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DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203136 Max B. Schäfer*, Sophie Weiland, Kent W. Stewart and Peter P. Pott Compact Microscope Module for High- Throughput Microscopy Abstract: Microscopy is an essential tool in research and cost additional components, already has led to promising science. However, it is relatively resource consuming results [1–3], not only in microscopy but also for other regarding cost, time of usage, and consumable supplies. methods such as immunoassays . Even though the Current low-cost approaches provide good imaging quality but mentioned low-cost microscopy approaches result in relatively struggle in terms of versatility or applicability to varying good imaging qualities, they are mostly focused on a dedicated setups. In this paper, a Compact Microscope Module for application, such as transmitted light microscopy of samples versatile application in custom-made setups or research on a conventional specimen slide. A more universal setup projects is presented. As a first application and proof of would provide benefits in terms of applicability to various concept, the use of the module in a High-Throughput fields not only regarding the way of illumination but also the Microscope for screening of samples in microtiter plates is implementation in different research projects or setups. shown. The Compact Microscope Module allows for simple A small and low-cost microscope module could be an and resource-efficient microscopy in various applications adequate solution to provide easy-to-use microscopy. With a while still enabling relatively good imaging qualities. small microscope module, providing sufficient imaging capabilities, numerous initial investigations and/or Keywords: Microscopy, Compact Microscope Module, experiments could be performed on a low level regarding costs High-Throughput Microscopy, Scanning, Microtiter Plate and complexity. However, this might have even more effect if the field of screening microscopy is considered. Modern day https://doi.org/10.1515/cdbme-2020-3136 biomedical research for example, requires frequent observation of cell populations which are cultivated in small cavities on microtiter plates to determine for example growth 1 Introduction and confluency of the cells . Screening of either a high number of samples and/or of samples over a certain time interval is a limited resource since relatively expensive 1.1 Motivation microscopes are occupied for the complete imaging procedure resulting in a tightly scheduled utilization. Aiming on a small In science, education, and medicine, light microscopy is one and low-cost microscope module could therefore lower the of the most prevalent used tools. However, microscopy is still barrier to entry and make screening microscopy more relatively resource consuming considering cost, time of usage, accessible. and consumable supplies. Applying low-cost approaches in In this paper, a Compact Microscope Module (CMM) for the field of microscopy might limit imaging quality, but also versatile application is presented. As a first application and has the potential to elevate this crucial tool to a new level of proof of concept, the use of the module in a High-Throughput dissemination or even to new applications. An example for an Microscope (HTM) is shown. approach, focusing on a more balanced trade-off between costs and imaging capabilities, are smartphone-based microscopes. Utilizing the potent camera systems of modern smartphones 1.2 Form Factor Requirements for analytic purposes in combination with only few and low- To ensure a high applicability of the CMM to various setups, an appropriate form factor is crucial besides imaging ______ requirements. To meet high standards, the limited dimensions *Corresponding author: Max B. Schäfer: Institute of Medical of a standard microtiter plate with up to 96 wells  are Device Technology, University of Stuttgart, Pfaffenwaldring 9, considered (see Fig. 1). Assuming the potential requirement of Stuttgart, Germany, firstname.lastname@example.org Sophie Weiland, Kent W. Stewart, Peter P. Pott: Institute of arranging 96 CMMs in the present area of such a microtiter Medical Device Technology, University of Stuttgart, Germany Open Access. © 2020 Max B. Schäfer et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. Max B. Schäfer et al., Compact Microscope Module for High-Throughput Microscopy — 2 plate, leads to maximum size of 9 mm in width and length of smartphone camera lens, a Samsung Galaxy S4 (Samsung the optical system with no specified limit in height. Electronics Co. Ltd., Suwon, KR) front camera lens with a Commonly, there is only limited space close to the sample, but focal length of 1.90 mm or an Apple iPhone 5S (Apple Inc., further off, slightly more space is available. This can lead to Cupertino, CA, US) rear camera lens with a focal length of an approach where the optical system is designed as small as 4.12 mm was used, aiming on varying working distances possible, whereas the electronic part is placed slightly away. depending on the application. 2.2 Evaluation of the Optical System A MIL-STD-150A US Air Force optical resolution test target was used to determine the resolution of the optical system. Expecting a Michelson contrast of at least 10 % in horizontal and vertical orientation between the white and black bar Figure 1: Standard microtiter plate with 96 wells . elements on the target [8, 9], analysis of the contrast gradient was performed to determine the achievable resolution. To assess the FOV, images of graph paper were taken and measured with knowledge of the respective pixel size of the 2 Materials and Methods camera sensor. In order to determine the distance between the front lens surface and the focal plane, referred to as working distance, the optical system was positioned relative to graph 2.1 Optical System paper on a specimen slide using a micrometer screw. Initially, the optical system was moved until the front lens had contact The optical principle described in  is used. Therefore, an with the graph paper. By moving back until the graph paper inverted smartphone camera lens is combined with an off-the- was in focus, the travelling range was obtained from the scale shelf camera module designed for use with a Raspberry Pi of the micrometer screw. single-board computer (Raspberry Pi Foundation, UK). This optical setup, shown in Fig. 2, enables microscopy with resolutions of a few micrometers while providing a relatively large field of view (FOV). 3 System Design Inverted Sm artphone Ras pberry Pi Cam era Lens Cam era Object 3.1 Compact Microscope Module A CMM was developed consisting of a small optical unit and a slightly larger handle mount to provide housing for the 𝑓 𝑓 electronics but more importantly to enable ease of handling. Figure 2: Optical principle of the Compact Microscope Module. The optical unit holds the camera and the additional lens in place and provides a dovetail connection to allow fast While both lenses have focal lengths in the same order of attachment to other setups (see Fig. 3a). Camera and lens can magnitude, only limited optical magnifications can be be mounted without any tools due to press-fit tolerances. A achieved (see Eq. 1). However, a digital magnification is rectangular and flat shaped handle mount allows for better enabled due to the high number of camera pixels in the handling of the small optical unit (see Fig. 3b). The optical unit Raspberry Pi camera. is attached by the dovetail joint and locked by a bolt. Additionally, the handle provides space for the camera board 𝒕𝒆𝒄𝒊 𝒍𝒋𝒃𝒗𝒆𝒐 𝒔𝒆𝒏 𝑴 = − (1) 𝒂𝒎𝒆𝒓𝒄𝒂 𝒍𝒔𝒆𝒏 and ventilation slots for heat dissipation of the electronics. Due to a sufficient cable length, the optical unit can be taken off the A camera module designed for the Raspberry Pi was used. It handle if space around the sample of interest is limited. features an 8-megapixel IMX219 CMOS sensor (3280 × Manufacturing of housing components was done with a 2464 pixels) and a focal length of 2.96 mm. A Raspberry Prusa i3 MK3 3D printer (Prusa Research s.r.o., Prague, CZ) Pi v2.1 camera board and a Raspberry Pi 3B+ single-board using PLA filament. computer were used to control the camera. For the inverted Max B. Schäfer et al., Compact Microscope Module for High-Throughput Microscopy — 3 is achieved. The optical unit solely has a size of 9 × 9 × 11 mm (length × width × height), whereas the handle with the attached optical unit has a size of 38 × 27.5 × 13.2 mm . The cable which connects the optical unit with the camera board in the handle has a length of 70 mm, allowing for versatile placement of the CMM. The Figure 3: The Compact Microscope Module (a) can be attached to setup is shown in Fig. 5. the handle mount (b), which is housing the camera electronics. 3.2 High-Throughput Microscope To demonstrate a first possible application of the principle of the CMM, an HTM for automated imaging of samples in a 96-well microtiter plate was designed. Since the HTM is only Figure 5: Handle mount containing electronics with Compact an example for application of the designed CMM, only Microscope Module (CMM) attached (a) and CMM removed for use when space is limited (b). technical details, which are necessary for the description of the working principle, are given. Instead of positioning the Manual positioning of the CMM relative to the sample by hand samples relative to a single microscope, eight optical units is possible if the heel of the hand is placed on a stable surface were used to reduce scanning time. Since some cell cultures while guiding the CMM between index finger and thumb. are sensitive to movement, the optical unit instead of the vessel Further, natural ambient light is sufficient to achieve a is positioned in all three dimensions. Positioning in x- and y- satisfying image quality. axes is accomplished by conventional linear spindle drives. To align the focal plane of the optical units with the structure of interest, z-axis positioning is achieved through a solid-state 4.2 High-Throughput Microscope joint lever mechanism . This focusing mechanism is actuated by a geared DC motor with a spindle. Based on the CMM, the HTM enables scanning of standard In order to ensure a congruent and robust alignment of all microtiter plates with up to eight optical units working optical units, the CMMs are manufactured as a connected line simultaneously. With overall dimensions of 294 × 178 × of eight optical modules (see Fig. 4). Since the 96-well 85 mm , the HTM is relatively compact. A sufficient working microtiter plate comprises eight rows with twelve cavities distance was achieved - overcoming the thickness of the glass each, a line of eight combined CMMs allows for simultaneous bottom of the microtiter plates and enabling in-focus images recording of a complete column of cavities. of larger samples in the cavities – by equipping each CMM with an iPhone 5S rear lens, resulting in a magnification of -0,72 which enables the identification of structures with a size of 3.48 µm in a FOV of 21.2 mm and a working distance of 1.0 mm. The HTM is shown in Fig. 6, whereas Fig. 7 shows exemplary images recorded with the CMMs used. Figure 4: Microscope assembly comprising eight Compact Microscope Modules. 4 Results 4.1 Compact Microscope Module For the evaluation, the CMM was equipped with a Samsung Galaxy S4 lens, resulting in a magnification of -1,56 which Figure 6: High-Throughput Microscope based on eight Compact Microscope Modules arranged in a line to record enables the identification of structures with a size of 2.46 µm. samples in microtiter plates. Further, a FOV of 3,5 mm and a working distance of 0.3 mm Max B. Schäfer et al., Compact Microscope Module for High-Throughput Microscopy — 4 required larger working distance, a remarkable trade-off in image resolution had to be accepted. Regarding the resulting limits in identification of small structures, further investigations are necessary to improve the optical setup. However, the accomplished implementation of the CMM in a complex research setup around scanning microscopy reveals its potential as a versatile module for various setups. Author Statement Research funding: The author state no funding involved. Conflict of interest: Authors state no conflict of interest. Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either Figure 7: Example images recorded with the Compact Microscope Module equipped with the iPhone 5S lens human or animals use. showing laser printed lines on graph paper (a), onion cells (b), spinal cord (c), and freshwater algae (d). Samples were illuminated by a LED array placed above References the microtiter plate.  M. B. Schäfer, D. Reichert, K. Stewart, A. M. Herkommer, C. Reichert, and P. P. Pott, “Smartphone-based low-cost microscope with monolithic focusing mechanism,” Current 5 Discussion Directions in Biomedical Engineering, vol. 4, no. 1, pp. 267– 270, 2018.  D. N. Breslauer, R. N. Maamari, N. A. Switz, W. A. Lam, A CMM was developed, enabling widespread use of and D. A. 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Schenk, “Hochdurchsatz-Mikroskopie von therefore the possibility of capturing a large FOV with a larger Mikrotiterplatten auf Basis einer kontinuierlichen working distance. The CMM can easily be equipped with the Objektbewegung,” Dissertation, Aachen, 2016. desired lens due to tool-free press-fit connections only. As a  ANSI American National Standards Institute: Footprint Dimensions for Microplates, ANSI SLAS 1-2004 (R2012). result, a fast and simple adaption of the CMM to different  Greiner Bio-One, “Customer Drawing 96 Microplate,” 2019. applications is possible.  Dorina Hasselbeck, “Untersuchung der Wang et al. present a microscope based on a similar Anwendungsmöglichkeiten eines Low-Cost Smartphone- camera module and therefore with comparable Mikroskops,” Studienarbeit, Institute of Medical Device dimensions . However, this system allows for Technology, University of Stuttgart, Stuttgart, 2019.  Katharina Höllerhage, “Das menschliche Kontrastsehen - identification of a line pair of 10 µm distance on a limited FOV Vergleich zweier Testverfahren,” Dissertation, Medizinische of 1 mm . A similar resolution compared to the CMM showed Fakultät, Albert-Ludwigs-Universität, Freiburg i.Br., 2011. in the present paper is achieved by Tristan-Landin et al. .  Z. Wang et al., “A High-Resolution Minimicroscope System Aiming for fluorescence microscopy, a high number of for Wireless Real-Time Monitoring,” IEEE transactions on bio-medical engineering, vol. 65, no. 7, pp. 1524–1531, components is necessary resulting in larger overall dimensions.  S. B. Tristan-Landin, A. M. Gonzalez-Suarez, R. J. For a proof of the versatility of the CMM, the usage in a Jimenez-Valdes, and J. L. Garcia-Cordero, “Facile custom designed HTM was performed. The challenges in assembly of an affordable miniature multicolor fluorescence microscope made of 3D-printed parts enables detection of space and alignment could be accomplished and a dataset of single cells,” PloS one, vol. 14, no. 10, e0215114, 2019. sample images was successfully recorded. However, due to the
Current Directions in Biomedical Engineering – de Gruyter
Published: Sep 1, 2020
Keywords: Microscopy; Compact Microscope Module; High-Throughput Microscopy; Scanning; Microtiter Plate
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