Because the initial substrate-attached cells are vertically oriented, the consecutive layers of cells in biofilm growth are also primarily vertical, as shown in Fig. window into biofilm formation that will prove invaluable to understanding the mechanics underlying biofilm development. Bacteria assemble into communities, termed biofilms, which are embedded in a secreted polymer matrix and often coat liquidCair or liquidCsolid interfaces (1C4). Some biofilms are beneficial to human ML604440 health, for example as part of the healthy gut and skin microbiota (5, 6) or in wastewater treatment systems (7). Other biofilms, however, cause serious problems in oral hygiene, chronic infections, and prosthetic contamination (8C10) and act as fouling agents in industrial flow systems (11). In all contexts, biofilms can be difficult to control due to their resilience against chemical and physical stresses (12, 13), including antibiotic treatment (14). Because of their ubiquity and their relevance to medicine and industry, the formation of biofilms has been studied intensively, with an emphasis on the genes, regulatory mechanisms, and transport properties that underlie transitions from planktonic growth to surface attachment (15C17), to proliferation and matrix secretion (2, 18), and finally to dispersal (19, 20). A basic understanding of several regulatory circuits and secreted matrix components governing biofilm formation has been developed (21C25). Nonetheless, the physical, biological, and chemical factors that interact to determine the biofilm architecture remain largely unknown. Internal and global biofilm architectures are presumably consequences of emergent interactions between individual cell growth, physiological differentiation, secreted proteins, polymers and small molecules, and microenvironmental heterogeneity (21, 26C34). Attempts to dissect the individual and combined contributions of these factors to biofilm growth have increasingly relied on examination of bacterial communities in microfluidic devices that mimic central features of natural environments (11, 35, 36). Although sophisticated methods for fabricating biofilm microenvironments are available, a significant barrier to progress has been the lack of techniques capable of resolving all individual cells residing inside biofilms. Thus, the vast majority of studies to date have been limited to visualizing 3D biofilms as connected clouds of biomass, although some studies have used fixed ML604440 samples to obtain cellular resolution (37C40). We therefore know little about the organizational principles that convert individual cell behavior into macroscopic growth and collective properties of biofilms. Here, we develop and use experimental techniques to investigate at single-cell resolution the 3D architecture of biofilms containing thousands of cells. By using a customized spinning-disk confocal microscope that enables 3D imaging at high axial resolution with low-light doses and by combining this instrument with bespoke image analysis software, we were able to visualize and segment all individual cells in thousands of biofilms grown on submerged glass surfaces under flow containing nutrients. From these data, we could construct ensemble averages of biofilm structure during every phase of growth. We discovered that the internal community architecture and global biofilm morphology undergo several distinct transitions, which manifest as changes in the relative arrangements of individual cells over the course of biofilm development. From these data, we identified four fundamental phases of biofilm growth, each characterized by its own unique architecture: 1D growth of 1C6 cells, 2D growth of 20C100 cells, 3D ML604440 growth of 200C1,000 cells with low local order, and highly ordered growth of communities with more than 2,000 cells. ML604440 These phases can be explained by transitions in the physical dimensionality of the particular biofilm combined with changes in local cell density. Of the three known matrix proteins RbmA, Bap1, and RbmC, only deletion of RbmA substantially perturbs cellular orientations and the overarching biofilm architecture. We thus provide, to our knowledge, the first steps toward resolving how the 3D biofilm architecture results from the interactions of the constituent cells. Results and Discussion Fluorescent proteins expressed from the chromosome do not provide a sufficient signal for live-cell imaging at single-cell resolution, even when the fluorescent proteins are expressed at levels just below those that inhibit growth. We therefore grew biofilms for different times in microfluidic flow channels and stained the biofilms in situ with a nucleic acid dye that we added to the medium immediately before imaging. This protocol makes it possible to investigate biofilm architecture ML604440 at different growth stages. Using spinning disk confocal microscopy, we were able to resolve all individual cells inside biofilms. By applying our Matlab-based 3D image analysis software, we could localize and distinguish all cells in biofilms up to 30 m in height, as shown in Fig. 1(see = 4,543 cells is shown in Fig. 1wild-type biofilm at single-cell resolution. CYFIP1 (= 0.6 m, 12.6 m, and 24.6.