Optical Trapping and Fast Discrimination of Label-Free Bacteriophages at the Single Virion Level.

There is a recent resurgence of interest in phage therapy (the therapeutic use of bacterial viruses) as an approach to eliminating difficult-to-treat infections. However, existing approaches for therapeutic phage selection and virulence testing are time-consuming, host-dependent, and facing reproducibility issues. Here, this study presents an innovative approach wherein integrated resonant photonic crystal (PhC) cavities in silicon are used as optical nanotweezers for probing and manipulating single bacteria and single virions with low optical power. This study demonstrates that these nanocavities differentiate between a bacterium and a phage without labeling or specific surface bioreceptors. Furthermore, by tailoring the spatial extent of the resonant optical mode in the low-index medium, phage distinction across phenotypically distinct phage families is demonstrated. The work paves the road to the implementation of optical nanotweezers in phage therapy protocols.

Interaction of pAsa5 and pAsa8 Plasmids in Aeromonas salmonicida subsp. salmonicida.

The plasmid known as pAsa5 is present in Aeromonas salmonicida subsp. salmonicida, a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium's virulence. Recombination events are known to occur in pAsa5, resulting in the loss of certain segments or the acquisition of additional genetic elements. For example, the transposon carried by the large pAsa8 plasmid was found to be inserted into the pAsa5 plasmid in the SHY16-3432 strain, enabling the addition of antibiotic resistance genes to this plasmid, which does not normally possess any. In this study, we present the isolation of additional strains carrying pAsa8. Further analyses of these strains revealed that a fusion between pAsa5 and the complete version of pAsa8 is possible. The pAsa8 transposon insertion in pAsa5 seen in the SHY16-3432 strain appears to be an aberrant event compared to the fusion of the two full-length plasmids. A 22-nucleotide sequence, present in both plasmids, serves as the site for the fusion of the two plasmids. Moreover, it is possible to introduce pAsa8 through conjugation into naive strains of A. salmonicida subsp. salmonicida and once the plasmid is within a new strain, the fusion with pAsa5 is detectable. This study reveals a previously unexplored aspect of pAsa5 plasmid biology, highlighting an additional risk for the spread of antibiotic resistance genes in A. salmonicida subsp. salmonicida.

MQM1, a bacteriophage infecting strains of Aeromonas salmonicida subspecies salmonicida carrying Prophage 3.

Aeromonas salmonicida subsp. salmonicida is a Gam-negative bacterium responsible for furunculosis in fish. Because this aquatic bacterial pathogen has a rich reservoir of antibiotic-resistant genes, it is essential to investigate antibacterial alternatives, including the use of phages. Yet, we have previously demonstrated the inefficiency of a phage cocktail designed against A. salmonicida subsp. salmonicida strains due to a phage resistance phenotype associated to a prophage, namely Prophage 3. To bypass this resistance, one of the solutions is to isolate novel phages capable of infecting Prophage 3-bearing strains. Here we report on the isolation and characterization of the new virulent phage vB_AsaP_MQM1 (or MQM1), which is highly specific to A. salmonicida subsp. salmonicida strains. Phage MQM1 inhibited the growth of 01-B516, a strain carrying Prophage 3, including when combined to the previous phage cocktail. MQM1 infected 26 out of the 30 (87%) Prophage 3-bearing strains tested. Its linear dsDNA genome contains 63,343 bp, with a GC content of 50.2%. MQM1 genome can encode 88 proteins and 8 tRNAs, while no integrase or transposase-encoding genes were found. This podophage has an icosahedral capsid and a non-contractile short tail. We suggest that MQM1 may be a good addition to future phage cocktails against furunculosis to resolve the Prophage 3-resistance issue.

Approaching the Geometric Limit of Bacteriophage Conjugation to Gold: Synergy of Purification with Covalent and Physisorption Strategies.

Bacteriophages represent a remarkably versatile probe for biosensing and a key component of a new class of bioactive surfaces. Chemical immobilization of bacteriophages is a key operation enabling such applications, yet despite this, rarely is a comparison made between immobilization chemistries or for multiple phages with the same parameters. Here, we report the immobilization of bacteriophages 44AHJD, P68, Remus, and gh-1 by physisorption and covalent cross-linking via a series of thiolated reagents: 11-mercaptoundecanoic acid (11-MUA), l-cysteine with 11-MUA, l-cysteine with glutaraldehyde, and dithiobis(succinimidyl propionate). Surprisingly, phage purification protocols showed significant impact on the phage immobilization efficiency. Indeed, purification of phages by density gradient (CsCl) ultracentrifugation and centrifugal ultrafiltration was found to have a dramatic determinant effect on the quality of the immobilized layer. Surface densities of 160 ± 13.9 phage/µm2 were observed when careful phage purification was combined with 11-MUA self-assembled monolayer functionalization of the surface. High-resolution scanning electron microscopy enabled direct confirmation of immobilization, along with calculation of phage densities on the surface, and even resolution of phage capsid substructures.

Deuterium isotope probing (DIP) on Listeria innocua: Optimisation of labelling and impact on viability state.

An innovative approach, Raman microspectroscopy coupled with deuterium isotope probing (Raman-DIP), can be used to evaluate the metabolism of deuterated carbon source in bacteria and also to presume different anabolic pathways. This method requires the treatment of cells with heavy water that could affect the bacterial viability state at higher concentration. In this study, we evaluated the effect of heavy water incorporation on the viability state of Listeria innocua cells. We exposed the L. innocua suspensions to different heavy water concentrations (0%, 25%, 50% and 75%) from 30 minutes to 72 h of incubation times at 37°C. The total, viable and viable culturable populations were quantified by qPCR, PMA-qPCR and plate count agar respectively. We analyzed heavy water incorporation by Raman-DIP. The exposure of L. innocua cells to different concentrations of heavy water did not alter their cell viability to 24 h incubation time. In addition, the maximum intensity for C-D band, specific for the incorporation of heavy water, was reached after 2 h of exposure in a media containing 75% v/v D2O but an early detection of the labelling was possible at t = 1 h 30 min. In conclusion, the use of D2O as a metabolic marker was validated and can be developed for the detection of L. innocua cell viability state.

Host Dependent-Transposon for a Plasmid Found in Aeromonas salmonicida subsp. salmonicida That Bears a catB3 Gene for Chloramphenicol Resistance.

Plasmids that carry antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen with severe consequences in salmonid farming. Here, we describe a 67 kb plasmid found in the A. salmonicida subsp. salmonicida Strain SHY15-2939 from Quebec, Canada. This new plasmid, named pAsa-2939 and identified by high throughput sequencing, displays features never found before in this bacterial species. It contains a transposon related to the Tn21 family, but with an unusual organization. This transposon bears a catB3 gene (chloramphenicol resistance) that has not been detected yet in A. salmonicida subsp. salmonicida. The plasmid is transferable by conjugation into Aeromonas hydrophila, but not into Escherichia coli. Based on PCR analysis and genomic sequencing (Illumina and PacBio), we determined that the transposon is unstable in A. salmonicida subsp. salmonicida Strain SHY15-2939, but it is stable in A. hydrophila trans-conjugants, which explains the chloramphenicol resistance variability observed in SHY15-2939. These results suggest that this bacterium is likely not the most appropriate host for this plasmid. The presence of pAsa-2939 in A. salmonicida subsp. salmonicida also strengthens the reservoir role of this bacterium for antibiotic resistance genes, even those that resist antibiotics not used in aquaculture in Québec, such as chloramphenicol.

Passive limitation of surface contamination by perFluoroDecylTrichloroSilane coatings in the ISS during the MATISS experiments.

Future long-duration human spaceflight will require developments to limit biocontamination of surface habitats. The MATISS (Microbial Aerosol Tethering on Innovative Surfaces in the international Space Station) experiments allowed for exposing surface treatments in the ISS (International Space Station) using a sample-holder developed to this end. Three campaigns of FDTS (perFluoroDecylTrichloroSilane) surface exposures were performed over monthly durations during distinct periods. Tile scanning optical microscopy (×3 and ×30 magnifications) showed a relatively clean environment with a few particles on the surface (0.8 to 7 particles per mm2). The varied densities and shapes in the coarse area fraction (50-1500 µm2) indicated different sources of contamination in the long term, while the bacteriomorph shapes of the fine area fraction (0.5-15 µm2) were consistent with microbial contamination. The surface contamination rates correlate to astronauts' occupancy rates on board. Asymmetric particles density profiles formed throughout time along the air-flow. The higher density values were located near the flow entry for the coarse particles, while the opposite was the case for the fine particles, probably indicating the hydrophobic interaction of particles with the FDTS surface.

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay.

AIMS: To measure the infectious titre (IT) decay rate for various bacteriophages as a function of storage container material. Additionally, parallel light scattering and infectious titre measurements reveal distinct mechanisms for IT loss, depending on phage. METHODS AND RESULTS: Suspensions of bacteriophages 44AHJD, P68 and gh-1 were stored in various labware. IT of each suspension was repeatedly measured over the course of 2 weeks. Large variability in IT decay was observed, with >4 log10 loss in glass and low-binding polypropylene. Incubation of polymer containers with Bovine Serum Albumin (BSA) resulted in a consistent reduction in IT decay. Aggregation state of phage suspensions was studied by nanoparticle tracking analysis (NTA), revealing highest aggregation in glass-stored suspensions and lowest after storage in BSA-treated containers. CONCLUSIONS: Glass and 'low-binding' containers may aggravate IT decay while BSA treatment may present an easy mitigation strategy. IT versus NTA titre diagrams highlight the importance of phage inactivation in combination with aggregation. SIGNIFICANCE AND IMPACT OF THE STUDY: Container material is a significant determinant of bacteriophage IT decay. It is therefore essential to confirm IT following storage and tailor choice of phage storage containers accordingly. Aggregation of phages and adsorption onto labware surfaces are not only the mechanisms accounting for IT loss but also biological instability.

On-Chip Optical Nano-Tweezers for Culture-Less Fast Bacterial Viability Assessment.

Because of antibiotics misuse, the dramatic growth of antibioresistance threatens public health. Tests are indeed culture-based, and require therefore one to two days. This long time-to-result implies the use of large-spectrum antibiotherapies as a first step, in absence of pathogen characterization. Here, a breakthrough approach for a culture-less fast assessment of bacterial response to stress is proposed. It is based on non-destructive on-chip optical tweezing. A laser loads an optical nanobeam cavity whose evanescent part of the resonant field acts as a nano-tweezer for bacteria surrounding the cavity. Once optically trapped, the bacterium-nanobeam cavity interaction induces a shift of the resonance driven by the bacterial cell wall optical index. The analysis of the wavelength shift yields an assessment of viability upon stress at the single-cell scale. As a proof of concept, bacteria are stressed by incursion, before optical trapping, at different temperatures (45, 51, and 70 °C). Optical index changes correlate with the degree of thermal stress allowing to sort viable and dead bacteria. With this disruptive diagnosis method, bacterial viability upon stress is probed much faster (typically less than 4 h) than with conventional culture-based enumeration methods (24 h).

Spatio-temporal based deep learning for rapid detection and identification of bacterial colonies through lens-free microscopy time-lapses.

Detection and identification of pathogenic bacteria isolated from biological samples (blood, urine, sputum, etc.) are crucial steps in accelerated clinical diagnosis. However, accurate and rapid identification remain difficult to achieve due to the challenge of having to analyse complex and large samples. Current solutions (mass spectrometry, automated biochemical testing, etc.) propose a trade-off between time and accuracy, achieving satisfactory results at the expense of time-consuming processes, which can also be intrusive, destructive and costly. Moreover, those techniques tend to require an overnight subculture on solid agar medium delaying bacteria identification by 12-48 hours, thus preventing rapid prescription of appropriate treatment as it hinders antibiotic susceptibility testing. In this study, lens-free imaging is presented as a possible solution to achieve a quick and accurate wide range, non-destructive, label-free pathogenic bacteria detection and identification in real-time using micro colonies (10-500 µm) kinetic growth pattern combined with a two-stage deep learning architecture. Bacterial colonies growth time-lapses were acquired thanks to a live-cell lens-free imaging system and a thin-layer agar media made of 20 µl BHI (Brain Heart Infusion) to train our deep learning networks. Our architecture proposal achieved interesting results on a dataset constituted of seven different pathogenic bacteria-Staphylococcus aureus (S. aureus), Enterococcus faecium (E. faecium), Enterococcus faecalis (E. faecalis), Staphylococcus epidermidis (S. epidermidis), Streptococcus pneumoniae R6 (S. pneumoniae), Streptococcus pyogenes (S. pyogenes), Lactococcus Lactis (L. Lactis). At T = 8h, our detection network reached an average 96.0% detection rate while our classification network precision and sensitivity averaged around 93.1% and 94.0% respectively, both were tested on 1908 colonies. Our classification network even obtained a perfect score for E. faecalis (60 colonies) and very high score for S. epidermidis at 99.7% (647 colonies). Our method achieved those results thanks to a novel technique coupling convolutional and recurrent neural networks together to extract spatio-temporal patterns from unreconstructed lens-free microscopy time-lapses.

Strategies for Surface Immobilization of Whole Bacteriophages: A Review.

Bacteriophage immobilization is a key unit operation in emerging biotechnologies, enabling new possibilities for biodetection of pathogenic microbes at low concentration, production of materials with novel antimicrobial properties, and fundamental research on bacteriophages themselves. Wild type bacteriophages exhibit extreme binding specificity for a single species, and often for a particular subspecies, of bacteria. Since their specificity originates in epitope recognition by capsid proteins, which can be altered by chemical or genetic modification, their binding specificity may also be redirected toward arbitrary substrates and/or a variety of analytes in addition to bacteria. The immobilization of bacteriophages on planar and particulate substrates is thus an area of active and increasing scientific interest. This review assembles the knowledge gained so far in the immobilization of whole phage particles, summarizing the main chemistries, and presenting the current state-of-the-art both for an audience well-versed in bioconjugation methods as well as for those who are new to the field.

Phage susceptibility testing and infectious titer determination through wide-field lensless monitoring of phage plaque growth.

The growing number of drug-resistant bacterial infections worldwide is driving renewed interest in phage therapy. Based on the use of a personalized cocktail composed of highly specific bacterial viruses, this therapy relies on a range of tests on agar media to determine the most active phage on a given bacterial target (phage susceptibility testing), or to isolate new lytic phages from an environmental sample (enrichment of phage banks). However, these culture-based techniques are still solely interpreted through direct visual detection of plaques. The main objective of this work is to investigate computer-assisted methods in order to ease and accelerate diagnosis in phage therapy but also to study phage plaque growth kinetics. For this purpose, we designed a custom wide-field lensless imaging device, which allows continuous monitoring over a very large area sensor (3.3 cm2). Here we report bacterial susceptibility to Staphylococcus aureus phage in 3 hr and estimation of infectious titer in 8 hr 20 min. These are much shorter time-to-results than the 12 to 24 hours traditionally needed, since naked eye observation and counting of phage plaques is still the most widely used technique for susceptibility testing prior to phage therapy. Moreover, the continuous monitoring of the samples enables the study of plaque growth kinetics, which enables a deeper understanding of the interaction between phage and bacteria. Finally, thanks to the 4.3 µm resolution, we detect phage-resistant bacterial microcolonies of Klebsiella pneumoniae inside the boundaries of phage plaques and thus show that our prototype is also a suitable device to track phage resistance. Lensless imaging is therefore an all-in-one method that could easily be implemented in cost-effective and compact devices in phage laboratories to help with phage therapy diagnosis.

Sleep quality and sleep disturbances among volunteer and professional French firefighters: FIRESLEEP study.

BACKGROUND: Although insufficient sleep among firefighters reduces work efficiency and increases the risk of injury, little is known about the sleep quality of French firefighters in the Loire department. The aim of the FIRESLEEP study was to evaluate sleep quality and sleep disturbances among professional and voluntary French firefighters. METHODS: A cross-sectional observational study was conducted between November 2018 and May 2019. Firefighters were invited to complete a questionnaire on a secure platform including clinical questions and validated questionnaires (Pittsburgh Sleep Quality Index [PSQI], Epworth sleepiness scale [ESS], Insomnia Severity Index [ISI] and the STOP-Bang score) during their periodic medical examination. RESULTS: 193 firefighters were included in this study, of which 29% were of professional status and 71% were volunteer firefighters. Among them, 26.9% had poor sleep quality, 27.7% showed excessive daytime sleepiness (EDS), 18.8% reported moderate-to-severe symptoms of insomnia, and 1.6% had moderate-to-high risk of obstructive sleep apnea. Subgroup analysis revealed that professional firefighters had poorer sleep quality and higher sleep disturbances than volunteer firefighters. The independent risk factors associated with poor sleep quality were known sleep disorder, treated anxiety/depression, night calls, and insomnia symptoms. Moreover, the independent risk factors associated with EDS were short sleep duration, taking a nap, and insomnia symptoms; while older age was a protective factor for EDS. CONCLUSIONS: Poor sleep quality and sleep disturbances are highly frequent in French firefighters and underdiagnosed. Prevention through education and systematic screening could limit the impact of sleep disturbances on firefighters' global health.

Towards a passive limitation of particle surface contamination in the Columbus module (ISS) during the MATISS experiment of the Proxima Mission.

Future long-duration human spaceflight calls for developments to limit biocontamination of the surface habitats. The MATISS experiment tests surface treatments in the ISS's atmosphere. Four sample holders were mounted with glass lamella with hydrophobic coatings, and exposed in the Columbus module for ~6 months. About 7800 particles were detected by tile scanning optical microscopy (×3 and ×30 magnification) indicating a relatively clean environment (a few particles per mm2), but leading to a significant coverage-rate (>2% in 20 years). Varied shapes were displayed in the coarse (50-1500 µm2) and fine (0.5-50 µm2) area fractions, consistent with scale dices (tissue or skin) and microbial cells, respectively. The 200-900 µm2 fraction of the coarse particles was systematically higher on FDTS and SiOCH than on Parylene, while the opposite was observed for the <10 µm2 fraction of the fine particles. This trend suggests two biocontamination sources and a surface deposition impacted by hydrophobic coatings.

Systematic Analysis of the Stress-Induced Genomic Instability of Type Three Secretion System in Aeromonas salmonicida subsp. salmonicida.

The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity.

Antibacterial Cellulose Nanopapers via Aminosilane Grafting in Supercritical Carbon Dioxide.

In this work, we present an innovative strategy for the grafting of an antibacterial agent onto nanocellulose materials in supercritical carbon dioxide (scCO2). Dense cellulose nanofibril (CNF) nanopapers were prepared and subsequently functionalized in supercritical carbon dioxide with an aminosilane, N-(6-aminohexyl)aminopropyltrimethoxysilane (AHA-P-TMS). Surface characterization (X-ray photoelectron spectroscopy, contact angle, ζ-potential analysis) evidenced the presence of the aminosilane. The results show that the silane conformation depends on the curing process: a nonpolycondensed conformation of grafted silane with the amino groups facing outwards was favored by curing in an oven, while the curing step performed in scCO2 yielded CNF structures with the alkyl chain facing outwards. The grafted nanopapers exhibited antibacterial activity, and no antibacterial agent was released into the media. Furthermore, these materials proved to benefit from low cytotoxicity. This study offers a proof of concept for the covalent grafting of active species on nanocellulose structures and the control of aminosilane orientation using a green and controlled approach. These newly designed materials could be used for their antibacterial activity in the biomedical field. Thus, perspectives for topical administration and design of wound dressing could be envisaged.

Antimicrobial Cellulose Nanofibril Porous Materials Obtained by Supercritical Impregnation of Thymol.

This study presents the impregnation in supercritical carbon dioxide (scCO2) of nanocellulose-based structures with thymol as a natural antimicrobial molecule to prepare bioactive, biosourced materials. First, cellulose nanofibrils (CNFs) were used to produce four types of materials (nanopapers, cryogels from water or tert-butyl alcohol suspensions, and aerogels) of increasing specific surface area up to 160 m2·g-1, thanks to the use of different processes, namely, vacuum filtration, freeze-drying, and supercritical drying. Second, these CNF-based structures were impregnated with thymol in the scCO2 medium using a relatively low temperature and pressure of 40 °C and 100 bar during 1 h. The amount of impregnated thymol in the different CNF materials was investigated by fluorescence spectroscopy, 13C NMR analysis, and gas chromatography. All three methods consistently showed that the amount of impregnated thymol increases with the specific surface area of the material. The antimicrobial activity of the impregnated CNF-based materials was then measured against three reference strains of microorganisms: the Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis bacteria, and the yeast Candida albicans using the disk diffusion test method. The latter revealed the leaching of thymol in sufficient amounts to generate antimicrobial activity against the three strains in the case of the cryogel derived from a tert-butyl alcohol suspension and the aerogel, which are the two materials exhibiting the highest specific surface areas. The proposed strategy, therefore, enabled us to precisely steer the amount of active molecule loading and the related antimicrobial activity by adjusting the specific surface area of the biosourced material impregnated in green supercritical conditions. These results are very promising and confirm that supercritical impregnation of active molecules onto nanocellulose three-dimensional (3D) structures can be an interesting solution for the design of active medical devices such as wound dressings.

Optical forward-scattering for identification of bacteria within microcolonies.

The development of methods for the rapid identification of pathogenic bacteria is a major step towards accelerated clinical diagnosis of infectious diseases and efficient food and water safety control. Methods for identification of bacterial colonies on gelified nutrient broth have the potential to bring an attractive solution, combining simple optical instrumentation, no need for sample preparation or labelling, in a non-destructive process. Here, we studied the possibility of discriminating different bacterial species at a very early stage of growth (6 h of incubation at 37 °C), on thin layers of agar media (1 mm of Tryptic Soy Agar), using light forward-scattering and learning algorithms (Bayes Network, Continuous Naive Bayes, Sequential Minimal Optimisation). A first database of more than 1,000 scatterograms acquired on 7 gram-negative strains yielded a recognition rate of nearly 80%, after only 6 h of incubation. We investigated also the prospect of identifying different strains from a same species through forward scattering. We discriminated, thus, four strains of Escherichia coli with a recognition rate reaching 82%. Finally, we show the discrimination of two species of coagulase-negative Staphylococci (S. haemolyticus and S. cohnii), on a commercial selective pre-poured medium used in clinical diagnosis (ChromID MRSA, bioMérieux), without opening lids during the scatterogram acquisition. This shows the potential of this method--non-invasive, preventing cross-contaminations and requiring minimal dish handling--to provide early clinically-relevant information in the context of fully automated microbiology labs.

Facile and fast detection of bacteria via the detection of exogenous volatile metabolites released by enzymatic hydrolysis.

A low-cost, innovative and non-invasive colorimetric test, which can be universally used, is proposed to detect pathogenic bacteria via the simple and fast detection of volatile metabolites released by enzymatic hydrolysis. The proof of concept is shown via three sets of experiments studying the release of the p-nitrophenol metabolite in solution in the E. coli cultures containing 4-nitrophenyl-ß-d-glucuronide, the trapping efficiency of the gaseous metabolite by various tailored and functionalized xerogels, and the trapping and detection of gaseous p-nitrophenol released by E. coli bacteria.

Label-free analysis of prostate acini-like 3D structures by lensfree imaging.

We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.