Our results in the second year corroborated those of the first year, showing that the separation in both plant water status and leaf gas exchange between the two zones were consistent. Leaf gas exchange was closely related to plant water status, and this relationship was shown in previous research . The relationships between leaf gas exchange and plant water status were evident in our study, where a higher 9 stem would promote a greater stomatal conductance to increase carbon assimilation capacity and decrease intrinsic water use efficiency. In our study, the lowest 9 stem we observed were around harvest with 9 stem of -1.6 MPa and gs of around 50 mmol H2O m−2 ·s −1 , which were not severe enough to impair berry ripening although the photosynthetic activities were still affected. Overall, the gs and AN reached the maximum values at veraison and declined with decreasing plant water status and leaf age toward the end of the season. This further affirmed that the continuous water deficits during the growing season, especially being more pronounced after irrigation was ended after veraison, would reduce stomatal conductance. The water deficits would act as passive hydraulic signals or active hormonal signals with the upregulation in abscisic acid synthesis to limit plant photosynthetic activities, hence lower gs and AN values .According to the previous research, components of yield may be affected by plant water status, where higher water deficits would result in reductions of yield, berry skin weight, and berry weight . In our study, blueberry production we observed constant separation in plant water status after veraison. However, there was no difference shown in cluster number, yield, berry number, or pruning weight.

The only difference measured in yield components was that berry skin weight was higher in Zone 1 in the second season. Early season water deficit irrigation had higher probability to decrease yield than later season water deficit irrigation . However, a season-long water deficit irrigation would have the lowest yield even despite the season-long water deficit irrigation regime applying double amount of water than the other regimes . Some other studies did not have the same results, as early water deficit irrigation did not show significant influences on yield compared to late water deficit irrigation . Another possible explanation was that Zone 1 had greater water amount held in the soil due to the higher clay content. The clay soil with higher water-holding capacity had a better water status at the early season compared to Zone 2, even though the sandy soil in Zone 2 would benefit the plant growth with irrigation when the season progressed . The later season water deficit was exacerbated in Zone 1 due to its higher clay content, causing Zone 1 lost the benefits from the high water status in the early season, and eventually had similar yield components with Zone 2 at harvest. In our work, we did not see any evidence of Ravaz index being affected by spatial variability of plant water status. These results were corroborated by Terry and Kurtural when grapevine cultivar ‘Syrah’ was exposed to post-veraison water deficits in comparable severity of -1.4 MPa .Water deficits affect advancement of grape berry maturity, they promote TSS accumulation and TA degradation in grape berries . Two factors contributed to these differences between the two zones. First, a greater water deficit advanced the berry maturation, leading to a higher TSS and lower TA . Second, berry dehydration may have occurred and the TSS concentration increased in the berries. In our study, smaller berries were observed in Zone 1, which can confirm the berry dehydration could have led to higher TSS in Zone 1. As for berry TA, one study showed that grape organic acids biodegradation would be faster with more solar radiation and higher temperature .

Although the acid degradation was not related to water deficits, like mentioned above, water deficits would limit the grapevines’ ability to regulate temperature . Thus, waterdeficits could promote the organic acid degradation and this effect was observed in this study.Mild water deficits increased the flavonoid content and concentration of red-skinned grape berry due to the upregulation in flavonoid synthesis and the advancement of berry dehydration during growing season . A positive relationship was noticed between soil bulk EC and total skin anthocyanins in 2017 at both depths of soil bulk EC measurements. A more prolonged severe water deficit would lead to deleterious stomatal and temperature regulation and eventually resulted in flavonoid degradation, specifically anthocyanins . This was a plausible explanation for the non-significant relationship between soil bulk EC and total skin anthocyanins in 2016, wherein harvest took place at higher soluble solids and Zone 1 berry skin anthocyanins were presumably in decline. Furthermore, the berry weights were higher in Zone 2, which was similar to the observations in our previous work , indicating there was less berry dehydration. Thus, the higher anthocyanins in Zone 2 was mainly due to the upregulation in anthocyanins other than anthocyanins degradation. These effects were also observed in the wines of 2016, where Zone 2 had higher anthocyanin concentrations. However, in the second season, the differences in berry skin anthocyanins at harvest did not carry over into the wines. We contributed this to the more advanced berry maturity levels at harvest in the first season, the skin cell walls could have become more porous during ripening and increased the extractability of flavonoid compounds . With relatively greater amounts of flavonoids extracted, there was a higher chance to pass on the separations of anthocyanins from the berries to the wines.

Grape berry skin proanthocyanidins are less sensitive toward water deficits than anthocyanins . Nevertheless, their biosynthesis and concentration may be modified by water deficits . In 2016, wine total proanthocyanidins and all the subunits were greater in Zone 2. These differences were not observed in the second season. We attributed this lack of consistency in proanthocyanidin disparities between the two zones to the more advanced maturity of the berries were harvested in 2016 than in 2017. We suggest that similar to skin anthocyanins, the more advanced berry maturity in 2016 could have promoted the proanthocyanidin extractability in the skin tissues , which may augment the separations in the concentration of all the subunits between the two zones.Monitoring and handling live tissues and cell cultures as well as analyzing their secreted contents are essential tasks in experimental biology and bio-medicine. Advances in microscopy have revolutionized biological studies, allowing scientists to perform observations of cellular processes and organisms’ development and behaviors. Imaging has been pivotal to uncovering cellular mechanisms behind biological processes. Several options exist on the market to perform longitudinal imaging of biological materials. These range from super-resolution microscopes, that allow the imaging of individual bio-molecules, to conventional benchtop microscopes, which are common in academic research, industrial, and teaching laboratories. When deciding between the different technologies for longitudinal live tissue imaging, several factors need to be considered in the experimental design. The image acquisition speed of the microscope should be sufficient for the phenomenon being studied. The microscope should be able to acquire images without damaging or disturbing the specimen, such as photo bleaching. The microscope should be capable of imaging in the environmental conditions needed for the desired experiment, including temperature, light, and humidity. The resolution of the microscope should be sufficient to view the phenomenon being studied. When scaling to simultaneous multi-well longitudinal tissue imaging it is also important that the apparatus not be bulky or expensive. It has been challenging to meet all of these criteria. The use of open-source technology, including 3D printers, laser cutters, blueberry in container and low-cost computer hardware, has democratized access to rapid prototyping tools and dramatically increased the repertoire of biomedical equipment available to laboratories around the world. Through rapid prototyping and the use of open-source platforms, the technology can be replicated and quickly improved. 3D printer technology has been applied to several fields in bio-medicine, including biotechnology, bioengineering, and medical applications including fabrication of tissues and organs, casts, implants, and prostheses. Existing 3D printed microscopes range in complexity from simple low-cost systems with pre-loaded imaging modules to portable confocal microscopes capable of imaging individual molecules and even 3D printed microfluidic bioreactors. The majority of low-cost 3D printed microscopes are not intended for longitudinal imaging of simultaneous biological cultures . They usually have a single imaging unit or perform confocal, and even light-sheet imaging. Other systems have taken advantage of one camera attached to a gantry system to perform imaging of multiple experimental replicates. Few 3D-printed microscopes have been developed that perform multi-well imaging with medium throughput.

Several biological applications exist that would greatly benefit from multi-well, multi-week simultaneous imaging, as it allows for concurrent interrogation of different experimental conditions and the inclusion of biological replicates. These include cell culture applications, in which 2D and 3D culture models can be tracked over multi-week periods, as well as developmental and behavioral biology experiments in which multi-week tracking could be performed on whole organisms. Here, we report a simultaneous multi-well imaging system , which features a low-cost per well and performs longitudinal bright field z-stack imaging of 24-well cell culture plates. Images are uploaded to a server as they are captured allowing the users to view the results in near real time. We used this system to longitudinally track different animal models of development and regeneration, including Xenopus tropicalis , Danio rerio , and planaria worms. Finally, we demonstrate this system’s versatility by imaging human embryonic stem cells and 3D cortical organoids inside a standard tissue culture incubator. We demonstrate that the Picroscope is a robust low-cost, versatile multi-well imaging system for longitudinal live imaging biological studies.System design. The Picroscope is a programmable, data rich, sensor-per-well simultaneous imaging system for longitudinal bright field imaging to automate microscopy . The system simultaneously images in each one of the 24 wells multiple focal planes several times every hour for weeks, a frequency impractical to perform manually. The instrument is made using off-the-shelf components , and 3D printed Polylactic acid components with 100% infill . Cost comparison with other open-source microscope projects can be found in Table 1. A cost breakdown of the materials required can be found in Table 2. The Picroscope has been used to image Planaria worms , Xenopus tropicalis , as well as zebrafish . The system was developed to be operated remotely through the internet. Users can set and change the device settings to modify experiments on the fly. Images captured by the system are uploaded to a server where they become visible on a viewer website. We have also created several image analyses scripts that can directly access images on the server, allowing us to generate timelapse videos and composite images in an automated fashion. While the system receives commands and transmits results through the Internet, it is also capable of running on a Local Area Network if internet access is not available. Figure 2 shows the basic workflow from control console to image viewer. Further details about the software and network architecture developed to implement these features can be found in .Diffused illumination from below results in images that show contours and surface features, this is particularly useful when the sample is opaque. Illumination from above typically works best for samples that are sufficiently translucent and can show internal structures as the light can pass through the sample. The flexibility of using different illumination techniques emulates commercial bright field microscopes. The difference from over and under light is best shown in Supplementary Fig. 1. The 3D printed plate holder supports the biological sample during an experiment. For easy alignment, the holder is attached to a xy sliding stage that consists of two interconnected linear stages . The inner stage translating along the y-axis uses 8 leaf springs to connect a central piece holding the 24-well plate with four rigid elements surrounding it. The outer stage translating along the x-axis uses 8 additional leaf springs to connect the inner stage with the outside 4 rigid elements, two of them being connected to the Picroscope frame using 4 screws . While each stage is flexible along one axis , together they can slide along both, x and y axes. Each stage is actuated by two adjustment screws depicted as gray arrows in Fig. 3f.