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Parallel Fourier Ptychographic Microscopy for High-Throughput Screening Antony C

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Parallel Fourier Ptychographic Microscopy for High-Throughput Screening Antony C bioRxiv preprint doi: https://doi.org/10.1101/547265; this version posted April 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 96 Eyes: Parallel Fourier Ptychographic Microscopy for high-throughput screening Antony C. S. Chan1, Jinho Kim1,+, An Pan1,2, Han Xu3,++, Dana Nojima3,+++, Christopher Hale3, Songli Wang3, and Changhuei Yang1,∗ 1Division of Engineering & Applied Science, California Institute of Technology, 1200 E California Blvd, Pasadena, CA 91125 2State Key Laboratory of Transient Optics and Photonics, Xi’an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, Xi’an 710119, China 3Amgen South San Francisco, 1120 Veterans Blvd, South San Francisco, CA 94080 +Current address: Samsung Engineering, 26 Sangil-ro 6-gil, Gangdong-gu, Seoul, Korea ++Current address: A2 Biotherapeutics, 2260 Townsgate Rd, Westlask Village, CA 91361 +++Current address: Merck, 630 Gateway Blvd, South San Francisco, CA 94080 ∗Corresponding author: [email protected] ABSTRACT We report the implementation of a parallel microscopy system (96 Eyes) that is capable of simultaneous imaging of all wells on a 96-well plate. The optical system consists of 96 microscopy units, where each unit is made out of a four element objective, made through a molded injection process, and a low cost CMOS camera chip. By illuminating the sample with angle varying light and applying Fourier Ptychography, we can improve the effective brightfield imaging numerical apertuure of the objectives from 0.23 to 0.3, and extend the depth of field from ±5µm to ±15µm. The use of Fourier Ptychography additionally allows us to computationally correct the objectives’ aberrations out of the rendered images, and provides us with the ability to render phase images. The 96 Eyes acquires raw data at a rate of 0.7 frame per second (all wells) and the data are processed with 4 cores of graphical processing units (GPUs; GK210, Nvidia Tesla K80, USA). The system is also capable of fluorescence imaging (excitation = 465nm, emission = 510nm) at the native resolution of the objectives. We demonstrate the capability of this system by imaging S1P1-eGFP-Human bone osteosarcoma epithelial (U2OS) cells. 1 Introduction data rate and the mechanical scanning speed of the scanner 27 itself. As a reference point, the state-of-the-art commercial 28 2 e multi-well plate reader is extensively used in large for- well plate imager operating at an optical resolution of 1.2µm 29 3 mat cell culture experiments. A typical well plate reader is (corresponds to a typical 20× objective) can scan a complete 30 4 designed to operate on either a 96-well or 384-well plate, and well plate in 8min. Yet, in a per plate processing speed com- 31 5 is usually used to perform uorescence or absorbance mea- parison, the per plate processing time of a well plate imager 32 6 surements on the contents of the wells. ese measurements is ≈ 50 times longer than that of a non-imaging well plate 33 7 can be performed quickly — readers with a throughput rate 1,2 reader. 34 8 of 1 well plate per 10 seconds are commercially available . 9 However, by their nature, well plate readers can only provide e paper reports on our work at addressing this per plate 35 10 gross characterization of the samples. throughput gap between a well plate reader and a well plate 36 11 To address this decit, a number of imaging well plate imager through the use of parallel imaging. e idea of us- 37 12 microscopy systems have been developed over the past two ing 96 objectives to simultaneously image all the wells on a 38 3–5 13 decades . ese systems are typically designed to use a sin- 96 well plate may seem like an obvious and straightforward 39 14 gle microscope column to scan the entire well plate. e abil- way to boost a well plate imager throughout. However, a 40 15 ity to collect microscopy images of the cell cultures provides more detailed analysis reveals several dicult engineering 41 16 a wealth of information that simple well plate reader cannot challenges that need to be overcome. First, ensuring that 42 17 oer. With such a microscopy system, individual cells within all the wells are simultaneously in focus during the imaging 43 18 a culture can be examined for their individual morphology, process is a very tall order, as each multi-well plate has its 44 19 integrity, vitality, and their connections to neighboring cells. own unique warps. While the warp may be gradual, their 45 20 e range of experiments can be further expanded with the cumulative eect across the wells can easily put a signi- 46 21 addition of uorescence imaging functionality. For example, cant fraction of the wells outside the depth of eld of the 47 22 uorescence imaging provides the capability to track gene objectives. Second, the physical size of the objectives for 48 23 expression in individual cells through the use of uorescence parallel 96-well imaging is necessarily restricted due to their 49 6 24 biomarker methods . Well plate imagers do come with a sig- closely packed geometry in the imaging system — each ob- 50 25 nicant compromise. A single scanning microscope column jective cannot be larger than 6mm in diameter. Designing 51 26 has a nite data throughput rate that is set by the camera a scientic-quality objective with this size constraint can 52 1/14 bioRxiv preprint doi: https://doi.org/10.1101/547265; this version posted April 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 53 be a daunting task. Worse, each objective can be expected enabling all wells to simultaneously be within focus. 108 54 to have slightly dierent aberration characteristics due to In addition to solving these challenges, the FPM approach 109 55 variance in the manufacturing process. In principle, one also brings the following advantages: (i) FPM’s inherent 110 56 can conceivably solve some of these problems by tightening ability to improve resolution allows us to perform large eld 111 57 the manufacturing tolerances and reducing residual aberra- of view (FOV) imaging of cell cultures at improved resolution. 112 58 tions by using more optical surfaces in the objective design. 7 9 (ii) Computational refocusing and aberration correction 113 59 However, these engineering xes would sharply drive up the can occur post data acquisition. is is a marked benet as 114 60 optical system cost, especially when one considers the fact users of conventional microscopy systems generally have no 115 61 that 96 such objectives are needed in total. ird, further recourse for image correction post acquisition. (iii) e FPM 116 62 compounding the aberration issue, the slight dierence in image data contains phase information and we can render 117 63 well curvature and well-to-lens distance for each well in a phase images of the sample with ease. is enables rapid, 118 64 well plate introduces additional aberration variations to the live, and label-free imaging of cell cultures. 119 65 imaging problem for each well. In aggregate, performing 66 parallel well plate imaging in a conventional microscopy con- We have previously demonstrated that the strategy of us- 120 67 text is particularly challenging due to the various aberrations ing FPM to implement parallel microscopy is feasible for 121 10 68 involved in the task. 6 well plate imaging . In this work, we tackle parallel imag- 122 69 Our approach to parallel well imaging implementation de- ing of 96-well plates, hence the name of our prototype — the 123 70 parts from the conventional microscopy system design. Here, 96 Eyes system. Dierent from our previous 6 well version 124 71 we rely heavily on the Fourier ptychographic microscopy where we use research-grade objectives with near identical 125 72 (FPM) method to overcome the three challenges we have just aberrations characteristics, we custom-designed our own 126 73 discussed. objectives due to the high packing density required for 96 127 well simultaneous imaging. ese objectives exhibit signif- 128 74 e FPM method operates by collecting a sequence of icant aberration variations due to surface defects from the 129 75 transmission images of the sample through a microscope ob- platic injection molding process, as well as the lens alignment 130 76 jective. e orientation of the illumination is varied between errors from the assembly process. As such, the completed 131 77 each image. Post image acquisition, we then computation- system relies much more signicantly on the robustness of 132 78 ally stitch the collected image data together in the spatial the FPM technique in order to work. e 96 Eyes system also 133 79 frequency domain using the Fourier ptychographic phase 7,8 requires signicant engineering work on the data transfer 134 80 retrieval algorithm . A higher resolution image of the sam- and processing as the data throughput rate is much higher. 135 81 ple can then be recovered by inverse Fourier transform of During operation, the system prototype acquires and trans- 136 82 the fused spatial frequency spectrum. e resolution of the fers the raw data at 340MB/s. e system captures a batch 137 83 nal image is related to the sum of the objective numerical 7 of 64 frames of all the wells on the well plate in 90 seconds, 138 84 aperture and illumination numerical aperture . As such, it is 2 equivalent to an imaging coverage of 3.6mm per second. 139 85 possible for the FPM rendered image’s resolution to be signif- 7 Equipped with a graphical processing unit (GPU) array to ac- 140 86 icantly improved over the native resolution of the objective .
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