The imaging unit consists of 24 independent objectives attached to a vertical sliding stage using 4 maker beam vertical columns and 2 Nema-11 stepper motors , an example of a row can be seen in Fig. 4a. The fine threads are necessary for focusing on specific biological features and collecting z-stack imaging . With this fixed lens system, the system has a field of view of approximately 5 mm. The Picroscope is able to resolve Group 7, Element 1 targets , corresponding to a resolution of 7 μm . If higher resolution is needed the lens can be swapped out for more magnification . The lens currently on the system was chosen due to our interest in imaging whole organisms. The objectives are distributed on 4-rows and 6-columns to match a standard 24-well culture plate. Each objective consists of a 3D printed camera body that hosts a 5 MegaPixel camera and an off-the-shelf Arducam 1/2” M12 Mount 16 mm Focal Length. Each objective is controlled by a single-board computer , which is connected to an individual slot on one of the three custom-made power distribution boards . All 24 single-board computers computers communicate to a hub board computer that manages the images and autonomously uploads them to a remote server. The hub single-board computer has the MIPI CSI-2 camera port and is connected to an Arduino Uno, which has a motor shield attachment, to control the motors and lift the elevator piece . As a safety feature, the system also includes a custom-made Relay Board that is attached to the Arduino and motor driver stack. The relay board provides control of the LED boards and in the event of an overheat allows us to shut down the system, protecting the system and the biological sample. After each set of pictures, the imaging unit returns to the lowest position, plastic planters bulk which is determined by a limiting switch attached to the elevator unit.
The entire system sits on a 3D printed base, that includes a fan for heat dissipation. Supplementary Fig. 3 shows thermal images of the Picroscope to demonstrate that heat from the system does not impact the experiment. A guide on how to assemble the Picroscope and components needed can be found in Table 2 and Supplementary Note 1. During the course of an experiment, the pictures are autonomously uploaded on a remote computer/server using the ethernet connection of the hub computer board, where they can be viewed or processed in near real time .As proof of principle of the longitudinal live imaging capabilities of the Picroscope, we imaged the development of Xenopus tropicalis embryos from the onset of gastrulation through organogenesis . The fertilization and development of Xenopus occur entirely externally, which allows scientists to easily observe and manipulate the process. For decades, Xenopus have been heavily used in biology studies to model a variety of developmental processes and early onset of diseases, particularly those of the nervous system. While several species of Xenopus are used in different laboratories around the world, Xenopus tropicalis is one of the preferred species due to its diploid genomic composition and fast sexual maturation. Normal development and optimal husbandry of Xenopus tropicalis occur at 25∘ –27 ∘ C, closely approximating standard room temperature, which eliminates the need of special environmental control for most experiments. Given these convenient experimental advantages and their large size, Xenopus embryos have been used extensively to understand the development of the vertebrate body plan, with particular success in elaborating the complex cellular rearrangements that occur during gastrulation and neural tube closure.
These experiments rely on longitudinal imaging of developing embryos, often at single-embryoscale with dyes, fluorescent molecules, and computational tracking of single cells. These studies have elucidated key cellular mechanical properties and interactions critical to vertebrate development, often replayed and co-opted during tumorigenesis. There exists an opportunity to scale these experiments to be more high-throughput with the Picroscope, as one could image hundreds of developing embryos simultaneously, rather than having to move the objective from embryo to embryo during development, or repeating the experiment many times. We imaged Xenopus tropicalis embryos over a 28 h time period. Four embryos were placed in each of the 23 wells used in a 24-well plate, and we used an extra well as calibration . The embryos were grown in simple saline solution and the experiment took place at room temperature. Imaging was performed hourly starting at gastrulation . Then, we visually inspected each image and mapped the embryos to the standard stages of frog development, categorizing their development in gastrulation, neurulation, and organogenesis . Finally, we took a subset of 27 embryos and measured the diameter of the blastopore as the embryos underwent gastrulation . Only 27 embryos were used because those were the only embryos with their blastopores clearly visible throughout the image set. We observed a progressive reduction of blastopore diameter over a 6 h time period, consistent with progression through gastrulation and the start of neurulation. This simple experiment demonstrated that the Picroscope can be used for longitudinal sequential imaging and tracking of biological systems.
While many biological systems including zebra- fish, planaria, and frogs develop at room temperature and atmospheric gas concentrations, mammalian models require special conditions requiring an incubator enclosure. Mammalian models include 2D monolayer cell cultures, as well as 3D organoid models of development and organogenesis. They have been used to assess molecular features and effects of drugs for a variety of phenotypes including cell proliferation, morphology, and activity, among others. Deploying electronics and 3D printed materials inside tissue culture incubators presents some unique challenges. The temperature and humidity conditions can cause electronics to fail and cause certain plastics to off gas toxins. Plastics can also be prone to deformation in these conditions. A common solution for protecting electronics and preventing off gassing is to use inert protective coatings e.g., Parylene C. This requires expensive clean room equipment. Instead, we print all of the components with PLA, a nontoxic and biodegradable material, to prevent deformation we print using 100% infill and reinforce vulnerable elements with aluminum MakerBeam profiles. We coat all electronic components with Corona Super Dope Coating to protect the electronics from the conditions of an incubator. We tested the functionality of the Picroscope inside a standard tissue culture incubator by imaging 2D-monolayers of human embryonic stem cells . To demonstrate the capacity of our system to perform longitudinal imaging across the z-axis, we imaged human cortical organoids embedded in Matrigel . Using this system, we could monitor and measure the growth of the organoids over 86 h . Tracking of individual cells within organoid outgrowths allowed us observe their migration patterns and behavior . Altogether, we show the feasibility of using our system for longitudinal imaging of mammalian cell and organoid models.The combination of 3D printed technology and open-source software has significantly increased the accessibility of academic and teaching laboratories to biomedical equipment. Thermocyclers, for example, were once an expensive commodity unattainable for many laboratories around the world. Now, lowcost thermocyclers have been shown to perform as well as high end commercially available equipment. Inexpensive thermocyclers can be used in a variety of previously unimaginable contexts, including conservation studies in the Amazon, collection pot diagnostics of Ebola, Zika and SARS-CoV-2, teaching high-school students in the developing world and epigenetic studies onboard the International Space Station. Simultaneous imaging of biological systems is crucial for drug discovery, genetic screening, and high-throughput phenotyping of biological processes and disease. This technique typically requires expensive multi-camera and robotic equipment, making it inaccessible to most. While the need for a low-cost solution has long been appreciated, few solutions have been proposed. Currently, the low-cost solutions can be grouped in two categories: those that use gantry systems that move an individual camera through multiple wells, performing “semi-simultaneous” imaging or those that use acquisition of large fields of view encompassing multiple wells , where they can be viewed and/or processed , with minimal intervention. Commercial electronic systems for simultaneous imaging of biological samples are typically designed to image cells plated in monolayers. Yet, significant attention has been given to longitudinal imaging-based screens using whole organisms. These have included zebrafish, worms, and plants. Many times, the results of the screens are based on single-plane images or in maximal projections obtained from external microscopes.
This is accomplished with fine adjustment by two stepper motors that lift the elevator unit that holds all 24 camera objectives .To date, few 3D printed microscopes are designed to function inside incubators. We have run the Picroscope in the incubator for three weeks. This makes the Picroscope compatible with screens in 3D mammalian models including organoids. We have shown a proof of principle of this function by performing longitudinal imaging of human cortical organoids and analyzing the behavior and movement of individual cells . We anticipate many useful applications of the Picroscope and derivatives of it. Here, we demonstrated the versatility of the Picroscope across animal and cell models in different environmental conditions. The modular nature of the system, allows for new features to be easily built and added. For example, defined spectrum LED light sources and filters for fluorescent imaging would enable longitudinal studies of the appearance and fate of defined sub populations of cells in a complex culture by taking advantage of genetically encoded fluorescent reporter proteins. Similarly, the use of fluorescent reporters or dyes that respond to dynamic cell states such as calcium sensors allow long-term imaging of cell activity.Most studies that monitor plants and their environment, whether it be in the field or in the laboratory, require sensors that convert physical or chemical energy into an electrical signal. Some examples of sensors commonly used in plant research are thermocouples, which convert temperature gradients into an electrical potential; photodiodes, which convert light into an electrical current; and strain gauges, which have an electrical resistance that changes when deformed. Many existing methods, such as sap flow measurement , measuring chloroplast movement , and lysimeters , utilize these types of sensors, and nearly all methods that use sensors require a data acquisition system to record measurements. Such systems usually have two basic components: an analog‐ to‐digital converter that converts the electrical signal from the sensor into digital information and a microcontroller or computer that records and processes the digital information from the ADC . There are many commercially available DAQ systems, but these products are often expensive and lack flexibility; a project may need a custom DAQ system to overcome these limitations. One ideal choice for a custom system is a Raspberry Pi computer paired with a high‐resolution ADC. The low cost, flexibility, and high resolution of such a system is ideal for improving existing plant research methods or for developing new ones. The Raspberry Pi is an inexpensive, single‐board computer that has many easily accessible and configurable input/output interfaces, including multiple serial peripheral interfaces and general‐purpose input/output pins , which allow it to be used with a wide variety of ADCs and other peripheral devices. It can run many different operating systems, but the most common is the Linux‐based Raspberry Pi OS, which supports most programming languages. The Raspberry Pi and other similar single‐board computers have many possible applications in life science research. Its small size and low cost make it suitable for data logging in a variety of environments. The easily accessed I/O interfaces can be connected to many different types of sensors for data acquisition, including cameras for high‐throughput plant imaging , microphones for bioacoustic data collection , or gas sensors for air quality monitoring . These same interfaces can also be used to control external components such as mechanical actuators, lighting, or temperature control. To use sensors for data logging with a Raspberry Pi, an ADC is needed to convert the analog output of a sensor into digital information that the computer can use. Many different ADCs are available for this purpose, and it is important to choose one that is appropriate for the application. A few important specifications to consider when choosing an ADC are bit resolution, sampling rate, and number of channels. There is a necessary tradeoff between an ADC’s sampling rate and effective resolution, in that ADCs with very high resolutions are limited to sampling rates in the kilohertz range or less and that as the sampling rate of a given ADC is increased the effective resolution declines . For applications where ultra‐high‐resolution is not critical, there are many ADCs on the market that have readily available open‐ source software libraries and schematics for interfacing with a Raspberry Pi.