Ly VT et al. (2021), Picroscope: low-cost system for simultaneous lo...

XB-ART-58758

Commun Biol 2021 Nov 04;41:1261. doi: 10.1038/s42003-021-02779-7.

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Picroscope: low-cost system for simultaneous longitudinal biological imaging.

Ly VT , Baudin PV , Pansodtee P , Jung EA , Voitiuk K , Rosen YM , Willsey HR , Mantalas GL , Seiler ST , Selberg JA , Cordero SA , Ross JM , Rolandi M , Pollen AA , Nowakowski TJ , Haussler D , Mostajo-Radji MA , Salama SR , Teodorescu M .

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Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods.

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	Fig. 1. Development of a low-cost system for simultaneous longitudinal biological imaging.a The Picroscope fits a standard 24-well plate, it is controlled remotely and images can be accessed through a web browser. b–d Applications of the Picroscope to longitudinal imaging of developmental biology and regeneration. b Regeneration of planaria worms Dugesia tigrina. c Zebrafish embryonic development at oblong stage. d Zebrafish embryo at 48 hours post fertilization. In complement see Supplementary Video 1.
	Fig. 2. Basic workflow from Control Console to Image Viewer.The Control Console passes commands and experiment parameters to Picroscope, which uploads results to a server allowing them to be viewed through the Image Viewer website.
	Fig. 3. The Picroscope.a Physical representation of the proposed imaging system. b One line of independent cameras. c An integrated rack of cameras and Raspberry Pi board computers. d The interlacing strategy of four independent racks of power distribution boards. e The Raspberry Pi Hub and Arduino Uno, Motor driver and custom relay board. f The XY adjustment stage. 1 = Over-the-plate illumination board, 2 = 3D printed Cell Culture Plate Holder and XY stage, 3 = Lenses, 4 = Illumination Board from below, 5 = 3D Printed Camera Bodies, 6 = 3D Printed Elevator, 7 = Raspberry Pi Zero W, 8 = Motors, 9 = Base, 10 = Raspberry Spy Cameras, 11 = Interface Board a. row 1, b. rows 2 and 3c. row 4, 12 = Pi Hub -- Raspberry Pi 4, 13 = Custom Relay Board, 14 = Adafruit Motor/Stepper/Servo Shield for Arduino v2, 15 = Arduino Uno, 16 = Leaf Springs, 17 = Rigid Elements, 18 = Relays, 19 = Limit switches connectors, 20 = Power distribution board connectors, 21 = Light board connectors, 22 = Motor power connector, 23 = 12 V power source, 24 = Voltage regulators, and 25 = Temperature & Humidity sensor.
	Fig. 4. Schematic representation of the z-stack function.a A single row of cameras demonstrating the z-stack function. 1.a = Over-the-plate illumination board, 1.b = Under-the-plate illumination board, 2 = Acrylic Light Diffuser, 3 = Lenses, 4 = Cell Culture Plate, 5 = LEDs, 6 = Raspberry Spy Cameras, 7 = 3D Printed Camera Bodies, 8 = Biological Sample (e.g., Frog Embryos), 9 = Individual Culture Well. b Four focal planes of a single z-stack. These photos at were taken at four planes, 0.3 mm apart. The blastopore is only in focus in Plane 1.
	Fig. 5. System architecture.a The images are autonomously collected and wirelessly transferred to a remote computer for viewing or post processing. b Image of 23 wells observing 57 frog embryos.
	Fig. 6. Longitudinal imaging of Xenopus tropicalis development.Images of a representative well in which 4 frog embryos developed over a 28 hour period. Images were taken hourly. White Balance adjusted for visibility.
	Fig. 7. Longitudinal imaging allows the tracking of individual developmental processes.a The images shown in Fig. 6 were taken hourly over a 28 h period and encompass three developmental stages: Gastrulation, neurulation, and organogenesis. Y-Axis represents the stages of frog embryonic development: 1 = Fertilization, 2 = Cleavage, 3 = Gastrulation, 4 = Neurulation, 5 = Organogenesis, and 6 = Metamorphosis. X-axis represents the timepoint at which it occurs. Each dot in the plot represents a timepoint in which the images were taken. Magenta = the beginning of each developmental process. Red = the end of the experiment at 28 h. Blue = intermediate timepoints. b Diameter of the blastopore is reduced over time from gastrulation to neurulation. Top right-hand panel shows an example of an individual blastopore. A total of 27 embryos were considered for the analyses. Error bars represent Standard Deviation (SD).
	Fig. 8. In-incubator imaging of mammalian cell and cortical organoid models.a The Picroscope inside a standard tissue culture incubator. b Imaging of human embryonic stem cells as a model of 2D-monolayer cell cultures. c Longitudinal imaging of human cortical organoids embedded in Matrigel. Zoomed images show cellular outgrowths originating in the organoids. d Tracking of cortical organoid development over 86 h. Images were taken hourly. On left. Images of the tracked organoid at timepoints 0, 43, and 86. On right. Measurement of organoid area at each timepoint analyzed. e Manual Longitudinal tracking of individual cells in embedded cortical organoids over 40 min. Images were taken every 10 min. Magenta = example of cell division, Red = example of cell migration, and Purple = example of morphological changes.

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Abe-Fukasawa, Novel 3D Liquid Cell Culture Method for Anchorage-independent Cell Growth, Cell Imaging and Automated Drug Screening. 2018, Pubmed