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Airyscanning Evoking the Full Potential of Confocal Microscopy

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Airyscanning Evoking the Full Potential of Confocal Microscopy COVER STORY Airyscanning Evoking The Full Potential of Confocal Microscopy Confocal laser scanning microscopes (CLSMs) are renowned for their sectioning capability. This fea- ture is enabled by the use of a pinhole, which rejects out-of-focus light. Less appreciated, on the other hand, is the gain in lateral resolution by this type of microscopes for one obvious reason. As the pinhole is closed to improve the resolution, less light can reach the detector leading to images with poor signal- to-noise ratios (SNR). Therefore CLSMs are operated with a pinhole diameter of around 1 Airy unit (AU), sacrificing resolution for SNR. But with a clever detector design one can solve this conundrum. By using a sub-Airy detector element array instead of a single point detector, it is possible to collect light more ef- ficiently boosting the strengths of a confocal. The extra photon budget can be diverted towards increas- ing the sensitivity, the scanning speed or the resolution of the microscope. In this article we will focus our discussion on how resolution is improved. Ralf Engelmann Klaus Weisshart Improving Resolution would be dramatically reduced and this It allows arranging various pinhole de- would yield images with poor signal-to- tectors in an array covering an area ex- The resolution in a widefield microscope noise ratios (SNR). Therefore, in practi- ceeding the conventional pinhole size by is limited by the diffraction of light, which cal terms, the pinhole radius will hardly approximately one quarter. Reassigning causes a point emitter to be imaged as an be closed below one Airy unit, sacrificing the detected photons from the shifted de- extended spot [1,2] (Fig. 1). resolution for SNR. tector to the central detection position An improvement in resolution in fluo- and summing up the back shifted signal rescence microscopy by a factor of 1.4 from all detectors will increase the detec- was achieved by confocal laser scan- Resolution and Signal Improvement tion efficiency as no light is rejected by a ning microscopy (CLSM) [3]. The sample closed pinhole but rather collected by the is hereby illuminated with a scanned fo- The insight that a displaced pinhole de- off-axis detector elements. Therefore an cused laser beam. The detected intensity tector produces an image of about equally increased signal level arises from the re- values of every scanning position are re- improved resolution as an aligned pinhole assignment of photons to a smaller spatial corded with an integrating detector like detector, however smaller in amplitude region [5]. a photomultiplier tube (PMT). The resolu- and shifted by half the displacement (fig. One way to realize off-set pinhole de- tion enhancement is achieved by collect- 2), is the key for constructing a compound tection would be to employ an array de- ing the emitted light through a pinhole, eye detector for a confocal microscope [4]. tector like a pixilated camera in place of which is aligned to the position of the ex- citation focus. That way all detected pho- tons will be assigned to the nominal exci- tation position. Fig. 1: Due to the diffraction nature of light, a Despite this slight resolution improve- point source will be imaged by a microscopic ment CLSM is more renowned for its op- system as a blurred spot surrounded by rings tical sectioning capabilities for one very of decreasing intensities (I) in the lateral plane clear reason. The increase in resolution (rx, ry), the so-called Airy disk (displayed in is only achieved by minimizing the pin- grey). The intensity distribution along a trans- hole diameter, theoretically down to zero. verse direction (v) is shown in blue. As a consequence detection efficiencies 20 • G.I.T. Imaging & Microscopy 3/2014 COVER STORY Fig. 2: The effective PSF (PSFeff; dark grey) in a confocal microscope with aligned illumination and detection is the product of the illumination PSF (PSFillu; dark blue) with the detection PSF (PSFdet; dark red). It will therefore be narrower. As the detector element is displaced in the transverse direc- Fig. 4: Confocal (CLSM) and Airyscan recording of a FluoCell #1 (Invitrogen) tion (v), the amplitude of the detection PSF (light red) will be shifted the imaged for MitoTracker to visualize mitochondria. Shown in the upper right same way. The resulting effective PSF (light grey) will shift by a smaller corner is a magnified view of the area indicated by the smaller squared increment and has a decreased amplitude. But it will be narrower than is box. The lower right corner shows an xz section along the indicated arrow the case for the on-axis detector (compare dark grey with light blue PSF, in the inset. Image conditions were identical with a pixel size of 58 nm and the latter being the effective PSF derived by the displaced detector, but a sectioning of 125 nm. Scales are indicated. shifted back and normalized for better visualization). A commercial realization would fail with other widefield of the concept is provided by based super-resolution tech- Airyscan from Zeiss. Here, niques. As such the method the detector design consists can offer an useful and com- of a 32 channel gallium arse- plementary tool to study cellu- nide phosphide (GaAsP) PMT lar structures to finer details. array arranged in a com- pound eye fashion. Each de- References Fig. 3: The object (grey spot) is illuminated by a point source (blue) and will tector element will represent [1] E. Abbe: Beiträge zur Theorie emit photons that will be recorded on an array detector. As the sample is one specific offset position of des Mikroskops und der mik- scanned (in the direction of the arrow), not only will each detector element the Airy function (see cover roskopischen Wahrnehmung, (squares) see the whole image, but they would probe the Airy disk (dis- image of this issue). The inte- Archiv Mikroskop Anat. 1873 played in orange) as well. The obtained detection PSFs (grey curves) would grated processing algorithms (9) 413–468 be shifted and their amplitudes decrease with increasing shift. enable online deconvolution [2] S. Hell and A. Schönle: Oxford for visualization of resolu- University Press, 237–264 tion enhanced images, given [3] J. Pawley: Handbook of bio- the pinhole. Indeed such a set- Isotropic Resolution In- proper 2.3 fold oversampling logical confocal microscopy, up has been applied in sin- crease in all Three Spatial has been performed to meet Springer (2010) gle and multi-spot excitation Directions the Nyquist criterion for the [4] C. J. Sheppard: Optik 80 (2), 53– schemes for imaging biologi- increased resolution (fig. 4). 54 (1988) cal samples [6,7]. Another pos- In Airyscan a PMT point de- With conventional resolution, [5] C. J. Sheppard et al.: Opt Lett. sibility would be an all-optical tector array is used with the a virtual pinhole effect and 38(15), 2889–2892 (2013) way where the detection beam benefit of a fast read out time sensitivity improvement can [6] C. B. Muller and J. Enderlein: after having been de-scanned compared to a camera and low be obtained as alternative im- Phys Rev Lett 104(19), 198101 is displaced from the optical dark counts. As the object is aging modes. (2010) axis by a defined amount via scanned all detector elements [7] A. G. York et al.: Nat Methods a re-scan [8, 9]. In all these will record the whole image 9(7), 749–754 (2012) methods based on pixel re- for each scanner position. Into Conclusion [8] S. Roth et al.: Optical Nanoscopy assignment a lateral resolu- the bargain, since at each scan 2(5), 1–6 (2013) tion enhancement by a factor position an image is taken by The concept evokes the full [9] A. G. York et al.: Nat Methods of 1.7 has been obtained com- the detector array the whole potential of a confocal micro- 10(11), 1122–1126 (2013) pared to a widefield image. Airy disk is acquired (fig. 3). scope obtaining resolutions Thus, most of the effect is the Pixel reassignment would comparable to a very small Contact result of a greatly improved now be one way to boost the pinhole, but with a much bet- Ralf Engelmann signal allowing the small pin- lateral resolution. But since ter SNR. The method works for Tiemo Anhut hole sizes for the individual el- each detector element acts like any dye and is live cell com- Ingo Kleppe ement of the detector; yet an- an individual pinhole, each im- patible, as low laser powers Klaus Weisshart other tiny part stems from a age can be individually decon- can be applied. Last not least slight improvement upon the volved. A proper weight can be as it is based on the confo- Carl Zeiss Microscopy GmbH true confocal resolution limit applied to the individual im- cal principle, thick samples Jena, Germany by narrowing the effective PSF. ages and photons properly as- are amenable to analysis that [email protected] In pure reassignment mode signed. As axial information is axial resolution is lost [5]. accessible and not lost, an iso- Read more on: Re-scan However, it is possible to even tropic 1.7 fold increase in res- http://bit.ly/ confocal microscopy: improve axial resolution with olution in all spatial directions Super-resolution http://bit.ly/re-scan proper algorithms. can be achieved. G.I.T. Imaging & Microscopy 3/2014 • 21.
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