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Pursuing the Diffraction Limit with Nano-LED Scanning Transmission Optical Microscopy

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Pursuing the Diffraction Limit with Nano-LED Scanning Transmission Optical Microscopy sensors Article Pursuing the Diffraction Limit with Nano-LED Scanning Transmission Optical Microscopy Sergio Moreno 1,* , Joan Canals 1 , Victor Moro 1, Nil Franch 1, Anna Vilà 1,2 , Albert Romano-Rodriguez 1,2 , Joan Daniel Prades 1,2, Daria D. Bezshlyakh 3, Andreas Waag 3, Katarzyna Kluczyk-Korch 4,5 , Matthias Auf der Maur 4 , Aldo Di Carlo 4,6 , Sigurd Krieger 7, Silvana Geleff 7 and Angel Diéguez 1,2 1 Electronic and Biomedical Engineering Department, University of Barcelona, 08028 Barcelona, Spain; [email protected] (J.C.); [email protected] (V.M.); [email protected] (N.F.); [email protected] (A.V.); [email protected] (A.R.-R.); [email protected] (J.D.P.); [email protected] (A.D.) 2 Institute for Nanoscience and Nanotechnology-IN2UB, University of Barcelona, 08028 Barcelona, Spain 3 Institute of Semiconductor Technology, Technische Universität Braunschweig, 38106 Braunschweig, Germany; [email protected] (D.D.B.); [email protected] (A.W.) 4 Department of Electronic Engineering, University of Rome “Tor Vergara”, 00133 Roma, Italy; [email protected] (K.K.-K.); [email protected] (M.A.d.M.); [email protected] (A.D.C.) 5 Faculty of Physics, University of Warsaw, 00-662 Warsaw, Poland 6 CNR-ISM, 00128 Rome, Italy 7 Department of Pathology, Medical University of Vienna, 1210 Wien, Austria; [email protected] (S.K.); [email protected] (S.G.) * Correspondence: [email protected] Abstract: Recent research into miniaturized illumination sources has prompted the development Citation: Moreno, S.; Canals, J.; of alternative microscopy techniques. Although they are still being explored, emerging nano-light- Moro, V.; Franch, N.; Vilà, A.; emitting-diode (nano-LED) technologies show promise in approaching the optical resolution limit in Romano-Rodriguez, A.; Prades, J.D.; a more feasible manner. This work presents the exploration of their capabilities with two different Bezshlyakh, D.D.; Waag, A.; prototypes. In the first version, a resolution of less than 1 µm was shown thanks to a prototype based Kluczyk-Korch, K.; et al. Pursuing the on an optically downscaled LED using an LED scanning transmission optical microscopy (STOM) Diffraction Limit with Nano-LED Scanning Transmission Optical technique. This research demonstrates how this technique can be used to improve STOM images by Microscopy. Sensors 2021, 21, 3305. oversampling the acquisition. The second STOM-based microscope was fabricated with a 200 nm https://doi.org/10.3390/s21103305 GaN LED. This demonstrates the possibilities for the miniaturization of on-chip-based microscopes. Academic Editor: Bruno Tiribilli Keywords: CMOS sensor; nano-LED; optical downscaling; nanopositioners; miniaturization Received: 4 March 2021 Accepted: 5 May 2021 Published: 11 May 2021 1. Introduction Optical microscopy systems have been diversified over past centuries with a va- Publisher’s Note: MDPI stays neutral riety of techniques (phase contrast [1–3], dark field [4,5], confocal [6,7], fluorescence with regard to jurisdictional claims in microscopy [8–10], etc.) that have allowed for the easy observation of most processes published maps and institutional affil- and cellular structures down to a few tenths of a micrometer. For the exploration of objects iations. on a smaller scale, photonic systems have been hindered by the light diffraction limit, theorized by Ernst Abbe to be approximately 200 nm. More recently, some super-resolution (SR) techniques, i.e., techniques with a resolving power beyond the diffraction limit, such as stimulated emission depletion (STED) [11–13], Copyright: © 2021 by the authors. structured illumination microscopy (SIM) [14,15], stochastic optical reconstruction mi- Licensee MDPI, Basel, Switzerland. croscopy (STORM) [14,15], photo-activated localization microscopy (PALM) [16–18], or This article is an open access article scanning near-field microscopy (SNOM) [19], have demonstrated resolutions of as small as distributed under the terms and a few tenths of a nanometer. However, the overall complexity and cost of these imaging conditions of the Creative Commons systems have increased significantly, limiting the widespread use of some of the more Attribution (CC BY) license (https:// advanced optical imaging techniques beyond well-equipped laboratories. creativecommons.org/licenses/by/ 4.0/). Sensors 2021, 21, 3305. https://doi.org/10.3390/s21103305 https://www.mdpi.com/journal/sensors Sensors 2021, 21, x FOR PEER REVIEW 2 of 15 Sensors 2021, 21, 3305 imaging systems have increased significantly, limiting the widespread use of some 2of of the 14 more advanced optical imaging techniques beyond well-equipped laboratories. Alternatively, cheaper and simpler techniques are being investigated. For instance, lenslessAlternatively, microscopy cheaper [20] takes and advantage simpler techniques of the large are field-of-view being investigated. (FOV) and For small instance, pixel lenslesssize provided microscopy by large [20 commercial] takes advantage cameras of the(CMOS large or field-of-view CCD). Various (FOV) methods and small of lensless pixel sizeimaging provided can be by found large commercialtoday, whether cameras based (CMOS on shadowing or CCD). [21–23], Various fluorescence methods of lensless[24–26], imagingholography can [27–29], be found or today, 3D imaging whether [30,31]. based The on shadowingrefinement [of21 high-resolution–23], fluorescence (HR) [24 –tech-26], holographyniques and the [27 –improvement29], or 3D imaging of computational [30,31]. The resources refinement [32–34] of high-resolution have boosted (HR)the spatial tech- niquesresolution and below the improvement the pixel size of limitation. computational Thanks resources to techniques [32–34] such have as boosted pixel super-reso- the spatial resolutionlution (PSR), below based the on pixel the displacement size limitation. of the Thanks image to sensor techniques [35–37], such the as sample pixel super-or the resolutionlight source, (PSR), they based can reach on the resolutions displacement of lower of the than image 1 µm sensor [38]. [ 35However,–37], the the sample diffraction or the lightlimit source,remains they a barrier can reach for low-cost resolutions SR alternatives. of lower than 1 µm [38]. However, the diffraction limit remainsA novel amicroscopy barrier for low-costimaging SRtechnique alternatives. has emerged, which harnesses the key fea- turesA of novel lensless microscopy microscopy imaging and technique scanning hastechniques. emerged, As which in lensless harnesses microscopy, the key features LEDs ofare lensless the basic microscopy elements. andHowever, scanning instead techniques. of fixing As its in position, lensless microscopy,the LED is moved LEDs areto scan the basic elements. However, instead of fixing its position, the LED is moved to scan the the sample, as in scanning microscopes. This scan can be performed with one LED alone, sample, as in scanning microscopes. This scan can be performed with one LED alone, by moving it over the sample or by moving the sample over the LED; alternatively, its by moving it over the sample or by moving the sample over the LED; alternatively, its apparent position can be electronically controlled using the LEDs available in an array or apparent position can be electronically controlled using the LEDs available in an array or in an LED micro-display (see Figure 1). The other key component of the microscope is an in an LED micro-display (see Figure1). The other key component of the microscope is optical photodetector used to measure the light traversing the sample, and a camera can an optical photodetector used to measure the light traversing the sample, and a camera typically be used. The first prototype using this technique was already presented in [39], can typically be used. The first prototype using this technique was already presented where every LED in an 8 × 8 gallium nitride (GaN) array (5 µm in size and 10 µm in pitch) in [39], where every LED in an 8 × 8 gallium nitride (GaN) array (5 µm in size and was sequentially turned on and off, mapping the sample to prove the principle of opera- 10 µm in pitch) was sequentially turned on and off, mapping the sample to prove the tion of nano-illumination microscopy (NIM). The main features of this technique are that principle of operation of nano-illumination microscopy (NIM). The main features of this the resolution is given by the LED pitch and that the FOV is given by the LED array size technique are that the resolution is given by the LED pitch and that the FOV is given by thewhen LED the array sample size is when placed the in sample direct contact is placed with in directthe array. contact Therefore, with the the array. sensing Therefore, device theused sensing does not device play usedas important does not a role play as as in important conventional a role lensless as in microscopy. conventional NIM lensless only microscopy.needs two chips NIM and only benefits needs from two chips being and relati benefitsvely close from together. being relatively On the other close hand, together. LED Onminiaturization the other hand, is a LED very miniaturization active field of research, is a very activewhich field seems of research,to allow for which sizes seems smaller to allowthan Abbe’s for sizes diffraction smaller than limit Abbe’s [40–42]. diffraction In this context, limit [40 the–42 fabrication]. In this context, of new the compact fabrication low- ofcost new microscopes compact low-cost based on microscopes nano-LED basedscanning on nano-LEDtransmission scanning optical transmissionmicroscopy (STOM) optical microscopyis pursued, capable (STOM) of is sub-micron pursued, capable resolutions. of sub-micron With the decrease resolutions. in LED With size, the further decrease res- inolutions LED size, can furtherbe achieved resolutions [43]. Nevertheless can be achieved, high-resolution [43]. Nevertheless, and large-FOV high-resolution microscopes and large-FOVremain a still-distant microscopes prospect remain because a still-distant of the prospectresearch becauserequired, of which the research is limited required, to ad- whichvancements is limited in both to advancements directions.
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