Article

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

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Abstract

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.

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... Endothelial cells cultured within a chamber capable of applying physiological shear stresses are induced to differentiate due to stimulation of specific integrin/endothelial cell-mediated signalling cascades [2]. Also, epithelial cells cultured on soft extracellular matrix gels organize themselves into polarized structures that strongly resemble functional tissue in vivo [3,4]. For in vitro studies of vascular tissue, lab-on-chip (LOC) systems [5] that incorporate a unique 3D and dynamic microenvironment with high spatiotemporal precision provide a physiologically relevant way to reproduce vascular tissues. ...
... For biological assays, the inside of the PDMS microchannels was coated with a thin film of either polyelectrolytes or Matrigel (BD Biosciences, San Jose, CA, Ref 356231), which is widely used for 3D culture of epithelial cells due to its ability to promote specific cellular organization [3] and in case of endothelial cells for sprouting experiments. The thin film of polyectrolytes was achieved using Layer-by-Layer (LbL) assembly of the polyelectrolytes PSS (poly (sodium 4-styrenesulphonate; Sigma 243051)) and PAH (poly (allylamine hydrochloride; Sigma 283223)). ...
... Acinar morphogenesis assay was performed according to the previously published top-coat protocol [3]. Matrigel was thawed overnight and poured into either 4-well (160 mL of Matrigel, 500 mL of culture medium) or 8-well Labtek (90 mL of Matrigel, 250 mL of culture medium) plates on ice. ...
Article
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We present a simple bench-top method to fabricate enclosed circular channels for biological experiments. Fabricating the channels takes less than 2 hours by using glass capillaries of various diameters (from 100 µm up to 400 µm) as a mould in PDMS. The inner surface of microchannels prepared in this way was coated with a thin membrane of either Matrigel or a layer-by-layer polyelectrolyte to control cellular adhesion. The microchannels were then used as scaffolds for 3D-confined epithelial cell culture. To show that our device can be used with several epithelial cell types from exocrine glandular tissues, we performed our biological studies on adherent epithelial prostate cells (non-malignant RWPE-1 and invasive PC3) and also on breast (non-malignant MCF10A) cells We observed that in static conditions cells adhere and proliferate to form a confluent layer in channels of 150 µm in diameter and larger, whereas cellular viability decreases with decreasing diameter of the channel. Matrigel and PSS (poly (sodium 4-styrenesulphonate)) promote cell adhesion, whereas the cell proliferation rate was reduced on the PAH (poly (allylamine hydrochloride))-terminated surface. Moreover infusing channels with a continuous flow did not induce any cellular detachment. Our system is designed to simply grow cells in a microchannel structure and could be easily fabricated in any biological laboratory. It offers opportunities to grow epithelial cells that support the formation of a light. This system could be eventually used, for example, to collect cellular secretions, or study cell responses to graduated hypoxia conditions, to chemicals (drugs, siRNA, …) and/or physiological shear stress.
... . 6 Effect of ActinomycinD on cell proliferation -EdU Proliferation assay Imaging is an inherent part of biology. Microscope allows researchers to peer through cells and sub-cellular structures. ...
... describing a rather flat gray value distribution. The distribution is peaked, due to the sharp change in the zero-order gray value during the initial attachment of the cell to the substrate: [3][4][5][6] fold increase in kurtosis is seen, with values going up to 40 a.u. As the cell spreads, the zero-order gray value decreases resulting in a flat distribution. ...
... On the complete contrary, average percentage population of dividing cells in the presence of the ActD (5μg/ml and 10μg/ml) was close to zero (Table 3B. 6). Hence similar to the results obtained by lensfree video proliferation assay, no difference could be observed between the two drug concentrations. ...
Article
Biological studies always start from curious observations. This is exemplified by description of cells for the first time by Robert Hooke in 1665, observed using his microscope. Since then the field of microscopy and cell biology grew hand in hand, with one field pushing the growth of the other and vice-versa. From basic description of cells in 1665, with parallel advancements in microscopy, we have travelled a long way to understand sub-cellular processes and molecular mechanisms. With each day, our understanding of cells increases and several questions are being posed and answered. Several high-resolution microscopic techniques are being introduced (PALM, STED, STORM, etc.) that push the resolution limit to few tens of nm, taking us to a new era where ‘seeing is believing'. Having said this, it is to be noted that the world of cells is vast, with information spread from nanometers to millimetres, and also over extended time-period, implying that not just one microscopic technique could acquire all the available information. The knowledge in the field of cell biology comes from a combination of imaging and quantifying techniques that complement one another.Majority of modern-day microscopic techniques focuses on increasing resolution which, is achieved at the expense of cost, compactness, simplicity, and field of view. The substantial decrease in the field of observation limits the visibility to a few single cells at best. Therefore, despite our ability to peer through the cells using increasingly powerful optical instruments, fundamental biology questions remain unanswered at mesoscopic scales. A global view of cell population with significant statistics both in terms of space and time is necessary to understand the dynamics of cell biology, taking in to account the heterogeneity of the population and the cell-cell variability. Mesoscopic information is as important as microscopic information. Although the latter gains access to sub-cellular functions, it is the former that leads to high-throughput, label-free measurements. By focussing on simplicity, cost, feasibility, field of view, and time-lapse in-incubator imaging, we developed ‘Lensfree Video Microscope' based on digital in-line holography that is capable of providing a new perspective to cell culture monitoring by being able to capture the kinetics of thousands of cells simultaneously. In this thesis, we present our lensfree video microscope and its applications in in-vitro cell culture monitoring and quantification.We validated the system by performing more than 20,000 hours of real-time imaging, in diverse conditions (e.g.: 37°C, 4°C, 0% O2, etc.) observing varied cell types and culture conditions (e.g.: primary cells, human stem cells, fibroblasts, endothelial cells, epithelial cells, 2D/3D cell culture, etc.). This permitted us to develop label-free cell based assays to study the major cellular events – cell adhesion and spreading, cell division, cell division orientation, cell migration, cell differentiation, network formation, and cell death. The results that we obtained respect the heterogeneity of the population, cell to cell variability (a raising concern in the biological community) and the massiveness of the population, whilst adhering to the standard cell culture practices - a rare combination that is seldom attained by existing real-time monitoring methods.We believe that our microscope and associated metrics would complement existing techniques by bridging the gap between mesoscopic and microscopic information.
... In addition, within the framework of in-line holography, the biological samples were preferably illuminated with a coherent or quasi-coherent incident light in most previous studies in order to generate non-blurred interference patterns 23,26,31,32,38,39,[41][42][43][44][45] . A monochromatic, spatially coherent light has been achieved by a laser beam focused through a pinhole by a microscope objective 31,32 . ...
... A monochromatic, spatially coherent light has been achieved by a laser beam focused through a pinhole by a microscope objective 31,32 . A quasi-coherent incident light is typically produced by a point source made by a pinhole 26,32,39,41,44,45 or an optical fibre 23,42,43 , back-illuminated by a colour LED with a low bandwidth (e.g., < 20 nm). Fewer works have explored the ability to capture images of non-labelled biological samples using partially non-coherent incident light 37,40 . ...
... In previous studies 23,26,31,32,[37][38][39][40][41][42][43][44][45] , contact imaging of cells was produced using coherent, quasi-coherent or partially non-coherent illumination. This work demonstrates that brightfield contact imaging of cell cultures can be successfully obtained even using incoherent incident light produced by a white, directional LED with no optical filter, spatial filter nor holographic reconstruction. ...
Article
Full-text available
Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.
... The resolution abilities, FOV and the imaging speed are similar to each other. For example, Cedric Allier et al. provide a method based on holographic lens-less video microscopy to measure important metrics for cell proliferation studies, e.g., cell cycle-duration and cell dry mass [35]. The microscopy imaging FOV is~29.4 ...
Article
Full-text available
In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.
... In the last two decades, on-chip microscopy based on computational imaging has received much attention due to its clear advantages as a low-cost biomedical research and diagnostic tool over conventional optical microscopy by providing high resolution and a large field of view (FOV) simultaneously [1]. Among the different computational microscopy implementations [2], lensless microscopy has been extensively used because of its versatility and flexibility to implement different techniques, from shadow imaging to fluorescence [3][4][5][6][7], while keeping the microscope implementation simpler. Lensless microscopy relies on the traditional microscopy principle, where the analyzed sample area is illuminated homogeneously by a single light source, and the scattered light from each point is collected by an area-selective detector providing the spatial resolution, commonly a high-resolution image sensor. ...
Article
Full-text available
The recent advances in chip-size microscopy based on optical scanning with spatially resolved nano-illumination light sources are presented. This new straightforward technique takes advantage of the currently achieved miniaturization of LEDs in fully addressable arrays. These nano-LEDs are used to scan the sample with a resolution comparable to the LED sizes, giving rise to chip-sized scanning optical microscopes without mechanical parts or optical accessories. The operation principle and the potential of this new kind of microscope are analyzed through three different implementations of decreasing LED dimensions from 20 µm down to 200 nm.
... In 3-dimensional Matrigel culture, RWPE-1 cells organize into acini, i.e., 3D clusters of cells that resemble to many-lobed berries. In previous work we have shown that the obtained holographic signature of RWPE-1 aggregates allows discriminating acini with lumen from spheroids which are tumoral like [24]. Spatial organization of RWPE-1 cells in a 3D volume was studied over time. ...
Conference Paper
Full-text available
Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.
... On the other hand, commercial CMOS imagers reduce the design time and provide good performance if integration is not one of the main priorities. [35][36][37][38][39][40] present CMOS imagers used in fluorescence and luminescence biodetection systems. Depending of the particular application, a certain characteristic is emphasized, such as dynamic range and color detection in reference [36], a high speed acquisition in reference [37], integration with microfluidic channels in reference [38], and the use of pnp phototransistors as photosensing element to generate larger photocurrents than photodiodes in [39,40]. ...
Article
This dissertation presents the design and implementation of a CMOS imager for use in integrated biosensors based on Surface Plasmon Resonance. First, the optimal conditions for plasmon resonance in a CMOS/Post-CMOS compatible interface are obtained by COMSOL modelling. Second, a 32x32-pixel CMOS-Active Column Sensor (CMOS-ACS) is implemented on 0.35 um CMOS technology. In a gold-water interface with prism excitation, it is found that for prisms showing refractive indexes of 1.55 and 1.46, optimal plasmon coupling is obtained for gold films with thicknesses of 50 and 45 nm respectively. Under these conditions, approximately 99.19% and 99.99% of the incident light's energy is transferred to the surface plasmon for both prism respectively, provided that the incident light, with a wavelength of 633 nm, arrives with incidence angles of 68.45° and 79.05° respectively. It is also obtained that a change of 10-4 RIU in the refractive index of the dielectric medium, produces a change of 0.01° in the plasmon resonance angle, which under a light intensity modulation scheme produces a change of 0.08% in the reflected light's energy reaching the photodetector. Concerning the CMOS imager, a n-well/p-substrate photodiode is selected as the photosensing element, due to its low junction capacitance, which results in high efficiency and high conversion gain compared to the n-diff/p-substrate photodiode. Computer simulations with Cadence and Silvaco produced a junction capacitance of 31 fF and 135 fF respectively. The imager's pixel is based on a three-transistor (3T) configuration and shows a fill factor of 61%. The readout circuitry employs an Active Column Sensor (ACS) technique to reduce the Fixed Pattern Noise (FPN) associated with traditional Active Pixel Sensors (APS). Additionally, Non-Correlated Double Sampling (NCDS) and Delta Double Sampling (DDS) are used as noise reduction techniques. An experimental optical setup is used to characterize the performance of the imager, obtaining a conversion gain of 7.3 uV/e-, a photodiode junction capacitance of 21.9 fF, a read noise of 324.5 uV, equivalent to ~45 e- and a dynamic range of 50.5 dB. The benefits of ACS and NCDS-DDS are observed in the low pixel and column FPN of 0.09% and 0.06% respectively. The work presented in this thesis is a first step towards the goal of developing a fully integrated SPR-biosensing platform incorporating light source, SPR interface, microfluidic channel, optical elements and CMOS imager.
... On the other hand, commercial CMOS imagers reduce the design time and provide good performance if integration is not one of the main priorities. [35][36][37][38][39][40] present CMOS imagers used in fluorescence and luminescence biodetection systems. Depending of the particular application, a certain characteristic is emphasized, such as dynamic range and color detection in reference [36], a high speed acquisition in reference [37], integration with microfluidic channels in reference [38], and the use of pnp phototransistors as photosensing element to generate larger photocurrents than photodiodes in [39,40]. ...
... Une fois de plus, l'imagerie sans lentille se révèle très adaptée pour passer ces difficultés. Nous avons mis en place une méthodologie innovante couplant la technique d'imagerie sans lentilles à la culture cellulaire 3D [10] (cf. figure 5). ...
... A global view of cell population with significant statistics both in terms of cell numbers, space and time is necessary to understand the dynamics of cell biology, taking in to account the heterogeneity of the population and the cellcell variability. By focusing on simplicity, cost, feasibility, field of view, and time-lapse in-incubator imaging, we developed a lensfree video microscope based on digital inline holography [11][12][13]. In a typical experiment, the lensfree video microscope is placed inside the cell incubator and the culture dish containing the cells is placed on top of the lensfree video microscope (Fig. 3a). ...
Conference Paper
Full-text available
Lensfree imaging is an emerging microscopy technique based on in-line holography as invented by Gabor in 1948. Albeit the existence of the method since decades, the recent development of digital sensors, helped the realization of its full potential. Over the recent years, the performance have tremendously increased while keeping the design simple, robust, and at a reasonable low cost. The detection ability improved from 10 µm (cell) in 2009, to 1 µm (bacteria) in 2010 , down to 100 nm beads in 2012, paving the way to the detection of viruses in 2013.
... Structured substrates have also been used in some studies to capture specific types of cells in a regular array [126,127]. In total, the list of different types of cells that have been imaged with shadow based on-chip imaging is quite extensive, including sperm cells, HeLa cells, MDCK cells, leukocytes, cardiomyocytes, human alveolar epithelial cells, human mesenchymal stem cells, Pseudomonas aeruginosa, Schizosaccharomyces pombe, A549 cells, NIH 3T3 cells, HepG2 cells, MCF-7 cells, RWPE1 prostate epithelial cells, and bioluminescent Escherichia coli [120,[123][124][125][128][129][130][131][132][133][134][135][136][137][138][139]. ...
Article
In the past two decades or so, there has been a renaissance of optical microscopy research and development. Much work has been done in an effort to improve the resolution and sensitivity of microscopes, while at the same time to introduce new imaging modalities, and make existing imaging systems more efficient and more accessible. In this review, we look at two particular aspects of this renaissance: computational imaging techniques and compact imaging platforms. In many cases, these aspects go hand-in-hand because the use of computational techniques can simplify the demands placed on optical hardware in obtaining a desired imaging performance. In the first main section, we cover lens-based computational imaging, in particular, light-field microscopy, structured illumination, synthetic aperture, Fourier ptychography, and compressive imaging. In the second main section, we review lensfree holographic on-chip imaging, including how images are reconstructed, phase recovery techniques, and integration with smart substrates for more advanced imaging tasks. In the third main section we describe how these and other microscopy modalities have been implemented in compact and field-portable devices, often based around smartphones. Finally, we conclude with some comments about opportunities and demand for better results, and where we believe the field is heading.
... This approach was initially used to perform blood cell counts to differentiate among red blood cells, fibroblasts, murine embryonic stem cells, hepatocytes, and polystyrene beads of similar sizes (36,39). Many other types of cells have since been imaged using lensless shadow imaging; these include leukocytes, HeLa cells, sperm cells, A549 cells, RWPE-1 prostate epithelial cells, Madin-Darby canine kidney cells, cardiomyocytes, human alveolar epithelial cells, human mesenchymal stem cells, Pseudomonas aeruginosa, Schizosaccharomyces pombe, NIH 3T3 cells, MCF-7 cells, bioluminescent Escherichia coli, and HepG2 cells (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53). The lensless imaging approach can monitor cell division, motility, and viability, among other properties (47,50,54), and can operate within standard cell culture incubators (Figure 3). ...
Article
High-resolution optical microscopy has traditionally relied on high-magnification and high-numerical aperture objective lenses. In contrast, lensless microscopy can provide high-resolution images without the use of any focusing lenses, offering the advantages of a large field of view, high resolution, cost-effectiveness, portability, and depth-resolved three-dimensional (3D) imaging. Here we review various approaches to lensless imaging, as well as its applications in biosensing, diagnostics, and cytometry. These approaches include shadow imaging, fluorescence, holography, superresolution 3D imaging, iterative phase recovery, and color imaging. These approaches share a reliance on computational techniques, which are typically necessary to reconstruct meaningful images from the raw data captured by digital image sensors. When these approaches are combined with physical innovations in sample preparation and fabrication, lensless imaging can be used to image and sense cells, viruses, nanoparticles, and biomolecules. We conclude by discussing several ways in which lensless imaging and sensing might develop in the near future.
... Therefore, the new challenging task is to extend lens-free microscopy techniques to the acquisitions and fully 3D reconstructions of large organoids structures. [4][5][6] The adaptation of lensless microscopy techniques to 3D organoid cultures imaging is the scope of the present paper. ...
... Unlike conventional 2D cell culture systems, 3D cultures allow to study biological processes closer to physiological reality such as tissue morphogenesis or tumour initiation [5][6][7]. Along with the standardisation of 3D culture protocols in cell biology, lens-free imaging techniques must also be extended to this new modality both in terms of acquisition methods and 3D reconstruction algorithms for large organoid structures [8][9][10][11]. ...
Article
Full-text available
We propose a 3D imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrices. Lens-free microscopy acquisitions present some inherent issues such as the lack of phase information on the sensor plane and a limited angular coverage. We developed and compared three different algorithms based on the Fourier diffraction theorem to obtain fully 3D reconstructions. These algorithms present an increasing complexity associated with a better reconstruction quality. Two of them are based on a regularised inverse problem approach. To compare the reconstruction methods in terms of artefacts reduction, signal-to-noise ratio and computation time, we tested them on two experimental datasets: an endothelial cell culture and a prostate cell culture grown in a 3D extracellular matrix with large reconstructed volumes up to 5 mm3 with a resolution sufficient to resolve isolated single cells. The lens-free reconstructions compare well with standard microscopy.
... It allows the observation of living cells, does not require any label, is time-efficient and enable HT analysis of hundreds of fixed or living organoids observed at the same time in a very large field of view (25 or 44 mm 2 ). We are hence able to discriminate control organoids from tumor-like spheroids upon an optical finger-print [5] (Fig. 3). Moreover, lensfree videomicroscopy combined with holographic reconstruction allowed us to monitor cell trafficking in between human 3D structures in real time, providing ways to observe the dynamics of organ development and carcinogenesis (Fig. 3). ...
... 22 Also, epithelial cells cultured on soft extracellular matrix gels organize themselves into polarized structures that strongly resemble functional tissue in vivo. 23,24 Lab-on-chip (LOC) systems, incorporating a unique 3D microenvironment with a high spatiotemporal precision, provide physiologically relevant models to study vascular tissues in vitro. 25 Microfluidic systems have already enabled various biological studies including protein crystallization, 26 collection of cellular secretions, 27 blood circulation, 28 angiogenesis, 29 and cellular cocultures. ...
Article
Full-text available
The prostate is a walnut-sized gland that surrounds the urethra of males at the base of the bladder comprising a muscular portion, which controls the release of urine, and a glandular portion, which secretes fluids that nourish and protect sperms. Here, we report the development of a microfluidic-based model of a human prostate gland. The polydimethylsiloxane (PDMS) microfluidic device, consisting of two stacked microchannels separated by a polyester porous membrane, enables long-term in vitro cocultivation of human epithelial and stromal cells. The porous separation membrane provides an anchoring scaffold for long-term culturing of the two cell types on its opposite surfaces allowing paracrine signaling but not cell crossing between the two channels. The microfluidic device is transparent enabling high resolution bright-field and fluorescence imaging. Within this coculture model of a human epithelium/stroma interface, we simulated the functional development of the in vivo human prostate gland. We observed the successful differentiation of basal epithelial cells into luminal secretory cells determined biochemically by immunostaining with known differentiation biomarkers, particularly androgen receptor expression. We also observed morphological changes where glandlike mounds appeared with relatively empty centers reminiscent of prostatic glandular acini structures. This prostate-on-a-chip will facilitate the direct evaluation of paracrine and endocrine cross talk between these two cell types as well as studies associated with normal vs disease-related events such as prostate cancer.
... On the other hand, commercial CMOS imagers reduce the design time and provide good performance if integration is not one of the main priorities. [35][36][37][38][39][40] present CMOS imagers used in fluorescence and luminescence biodetection systems. Depending of the particular application, a certain characteristic is emphasized, such as dynamic range and color detection in reference [36], a high speed acquisition in reference [37], integration with microfluidic channels in reference [38], and the use of pnp phototransistors as photosensing element to generate larger photocurrents than photodiodes in [39,40]. ...
Thesis
This dissertation presents the design and implementation of a CMOS imager for use in integrated biosensors based on Surface Plasmon Resonance. First, the optimal conditions for plasmon resonance in a CMOS/Post-CMOS compatible interface are obtained by COMSOL modelling. Second, a 32x32-pixel CMOS-Active Column Sensor (CMOS-ACS) is implemented on 0.35 um CMOS technology. In a gold-water interface with prism excitation, it is found that for prisms showing refractive indexes of 1.55 and 1.46, optimal plasmon coupling is obtained for gold films with thicknesses of 50 and 45 nm respectively. Under these conditions, approximately 99.19% and 99.99% of the incident light's energy is transferred to the surface plasmon for both prism respectively, provided that the incident light, with a wavelength of 633 nm, arrives with incidence angles of 68.45° and 79.05° respectively. It is also obtained that a change of 10-4 RIU in the refractive index of the dielectric medium, produces a change of 0.01° in the plasmon resonance angle, which under a light intensity modulation scheme produces a change of 0.08% in the reflected light's energy reaching the photodetector. Concerning the CMOS imager, a n-well/p-substrate photodiode is selected as the photosensing element, due to its low junction capacitance, which results in high efficiency and high conversion gain compared to the n-diff/p-substrate photodiode. Computer simulations with Cadence and Silvaco produced a junction capacitance of 31 fF and 135 fF respectively. The imager's pixel is based on a three-transistor (3T) configuration and shows a fill factor of 61%. The readout circuitry employs an Active Column Sensor (ACS) technique to reduce the Fixed Pattern Noise (FPN) associated with traditional Active Pixel Sensors (APS). Additionally, Non-Correlated Double Sampling (NCDS) and Delta Double Sampling (DDS) are used as noise reduction techniques. An experimental optical setup is used to characterize the performance of the imager, obtaining a conversion gain of 7.3 uV/e-, a photodiode junction capacitance of 21.9 fF, a read noise of 324.5 uV, equivalent to ~45 e- and a dynamic range of 50.5 dB. The benefits of ACS and NCDS-DDS are observed in the low pixel and column FPN of 0.09% and 0.06% respectively. The work presented in this thesis is a first step towards the goal of developing a fully integrated SPR-biosensing platform incorporating light source, SPR interface, microfluidic channel, optical elements and CMOS imager.
Conference Paper
Lensfree imaging is an emerging microscopy technique based on in-line holography as invented by Gabor in 1948. Albeit the existence of the method since decades, the recent development of digital sensors, helped the realization of its full potential. Over the recent years, innovations and improvements in CMOS imaging technology design and fabrication have allowed to decrease the pixel pitch down to ∼1μm and the number of pixels has dramatically increased up to 250 million of pixels. As a result, the performance of lensfree microscopy, which features a bare CMOS sensor without any magnification optics, have tremendously increased while keeping the design simple, robust, and at a reasonable low cost. The detection ability improved from 10 μm (cell) in 2009, to 1 μm (bacteria) in 2010, down to 100 nm beads in 2012, paving the way to the detection of viruses in 2013.
Article
Full-text available
New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup.
Article
Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D-a cell proliferation inhibitor.
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We discuss unique features of lens-free computational imaging tools and report some of their emerging results for wide-field on-chip microscopy, such as the achievement of a numerical aperture (NA) of ∼0.8-0.9 across a field of view (FOV) of more than 20 mm(2) or an NA of ∼0.1 across a FOV of ∼18 cm(2), which corresponds to an image with more than 1.5 gigapixels. We also discuss the current challenges that these computational on-chip microscopes face, shedding light on their future directions and applications.
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Background Conventional diagnosis and identification of bacteria requires shipment of samples to a laboratory for genetic and biochemical analysis. This process can take days and imposes significant delay to action in situations where timely intervention can save lives and reduce associated costs. To enable faster response to an outbreak, a low-cost, small-footprint, portable microbial-identification instrument using forward scatterometry has been developed. Results This device, weighing 9 lb and measuring 12 × 6 × 10.5 in., utilizes elastic light scatter (ELS) patterns to accurately capture bacterial colony characteristics and delivers the classification results via wireless access. The overall system consists of two CCD cameras, one rotational and one translational stage, and a 635-nm laser diode. Various software algorithms such as Hough transform, 2-D geometric moments, and the traveling salesman problem (TSP) have been implemented to provide colony count and circularity, centering process, and minimized travel time among colonies. Conclusions Experiments were conducted with four bacteria genera using pure and mixed plate and as proof of principle a field test was conducted in four different locations where the average classification rate ranged between 95 and 100%.
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We report a chip-scale lensless wide-field-of-view microscopy imaging technique, subpixel perspective sweeping microscopy, which can render microscopy images of growing or confluent cell cultures autonomously. We demonstrate that this technology can be used to build smart Petri dish platforms, termed ePetri, for cell culture experiments. This technique leverages the recent broad and cheap availability of high performance image sensor chips to provide a low-cost and automated microscopy solution. Unlike the two major classes of lensless microscopy methods, optofluidic microscopy and digital in-line holography microscopy, this new approach is fully capable of working with cell cultures or any samples in which cells may be contiguously connected. With our prototype, we demonstrate the ability to image samples of area 6 mm × 4 mm at 660-nm resolution. As a further demonstration, we showed that the method can be applied to image color stained cell culture sample and to image and track cell culture growth directly within an incubator. Finally, we showed that this method can track embryonic stem cell differentiations over the entire sensor surface. Smart Petri dish based on this technology can significantly streamline and improve cell culture experiments by cutting down on human labor and contamination risks.
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We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 µm spatial resolution over a large imaging area of ~24 mm(2). Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 µm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings.
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Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2).
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Lensless on-chip imaging is a promising technique to count and monitor cells and micro-objects in liquid sample. In this paper we apply this technique to the observation of µL sample containing bacteria evaporated onto a microscope slide. Compared with previously reported techniques, a large improvement in signal to noise ratio is obtained due to the presence of a few μm thick wetting film creating a micro-lens on top of each bacteria. In these conditions, standard CMOS sensor are able to detect micro-objects as small as few μm, e.g. E.coli and Bacillus subtilis bacteria and 1 μm polymer beads with a large signal to noise ratio of 45 ± 10. An overall detection efficiency of 85 ± 7% and a co-localization error of σ(1D) = 1.1μm compared with reference fluorescence microscopy images are achieved. This novel technique will be used as a pre-positioning tool prior to other optical identification methods, e.g. Raman spectroscopy.
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Iterative algorithms for phase retrieval from intensity data are compared to gradient search methods. Both the problem of phase retrieval from two intensity measurements (in electron microscopy or wave front sensing) and the problem of phase retrieval from a single intensity measurement plus a non-negativity constraint (in astronomy) are considered, with emphasis on the latter. It is shown that both the error-reduction algorithm for the problem of a single intensity measurement and the Gerchberg-Saxton algorithm for the problem of two intensity measurements converge. The error-reduction algorithm is also shown to be closely related to the steepest-descent method. Other algorithms, including the input-output algorithm and the conjugate-gradient method, are shown to converge in practice much faster than the error-reduction algorithm. Examples are shown.
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There are two predominant theories for lumen formation in tissue morphogenesis: cavitation driven by cell death, and membrane separation driven by epithelial polarity. To define the mechanism of lumen formation in prostate acini, we examined both theories in several cell lines grown in three-dimensional (3D) Matrigel culture. Lumen formation occurred early in culture and preceded the expression of cell death markers for apoptosis (active caspase 3) and autophagy (LC-3). Active caspase 3 was expressed by very few cells and inhibition of apoptosis did not suppress lumen formation. Despite LC-3 expression in all cells within a spheroid, this was not associated with cell death. However, expression of a prostate-secretory protein coincided with lumen formation and subsequent disruption of polarized fluid movement led to significant inhibition of lumen formation. This work indicates that lumen formation is driven by the polarized movement of fluids and proteins in 3D prostate epithelial models and not by cavitation.
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The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.
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Little is known about how the genotypic and molecular abnormalities associated with epithelial cancers actually contribute to the histological phenotypes observed in tumours in vivo. 3D epithelial culture systems are a valuable tool for modelling cancer genes and pathways in a structurally appropriate context. Here, we review the important features of epithelial structures grown in 3D basement membrane cultures, and how such models have been used to investigate the mechanisms associated with tumour initiation and progression.
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Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.
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The mammary gland undergoes a morphogenetic program during embryogenesis and puberty that leads to the development of hollow ductal system terminating in acinar units. It later expands to generate an elaborate network to deliver milk to newborn progeny. Previous studies in our laboratory using three-dimensional basement membrane cultures of mammary epithelial cells, in which acini-like structures form from single cells, have indicated that lumen formation requires clearance of the cells in the center of the acini by apoptosis. This apoptotic program in vitro requires the pro-death mediator BIM. Recently we found that BIM is also required in the mouse mammary gland for apoptosis during lumen formation, which correlates the 3D acinar model to mammary morphogenesis in vivo. Herein we put into perspective the relevance of our in vitro and in vivo findings to discuss luminal space formation and maintenance during mammary morphogenesis.
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In this contribution a real-time imaging system for cell cultures, adherently grown in standard disposable multi-well plates is presented. The compact system is designed to be operated in a lab incubator. The imaging system consists of a custom made platform in which a CCD image sensor is aligned and fixed directly at the bottom of the well of interest; a LED light source is positioned above the cell sample. The image sensor has a resolution of 640 × 480 pixels. A mini lens mounted on top of the CCD sensor allows close-up focusing. The field of view on the focal plane measures 3.26 mm × 2.45 mm, which corresponds with an overall optical resolution of 5.1 μm × 5.1 μm. The system has been validated by visualizing spherical beads of known size. With the presented imaging system we have successfully monitored collective (wound healing assay) and individual cell migration, as well as epithelial cell proliferation.
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Dynamic tracking of human sperms across a large volume is a challenging task. To provide a high-throughput solution to this important need, here we describe a lensfree on-chip imaging technique that can track the three-dimensional (3D) trajectories of > 1,500 individual human sperms within an observation volume of approximately 8-17 mm(3). This computational imaging platform relies on holographic lensfree shadows of sperms that are simultaneously acquired at two different wavelengths, emanating from two partially-coherent sources that are placed at 45° with respect to each other. This multiangle and multicolor illumination scheme permits us to dynamically track the 3D motion of human sperms across a field-of-view of > 17 mm(2) and depth-of-field of approximately 0.5-1 mm with submicron positioning accuracy. The large statistics provided by this lensfree imaging platform revealed that only approximately 4-5% of the motile human sperms swim along well-defined helices and that this percentage can be significantly suppressed under seminal plasma. Furthermore, among these observed helical human sperms, a significant majority (approximately 90%) preferred right-handed helices over left-handed ones, with a helix radius of approximately 0.5-3 μm, a helical rotation speed of approximately 3-20 rotations/s and a linear speed of approximately 20-100 μm/s. This high-throughput 3D imaging platform could in general be quite valuable for observing the statistical swimming patterns of various other microorganisms, leading to new insights in their 3D motion and the underlying biophysics.
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Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA-Rho kinase-myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear-centrosomal orientation and lumen initiation during 3D epithelial morphogenesis.
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Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges.
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Transforming Growth Factor Beta (TGF-β) is involved in regulating many biological processes and disease states. Cells secrete cytokine as a latent complex that must be activated for it to exert its biological functions. We previously discovered that the epithelial-restricted integrin α(v)β(6) activates TGF-β and that this process is important in a number of in vivo models of disease. Here, we show that agonists of G-protein coupled receptors (Sphingosine-1-Phosphate and Lysophosphatidic Acid) which are ligated under conditions of epithelial injury directly stimulate primary airway epithelial cells to activate latent TGF-β through a pathway that involves Rho Kinase, non-muscle myosin, the α(v)β(6) integrin, and the generation of mechanical tension. Interestingly, lung epithelial cells appear to exert force on latent TGF-β using sub-cortical actin/myosin rather than the stress fibers utilized by fibroblasts and other traditionally "contractile" cells. These findings extend recent evidence suggesting TGF-β can be activated by integrin-mediated mechanical force and suggest that this mechanism is important for an integrin (α(v)β(6)) and a cell type (epithelial cells) that have important roles in biologically relevant TGF-β activation in vivo.
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The formation of the basoapical polarity axis in epithelia is critical for maintaining the homeostasis of differentiated tissues. Factors that influence cancer development notoriously affect tissue organization. Apical polarity appears as a specific tissue feature that, once disrupted, would facilitate the onset of mammary tumors. Thus, developing means to rapidly measure apical polarity alterations would greatly favor screening for factors that endanger the breast epithelium. A Raman scattering-based platform was used for label-free determination of apical polarity in live breast glandular structures (acini) produced in three-dimensional cell culture. The coherent anti-Stokes Raman scattering signal permitted the visualization of the apical and basal surfaces of an acinus. Raman microspectroscopy subsequently revealed that polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca(2+)-chelator EGTA. This method overcame variation between different cultures by tracking the status of apical polarity longitudinally for the same acini. Therefore, the disruption of apical polarity by a dietary breast cancer risk factor, ω6 fatty acid, could be observed with this method, even when the effect was too moderate to permit a conclusive assessment by the traditional immunostaining method.
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We present a lens-free optical tomographic microscope, which enables imaging a large volume of approximately 15 mm(3) on a chip, with a spatial resolution of < 1 μm × < 1 μm × < 3 μm in x, y and z dimensions, respectively. In this lens-free tomography modality, the sample is placed directly on a digital sensor array with, e.g., ≤ 4 mm distance to its active area. A partially coherent light source placed approximately 70 mm away from the sensor is employed to record lens-free in-line holograms of the sample from different viewing angles. At each illumination angle, multiple subpixel shifted holograms are also recorded, which are digitally processed using a pixel superresolution technique to create a single high-resolution hologram of each angular projection of the object. These superresolved holograms are digitally reconstructed for an angular range of ± 50°, which are then back-projected to compute tomograms of the sample. In order to minimize the artifacts due to limited angular range of tilted illumination, a dual-axis tomography scheme is adopted, where the light source is rotated along two orthogonal axes. Tomographic imaging performance is quantified using microbeads of different dimensions, as well as by imaging wild-type Caenorhabditis elegans. Probing a large volume with a decent 3D spatial resolution, this lens-free optical tomography platform on a chip could provide a powerful tool for high-throughput imaging applications in, e.g., cell and developmental biology.
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We report a portable lensless on-chip microscope that can achieve <1 µm resolution over a wide field-of-view of ∼ 24 mm(2) without the use of any mechanical scanning. This compact on-chip microscope weighs ∼ 95 g and is based on partially coherent digital in-line holography. Multiple fiber-optic waveguides are butt-coupled to light emitting diodes, which are controlled by a low-cost micro-controller to sequentially illuminate the sample. The resulting lensfree holograms are then captured by a digital sensor-array and are rapidly processed using a pixel super-resolution algorithm to generate much higher resolution holographic images (both phase and amplitude) of the objects. This wide-field and high-resolution on-chip microscope, being compact and light-weight, would be important for global health problems such as diagnosis of infectious diseases in remote locations. Toward this end, we validate the performance of this field-portable microscope by imaging human malaria parasites (Plasmodium falciparum) in thin blood smears. Our results constitute the first-time that a lensfree on-chip microscope has successfully imaged malaria parasites.
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We experimentally illustrate a lensfree holographic imaging platform to perform on-chip cytometry. By controlling the spatial coherence of the illumination source, we record a 2D holographic diffraction pattern of each cell or micro-particle on a chip using a high resolution sensor array that has approximately 2 microm pixel size. The recorded holographic image is then processed by using a custom developed decision algorithm for matching the detected hologram texture to existing library images for on-chip characterization and counting of a heterogeneous solution of interest. The holographic diffraction signature of any microscopic object is significantly different from the classical diffraction pattern of the same object. It improves the signal to noise ratio and the signature uniformity of the cell patterns; and also exhibits much better sensitivity for on-chip imaging of weakly scattering phase objects such as small bacteria or cells. We verify significantly improved performance of this holographic on-chip cytometry approach by automatically characterizing heterogeneous solutions of red blood cells, yeast cells, E. coli and various sized micro-particles without the use of any lenses or microscope objectives. This lensless on-chip holography platform will especially be useful for point-of-care cytometry and diagnostics applications involving e.g., infectious diseases such as HIV or malaria.
Article
How do animal cells assemble into tissues and organs? A diverse array of tissue structures and shapes can be formed by organizing groups of cells into different polarized arrangements and by coordinating their polarity in space and time. Conserved design principles underlying this diversity are emerging from studies of model organisms and tissues. We discuss how conserved polarity complexes, signalling networks, transcription factors, membrane-trafficking pathways, mechanisms for forming lumens in tubes and other hollow structures, and transitions between different types of polarity, such as between epithelial and mesenchymal cells, are used in similar and iterative manners to build all tissues.
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Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.
Article
Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of "transgene toxicology," in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.
Article
We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.2-1.5mm colony size for a 1mm laser beam and brain heart infusion agar as substrate were used as fixed variables. The current study is focused on exploring identification of Listeria monocytogenes and other Listeria species exploiting the known differences in their phenotypic characters. Using diffraction theory, we could model the scattering patterns and explain the appearance of radial spokes and the rings seen in the scattering images of L. monocytogenes. Further, we have also demonstrated development of a suitable software for the extraction of the features (scalar values) calculated from images of the scattering patterns using Zernike moment invariants and principal component analysis and were grouped using K-means clustering. We achieved 91-100% accuracy in detecting different species. It was also observed that substrate variations affect the scattering patterns of Listeria. Finally, a database was constructed based on the scattering patterns from 108 different strains belonging to six species of Listeria. The overall system proved to be simple, non-invasive and virtually reagent-less and has the potential for automated user-friendly application for detection and differentiation of L. monocytogenes and other Listeria species colonies grown on agar plates within 5-10 min analysis time.
Article
Three-dimensional (3D) epithelial culture systems recreate the cardinal features of glandular epithelium in vivo and represent a valuable tool for modeling breast cancer initiation and progression in a structurally appropriate context. 3D models have emerged as a powerful method to interrogate the biological activities of cancer genes and oncogenic pathways, and recent studies have poignantly illustrated their utility in dissecting the emerging role of tensional force in regulating epithelial tissue homeostasis. We review how 3D models are being used to investigate fundamental cellular and biophysical mechanisms associated with breast cancer progression that have not been readily amenable to traditional genetic or biochemical analysis.
Article
We experimentally and theoretically demonstrate the proof-of-principle of a new lens-free cell monitoring platform that involves using an opto-electronic sensor array to record the shadow image of cells onto the sensor plane. This technology can monitor/count cells over a field-of-view that is more than two orders of magnitude larger than that of a conventional light microscope. Furthermore, it does not require any mechanical scanning or optical elements, such as microscope objectives or lenses. We also show that this optical approach can conveniently be combined with microfluidic channels, enabling parallel on-chip monitoring of various different cell types, e.g., blood cells, NIH-3T3 fibroblasts, murine embryonic stem cells, AML-12 hepatocytes. An important application of this approach could be a miniaturized point-of-care technology to obtain CD4 T lymphocyte counts of HIV infected patients in resource limited settings.
  • A Greenbaum
  • W Luo
  • T W Su
  • Z Göröcs
  • L Xue
  • S O Isikman
  • A F Coskun
  • O Mudanyali
Greenbaum, A., Luo, W., Su, T.W., Göröcs, Z., Xue, L., Isikman, S.O., Coskun, A.F., Mudanyali, O., Ozcan, A., 2012. Nature Methods 9, 889–895.
  • N Moscelli
  • S Van Den Driesche
  • W Witarski
  • S Pastorekova
  • M J Vellekoop
Moscelli, N., Van den Driesche, S., Witarski, W., Pastorekova, S., Vellekoop, M.J., 2011. Sensors and Actuators A: Physical 172 (1), 175-180.
  • O 'brien
  • L E Zegers
  • M M P Mostov
O'Brien, L.E., Zegers, M.M.P., Mostov, K.E., 2002. Nature Reviews 3, 531–537.
  • J Weidling
  • S O Isikman
  • A Greenbaum
  • A Ozcan
  • E Botvinick
Weidling, J., Isikman, S.O., Greenbaum, A., Ozcan, A., Botvinick, E., 2012. Journal of Biomedical Optics 17 (12) 126018.
  • S Seo
  • T W Su
  • D K Tseng
  • A Erlinger
  • A Ozcan
Seo, S., Su, T.W., Tseng, D.K., Erlinger, A., Ozcan, A., 2009. Lab on Chips 9 (6), 777-787.
  • W Bishara
  • U Sikora
  • O Mudanyali
  • T-W Su
  • O Yaglidere
Bishara, W., Sikora, U., Mudanyali, O., Su, T-W., Yaglidere, O., et al., 2011. Lab on a Chip 11 (7), 1276-1279.
  • A Tahmasbi
  • F Saki
  • S B Shoukouhi
Tahmasbi, A., Saki, F., Shoukouhi, S.B., 2011. Computers in Biology and Medicine 41, 726-735.
  • P P Banada
  • S Guo
  • B Bayraktar
  • E Bae
  • B Rajwa
  • J P Robinson
  • E D Hirleman
  • A K Bhunia
Banada, P.P., Guo, S., Bayraktar, B., Bae, E., Rajwa, B., Robinson, J.P., Hirleman, E.D., Bhunia, A.K., 2007. Biosensors and Bioelectronics 22 (8), 1664–1671.
  • S O Isikman
  • W Bishara
  • O Mudanyali
  • I Sencan
  • Su Ting-Wei
  • D K Tseng
Isikman, S.O., Bishara, W., Mudanyali, O., Sencan, I., Ting-Wei, Su., Tseng, D.K., 2012. IEEE Journal of Selected Topics in Quantum Electronics 18 (3), 1059-1072.
  • F Pampaloni
  • E G Reynaud
  • E H Stelzer
Pampaloni, F., Reynaud, E.G., Stelzer, E.H., 2007. Nature Reviews Molecular Cell Biology 8 (10), 839-845.
  • M M Zegers
  • L E O'brien
  • W Yu
  • A Datta
  • K E Mostov
Zegers, M.M., O'Brien, L.E., Yu, W., Datta, A., Mostov, K.E., 2003. Trends in Cell Biology 13 (4), 169-176.
  • O Mudanyali
  • E Mcleod
  • W Luo
Mudanyali, O, McLeod, E, Luo, W, et al., 2013. Nature Photonics 7, 247-254.
  • S Yue
  • J M Cardenas-Mora
  • L S Chaboub
  • S A Lelièvre
  • J-X Cheng
Yue, S., Cardenas-Mora, J.M., Chaboub, L.S., Lelièvre, S.A., Cheng, J-X., 2012. Biophysical Journal 102, 1215-1223.
  • C Hebner
  • V M Weaver
  • J Debnath
Hebner, C., Weaver, V.M., Debnath, J., 2008. Annual Review of Pathology 3, 313–339.
  • O Mudanyali
  • W Bishara
  • A Ozcan
Mudanyali, O., Bishara, W., Ozcan, A., 2011. Optics Express 19 (18), 17378–17389.
  • F Soulez
  • L Denis
  • C Fournier
  • E Thiebaut
  • C Goepfert
Soulez, F., Denis, L., Fournier, C., Thiebaut, E., Goepfert, C., 2007. Journal of the Optical Society of America A: Optics, Image and Vision 24 (4), 1164-1171.
  • J Debnath
  • J S Brugge
Debnath, J., Brugge, J.S., 2005. Nature Reviews Cancer 5 (9), 675–688.
  • M M Giacomini
  • M A Travis
  • M Kudo
  • D Sheppard
Giacomini, M.M., Travis, M.A., Kudo, M., Sheppard, D., 2012. Experimental Cell Research 318, 716-722.