot supply enough (due to photodamage and/or phototoxicity) fluorescence to yield images with sufficient SNR. As a result, the performance gains of the Airyscan detector afford researchers to study brain structure and circuitry with increased resolution and signal-to-noise all while maintaining the optical sectioning ability of confocal microscopy as initially conceived by Minsky .
4.4 Airyscan Detection for Fast Functional Imaging with Increased Resolution and SNR
The confocal microscopy concept was initially developed as a tool to help understand brain structure and how neurons are interconnected. The study of brain structure is still a primary application of traditional confocal imaging systems where the studied samples generally consist of excised tissue/brain specimens that have been fluorescently labeled and fixed on a microscope slide for repetitive study of their underlying structure. However, in addition to the advances in the commercial development of confocal microscope systems over the past decade, molecular biology tools sought to utilize the specificity of fluorescence labeling in combination with microscopy to observe and measure physiological changes as a means to study brain function in real time . More recently, major advances have been made by the utilization of light sensitive proteins (channelrhodopsin and halorhodopsin) that allow the opening and closing of ion channels with light . Subsequently, by combining channelrhodopsin or halorhodopsin with a suitable fluorescence reporter, a researcher will have much more precise control on brain function and response to further neuroscience function research. In order to adequately study and understand brain signaling and function, both the fluorescent reporter as well as the imaging system must respond fast enough to capture rapid and faint electrical signals as they transverse the brains neural circuitry. Thus, as molecular biology tools develop to provide better and better fluorescent reporters, confocal imaging systems have adapted to provide acquisition schemes that allow an increase in image acquisition rates to capture the change in the fluorescence emission of the reporter probes.
The acquisition speed of a conventional laser scanning confocal is determined by how fast a single diffraction-limited laser spot can be moved across a desired field of view with a desired resolution (i.e., pixel count). Therefore, to increase the achievable scan speeds of a conventional LSM, a researcher must decrease the amount of time the excitation laser spends on each pixel (pixel dwell time), reduce the resolution (i.e. pixel count), or reduce the image field of view. As a result, when using a conventional LSM, a researcher must compromise on image signal-to-noise by reducing pixel dwell time, on spatial resolution by reducing the pixel count, or on field of view by limiting structural context by restricting the image field of view by zooming into a portion of the structure of interest. Traditionally, for point-scanning LSMs, reduction of the pixel dwell time has been the preferred approach to maximize scan speeds in the form of resonant scanning.
4.4.1 The Fast Mode for Airyscan
Imaging time, dead time, and pixel dwell time for resonant scanner and Airyscan in fast mode
Airyscan in fast mode
Image acquisition time (7.5 FPS)
Accumulated pixel dwell time (imaging time × parallelization)
Average pixel dwell time (1024 × 1024 pixel image)
The growth and development of the laser scanning microscopy industry over the past 25 years has led to development improvements that have been designed to increase image contrast and instrument versatility. Utilizing the foundation laid by Bertero  and Sheppard , the Airyscan detector from Carl Zeiss Microscopy replaced the most fundamental part of a traditional laser scanning microscope: the pinhole. The Airyscan’s novel detector design dispenses with the classical physical pinhole and unitary detector assembly and utilizes a new pinhole-plane image detection assembly based on a collection of 32 detection elements. In the new assembly, each of the 32 detector elements acts as its own small pinhole with positional information. The positional information gained by the new approach allows for increased contrast of high spatial frequency information previously not available in traditional confocal systems. The increase in spatial frequency contrast enables the Airyscan to produce images with substantially increased SNR and resolution compared to an LSM acquiring images with a 1 AU pinhole without having to increase laser exposure or sampling. The afforded resolution increase is 2.0× in all three spatial dimensions and the obtained SNR increase is 4×–8× over traditional LSM images acquired with a 1 AU pinhole. Further, as a confocal detector Airyscan benefits from all advantages of confocal microscopy, above all out of focus light reduction. Hence, the Airyscan detection concept outperforms the traditional pinhole and unitary detector setup in every imaging metric for the study of neuroscience samples for both the study of structure and function.
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