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Confocal laser scanning microscopy is today’s standard method of obtaining detailed 2-D and 3-D information at the cellular level. The imaging technology has become popular in biomedical research because of its ability to produce precise, optically sectioned images, and address many types of samples and application demands.
Over the past 25 years, improvements have been made in sharpening image contrast and improving instrument adaptability to more laboratory settings and situations. However, until now, not as much improvement had been made in the optical section itself.
The new Airyscan detector from ZEISS is one of the first significant improvements in optical sectioning, resulting in better resolution and improved signal-to-noise ratios. ZEISS Airyscan, which is fully compatible with ZEISS LSM 8 family of confocal microscopes, detector fundamentally changes confocal microscopy by allowing the microscope to detect more light without sacrificing the signal-to-noise ratio.
An expanded pinhole
Typically, confocal microscopes allow light only from one point to reach the detector using a special kind of aperture, called a pinhole. Light coming from planes above or below the focal plane is out of focus when it reaches the pinhole. So, most of it cannot pass the pinhole, and does not contribute to the formation of the image.
Too large a pinhole, then too much light or “noise” gets through; too small a pinhole, then not enough light or “signal” gets detected. The pinhole size must maximize the signal- to-noise ratio of the image. By varying the pinhole diameter, the degree of confocality can be adapted to best fit – usually at one Airy Unit (1 AU).
There has always been a trade off in confocal microscopy among three parameters: sampling frequency (speed), sampling frequency and pinhole size (sensitivity) and pinhole size (resolution). This gives us this “triangle” of parameters, which are interconnected in such a way that no net gain can be made without a sacrifice. For example, if a smaller aperture is used and this is compensated by increased excitation, then other issues arise— such as photobleaching or phototoxicity.
Improving on the Airyscan Principle
Using a physical aperture for the pinhole has been a major limitation for the filed of confocal microscopy. However, an acentric and shifted pinhole detector can produce the same image as the normal pinhole, but smaller in amplitude and shifted in space. This led to the introduction of the ZEISS Airyscan detector; an array of detector elements that would use, and not reject, the light outside the area of 1 AU.
Figure 1. The ZEISS Airyscan Principle
Incident light is collected by a hexagonal micro lens array that connects directly to a 32 high transmission optical fibers. These fibers then project to a linear GaAsP-PMT array of the Airyscan detector. In this way, the Airy disk is imaged via zoom optics on to the detector array and no pinhole is needed, as the single detector elements act as separate pinholes. The detection efficiency is increased by reassigning the detected photons from the array elements to the central detection position and summing up the signals from all detector elements of the array. Instead of rejecting some of the light, as in conventional pinhole-based systems, all the light is collected and reassigned to the correct region, resulting in increased signal levels.
ZEISS Airyscan is the future of confocal microscopy
The new ZEISS Airyscan detector geometry maximizes the signal-to noise ratio and gives the same resolution benefits as a pinhole setting of 0.2 AU with the light collection efficiency of a 1.25 AU pinhole. The diameter of the Airyscan detector comprises 6 detector elements, each representing a pinhole of the size of the imaged Airy disk divided by 6. Hence, as the zoom optics are set to capture 1.25 AU, each single detector element act as a pinhole with a size of 1.25 AU/6 = 0.2 AU. This allows each image to be individually deconvolved, further enhancing resolution, and photons can be precisely reassigned by software, resulting in a lateral resolution enhancement by a factor of 1.7. Indeed, this results in overall increased resolution: 140 nm lateral and 400 nm axial resolution, at 488 nm. The resulting image signal-to-noise ratio is drastically improved when comparing to traditional confocal imaging with a 0.2 AU pinhole. In fact, it achieves a 4 fold increase over conventional confocal systems. Taken together, the new ZEISS Airyscan detector can allow you to work on previously challenging optically dense materials.
Figure 2. ZEISS LSM 880 with Airyscan: HeLa cells. Conventional confocal microscopy versus Airyscanning of HeLa cells. Sample courtesy of S. Traikov, BIOTEC, TU Dresden, Germany.
For the first time, the ZEISS Airyscan detector has been able to get past microscopy’s three-sided compromises of image resolution, by replacing the most fundamental part of a laser scanning microscope: the pinhole. You can have higher speed, higher resolution, or higher sensitivity—or any combination of the three to the desired degree. With the ZEISS Airyscan detector you can exploit the full potential of your confocal microscope.
Get your hands on ZEISS Airyscan technology
If you’d like to take advantage of Airyscan technology, click here to take a look at the ZEISS LSM 8 family of confocal microscopes, which comprises ZEISS LSM 800 and ZEISS LSM 880. If you already have a 3- or 34-channel ZEISS LSM 710/LSM 780 with Airyscan, we can upgrade your system right in your lab with minimal downtime. Contact ZEISS for more information on how you can get ZEISS Airyscan for your lab.