New white paper details confocal imaging with the Airyscan detector in Fast Acquisition Mode
In August 2014, ZEISS introduced Airyscan, a new detector concept for confocal laser scanning microscopy (LSM). Airyscan is a 32 channel GaAsP-PMT area detector, positioned at the pinhole-plane of an LSM. Using Airyscan, additional light and spatial information is collected beyond that of a typical LSM image, resulting in substantial and simultaneous improvements in spatial resolution and signal-to-noise ratio. The introduction of the Fast mode for Airyscan represents the next innovation step for LSM imaging. Airyscan detector technology is utilized along with an illumination shaping approach to enhance acquisition speeds by four times. Airyscan affords researchers access to superresolution, increased signal-to-noise ratio and increased acquisition speeds simultaneously without the traditional compromises.
Laser Scanning Microscopy
The Confocal Laser Scanning Microscope (LSM) has become one of the most popular instruments in basic biomedical research for fluorescence imaging. The main reason LSM has become so popular is that the technique affords researchers images with high contrast and a versatile optical sectioning capability to investigate three dimensional biological structures . The optical sectioning ability of an LSM is a product of scanning a diffraction limited spot, produced by a focused laser spot, across a sample to create an image one point at a time. The generated fluorescence from each point is collected by the imaging objective and results from fluorophores in the sample that reside both in the desired plane of focus and in out of focus planes. In order to separate the fluorescence emitted from the desired focal plane, an aperture (pinhole) is positioned in the light path to block all out of focus light from reaching the detector (traditionally a PMT) .
Based on the application needs, LSM offers tremendous flexibility to fit experimental requirements, such as the choice of the excitation laser wavelengths and scanner movement; magnification and resolution of objective lenses as well as the type and arrangement of the detectors. Hence LSMs can be used to image diverse samples from whole organisms to large tissue sections to single cells and their compartments, labeled with numerous fluorescent markers of diverse emission intensities. During the past couple of decades the LSM has undergone continuous improvement; both usability and technical capability of the instruments (to make use of the precious emission light) have been significantly enhanced. These improvements have been the result of constant technical advances, production of high class optical components and improvements in the design of the confocal beam path. But the one ultimate compromise of confocal laser scanning microscopy was not touched until 2014, when ZEISS introduced the Airyscan for its LSM 8 Family systems: the pinhole.
Until this point the pinhole would be generally set to a 1 Airy unit (AU) opening diameter, resulting in a good compromise between capturing the scarce emission light and achieving an effective resolution. In theory one can enhance the resolution of a confocal LSM by closing the pinhole below a 1 AU opening. However this is not usually an option, since too much light is rejected resulting in images with unusable signal-to-noise (SNR) ratios. For the first time, the Airyscan detector allowed to combine enhanced resolution and signal to noise for LSM imaging .
The Airyscan detector consists of 32 GaAsP PMT detector elements, which are arranged in a hexagonal array (Figure 1), positioned at a conjugated focal plane in the beam path the detector is functioning as the traditional LSM pinhole. For full flexibility an adjustable optical zoom is present in front of the Airyscan detector which enables adjustment of the number of Airy units that are projected onto the detector. This design made it possible to collect more light (equivalent to a pinhole opened to 1.25 AU), whilst at the same time dramatically enhancing the resolution, with every detector element acting as an efficient pinhole with a diameter of only 0.2 AU.
Instead of facing an either / or decision, a simultaneous enhancement of resolution by the factor of 1.7 x and signal-to-noise by 4 – 8x was introduced to LSM imaging. Superresolution imaging under gentle conditions, with low laser powers, became part of the confocal LSM repertoire. Flexibility was added with the zoom optic, which allowed researchers to decide if resolution or sensitivity was the priority for the experiment; adapting the Airyscan advantages to the specific experimental needs. Using either multiphoton or single photon excitation without altering the well-established LSM sample preparation and labelling protocols, further broadened the experimental prospects. Detailed descriptions of the theory and technology of Airyscanning can be found in these technology notes [4, 5].
Limitations of acquisition speed in conventional LSM
Research objectives can dictate the acquisition of fast, dynamic processes or the quick capture of many fields-of-view (FOV). In both cases, the challenge for the imaging system is to collect sufficient fluorescence for an image with good SNR but in a very limited period of time. Conversely, because traditional LSMs create images one point at a time, image acquisition can be relatively slow. To improve the acquisition speed of LSM instruments, several strategies can be pursued; such as limiting the field of view, sacrificing image resolution (using fewer image pixels) and scanning the laser spot faster. When scanning the laser spot faster across a FOV, the pixel dwell time is shortened. Consequently, the amount of time per pixel spent collecting fluorescence is also shorted which impacts the resulting SNR of the image.
As the acquisition speed is increased, fewer and fewer photons will be available resulting in a deterioration of image SNR. The outcome is not only a noisy image but also a compromised spatial resolution, in which fine structures cannot be properly resolved. To compensate for the deteriorating SNR the laser power can be increased but this too has disadvantages; the danger of bleaching the fluorophore and / or damaging live samples by phototoxic effects (e.g. free oxygen radicals) becomes more prevalent at higher laser powers and thus the risk of influencing experimental outcomes is increased [6, 7, 8,]. Therefore, traditional techniques to improve image acquisition speeds demand that a researcher compromises image SNR, resolution, FOV and laser exposure, all of which will likely impede the research goal.
ZEISS LSM 880 with Airyscan: Principle of Beampath & Detection in Fast Mode
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-  Huff, J., The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nature Methods, 2015. 12.
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