Using smaller zstacks, or less cameras allows higher maximum throughput while maintaining parallel image capture for each well. This is an important consideration for imaging samples displaying higher frequency dynamics. With 24 cameras capturing 10 layer z-stacks, we are able to capture an entire new z-stack approximately 4 times per hour. If it is necessary to capture higher frequency dynamics, the picroscope can be set to capture short videos instead of zstacks. Once uploaded to s3, the images are accessible over the Internet. When an experiment is started, the picroscope generates a file called “manifest.json”. The manifest serves as a text based map indicating where in the s3 object store each image is stored. The manifest can be interpreted in such a way that you can generate the URLs for every picture without needing to query the object store . The image viewer website interprets the manifest to generate an interactive display of the images from a given experiment id. The manifestis also used when pulling the data into our dockerized scripts which we use to generate timelapse videos, focus stacked composite images and perform other image analysis.In addition to being viewable through our web interface, plastic seedling pots the pictures on the server are can be fed into scripts for generating focal plane stacked composite images with FIJI using the Extended Depth of Field plugin. This plugin allows us to generate a single image containing the best focused features from each layer of the z-stack.

We can also use a script to generate timelapse videos from experiments. Generating timelapse videos with focus stacked frames allows for easy visual analysis of longitudinal changes in 3 dimensional samples. The containerization of these programs with Docker allows us to run them in an automated fashion and easily deploy them with cloud service providers.Cerebral cortex organoids generated from aggregates of pluripotent stem cells have been shown to recapitulate aspects of early embryonic brain development, providing an in vitro model for studying species specific brain development in organisms including humans . Here, human cerebral organoids were generated and plated on laminin coated 24 well plates and allowed to adhere to the surface. Outgrowths and cellular migration were restricted to the 2D plane of the plate’s surface and captured/monitored over the course of 21 days with the entire Picroscope device inside a temperature and humidity controlled CO2 incubator. Samples from that experiment can be seen in Figure 8.In a small pilot program, we participated in a project to implement a simple experiment with live zebrafish to run remotely on the Picroscope for a high school AP biology lab. The students used the Picroscope to measure survival and behavioral changes of zebrafish under the influence of varying concentrations of exogenous chemicals including caffeine and ammonia. In the set up phase of this experiment, we captured video showing fluid circulation inside a live zebrafish . This demonstrates the video capture capability which can be used to observe higher frequency dynamics than would be possible with z-stack image capture.Feedback regarding the student experience was gathered using surveys with free response answers to questions. All users responded very positively to questions regarding usability, interest, and excitement in doing remote scientific experiments and indicated interest in pursuing future projects with the Picroscope.

This experiment demonstrates the educational potential of remotely managing live whole organism studies on the Picroscope.In another study, we monitored development of Xenopus Tropicalis frog embryos into larvae. We were able to image all stages of development as the embryo grew into a moving larva. Figures 10 and 11 show 2 interesting periods of development with different time scales of change. At the conclusion of this experiment, each z-stack time step was fed through the Extended Depth of Field plugin in FIJI. Running FIJI through a docker container allowed the process to be scripted and run on a remote server. We then generated a timelapse video of these composite images. Each frame contains the in focus pieces of each of the 10 layers in the z-stack. When the embryo develops into the larval stage, it starts to move. The movement causes visible artifacts to appear in that section of the video, since the larvae moves between layer captures, demonstrating a drawback of this approach when imaging moving organisms.The Covid-19 pandemic changed the work landscape for many of us. The development of the picroscope was highly motivated by the access limitations we were presented with. The resulting system has helped our research group continue to produce work during this difficult time period. The picroscope was developed as a modular device and can be deployed in a number of configurations optimized for different experimental settings. We have run experiments inside a standard CO2 incubator for up to 3 weeks at a time. We have demonstrated that our hardware is robust and minimally interferes with incubator environments. The picroscope has been designed from the ground up as an extensible platform. Development of various compatible add-ons are in progress for new features including fluoresence microscopy. Our end goal is a general use parallel experiment system allowing remote control, sample manipulation, feeding, and imaging. With this system we have provided a low cost solution for biologists to work remotely with greater ease. We have developed a sensor-per-well parallel imaging system capable of bright field microscopy that can be deployed inside a standard CO2 incubator. By having one camera per well, we have an array of microscopes available to researchers allowing them to remotely monitor the development of the biological samples over a long period of time.

Having access to this system allows researchers to easily monitor long term morphological changes in their cell cultures without needing to interfere with their incubator environments. Using Picroscopes also allows for seamless collaboration between researchers at different institutions, allowing them to easily compare cultures as they grow. We envision deployment of many of these systems at once in our lab and collaborator’s labs to help push us into an interconnected open source bio-lab of the future.Grape berries undergo a series of complex physiological and biochemical changes during their development that determine their characteristics at harvest . Genome-wide expression studies using microarray and, more recently, RNA sequencing revealed that berry development involves the expression and modulation of approximately 23,000 genes and that the ripening transition is associated with a major transcriptome shift . Transcriptomic studies characterized the ripening program across grapevine cultivars , identifying key ripening-related genes and determining the impact of stress and viticultural practices on ripening . This knowledge increases the possibility of exerting control over the ripening process, improving fruit composition under suboptimal or adverse conditions, and enhancing desirable traits in a crop with outstanding cultural and commercial significance . These genome-wide expression analyses were possible because a highly contiguous assembly for the species was produced ;this first effort used a grape line created by several rounds of back crossing to reduce heterozygosity, facilitating genome assembly . Though poor by current standards, this pioneering, chromosome-resolved assembly served as the basis for numerous publications. However, the structural diversity of grape genomes makes using a single one-size-fits-all reference genome inappropriate . There is substantial unshared gene content between cultivars, with 8–10% of the genes missing when two cultivars are compared . Although many of these genes are not essential for plant survival, they can account for 80% of the expression within their respective families and expand key gene families possibly associated with cultivar-specific traits . Assembling genome references for all interesting cultivars is impractical, containers size for raspberries in part because its cost remains prohibitive and because of genomic features that impede the development of high-quality genome assemblies for any grape cultivar. Although the V. vinifera genome is relatively small  and as repetitive as other plant genomes of similar size , it is highly heterozygous . Most domesticated grape cultivars are crosses between distantly related parents; this and clonal propagation cause the high heterozygosity observed in the species . Earlier attempts using short reads struggled to resolve complex, highly heterozygous genomes . A limited ability to call consensus polymorphic regions yields highly fragmented assemblies where structural ambiguity occurs and alternative alleles at heterozygous sites are excluded altogether . Single Molecule Real Time DNA sequencing has emerged as the leading technology for reconstructing highly contiguous, diploid assemblies of long, repetitive genomes that include phased information about heterozygous sites . Recently, we used Vitis vinifera cv. Cabernet Sauvignon to test the ability of SMRT reads and the FALCON-Unzip assembly pipeline to resolve both alleles at heterozygous sites in the genome . The assembly produced was significantly more contiguous than the original PN40024 assembly and provided the first phased sequences of the diploid V. vinifera genome . Despite recent advances in genome reconstruction methodologies, assembling a complex plant genome is still costly. Transcriptome reconstruction is the only alternative strategy to depict known and unknown gene content information .

De novo assembly of RNA-seq reads is widely used for this purpose . SMRT technology was recently deployed to investigate expressed gene isoforms in a variety of organisms, including a handful of plant species . Long reads delivered by this methodology report full-length transcripts sequenced from their 59-ends to polyadenylated tails , making Iso-Seq an ideal technology for reconstructing a transcriptome without a reference genome sequence and without assembling fragments to resolve the complete isoform sequence . Moreover, alternative transcripts that contribute to the gene space complexity and vary with cell type , developmental stage , and stress cannot be definitively characterized without full-length transcript information. The objective of this study was to test whether full-length cDNA sequencing with Iso-Seq technology is a suitable alternative to traditional genome sequencing, assembly, and annotation for reconstructing a grape transcriptome reference for transcriptional profiling. We compared how Cabernet Sauvignon’s Iso-Seq transcriptome fares as a reference for RNA-seq analysis vs. its annotated genome. We sequenced the full-length transcripts of ripening berries with Iso-Seq and Illumina RNA-seq reads. The high-coverage short-read data were used to profile gene expression and to error-correct low-expression isoforms that would have been otherwise lost by the standard Iso-Seq pipeline. The transcriptome reference built with Iso-Seq data represented most of the expressed genes in the grape berries and included cultivarspecific or “private” genes. When used as the reference for RNAseq, Iso-Seq generated transcriptome profiles quantitatively similar to those obtained by mapping on a complete genome reference. These results support using Iso-Seq to capture the gene space of a plant and build a comprehensive reference for transcriptional pro- filing without a pre-defined reference genome.Grape berries from Cabernet Sauvignon FPS clone 08 were collected in Summer 2016 from vines grown in the Foundation Plant Services Classic Foundation Vineyard . Between 10 and 15 berries were sampled at pre-véraison, véraison, post-véraison, and at commercial maturity. Table S1 provides weather information for the sampling days. The ripening stages were visually assessed based on color development and confirmed by measurements of soluble solids . On the day of sampling, berries were deseeded, frozen in liquid nitrogen, and ground to powder . Total RNA was isolated using a Cetyltrimethyl Ammonium Bromide -based extraction protocol as described in Blanco-Ulate et al. . RNA purity was evaluated with a Nanodrop 2000 spectrophotometer . RNA was quantified with a Qubit 2.0 Fluorometer using the RNA broad range kit . RNA integrity was assessed using electrophoresis and an Agilent 2100 Bioanalyzer . Only RNA with integrity number greater than 8.0 was used for SMRTbell library preparation.Full-length cDNA sequencing with SMRT technology can be used to rapidly reconstruct the grape berry transcriptome, enabling the identification of cultivar-specific isoforms, refinement of the CabernetSauvignon genome annotation, and the creation of a reference for transcriptome-wide expression profiling. In contrast to transcriptome reconstruction using short-read sequencing that requires de novo assembly, Iso-Seq delivers full-length transcripts that eliminate the introduction of assembly errors and artifacts like chimeric transcripts and incomplete fragments due to PolyA capture . The incorporation of high-coverage short-read sequencing is still necessary to benefit from the complete transcript sequencing enable by Iso-Seq. Although Iso-Seq provides much longer reads than second-generation sequencing platforms and as a result is excellent in resolving transcript structure, its sequencing error rate is high and throughput is still relatively low .