A Fluorescent Kinase Inhibitor that Exhibits Diagnostic Changes in Emission upon Binding

The development of a fluorescent LCK inhibitor that exhibits favourable solvatochromic properties upon binding the kinase is described. Fluorescent properties were realised through the inclusion of a prodan-derived fluorophore into the pharmacophore of an ATP-competitive kinase inhibitor. Fluorescence titration experiments demonstrate the solvatochromic properties of the inhibitor, in which dramatic increase in emission intensity and hypsochromic shift in emission maxima are clearly observed upon binding LCK. Microscopy experiments in cellular contexts together with flow cytometry show that the fluorescence intensity of the inhibitor correlates with the LCK concentration. Furthermore, multiphoton microscopy experiments demonstrate both the rapid cellular uptake of the inhibitor and that the two-photon cross section of the inhibitor is amenable for excitation at 700 nm

Posicionamiento visual con resolución subpixel de objetos marcados que se desplazan en un plano: conceptos básicos y aplicaciones

Vision is a convenient tool for position measurements. In this paper, we present several applications in which a reference pattern can be defined on the target for a priori knowledge of image features and further optimization by software. Selecting pseudoperiodic patterns leads to high resolution in absolute phase measurements. This method is adapted to position encoding of live cell culture boxes. Our goal is to capture each biological image along with its absolute highly accurate position regarding the culture box itself. Thus, it becomes straightforward to find again an already observed region of interest when a culture box is brought back to the microscope stage from the cell incubator where it was temporarily placed for cell culture. In order to evaluate the performance of this method, we tested it during a wound healing assay of human liver tumor-derived cells. In this case, the procedure enabled more accurate measurements of the wound healing rate than the usual method. It was also applied to the characterization of the in-plane vibration amplitude from a tapered probe of a shear force microscope. The amplitude was interpolated by a quartz tuning fork with an attached pseudo-periodic pattern. Nanometer vibration amplitude resolution is achieved by processing the pattern images. Such pictures were recorded by using a common 20x magnification lens.La visión es una herramienta conveniente para mediciones de posición. En este artículo, presentamos aplicaciones en las que un patrón de referencia puede ser adherido al objeto de interés. Ésto permite tener un conocimiento a priori de las características de la imagen y así poder optimizar el software. Como patrón de referencia se usan patrones pseudo-periódicos, los cuales permiten una alta resolución en las mediciones de fase absoluta. El método es adaptado para codificar la posición de soportes de cultivos celulares, con el fin de documentar cada imagen biológica registrada con su posición absoluta. Por lo tanto, resulta sencillo encontrar de nuevo una región de interés, observada previamente, cuando una caja de cultivo es traída de nuevo al microscopio luego de estar en una incubadora. Para evaluar el método, éste se utilizó durante un ensayo de “cicatrización de herida” de un cultivo celular derivado de tumores hepáticos. En este caso, el método permite obtener mediciones más precisas de la tasa de “cicatrización”, comparado a los resultados obtenidos con el método usual. El método propuesto también se aplica a la caracterización de la amplitud de vibración en el plano de una sonda de un microscopio de fuerza atómica. La amplitud fue interpolada por medio de un diapasón de cuarzo al cual se la adhirió un patrón pseudo-periódico. A partir del procesamiento de las imágenes del patrón, se logra obtener resolución nanométrica en la medida de la amplitud de la vibración. Estas imágenes fueron obtenidas con un microscopio óptico con magnificación 20x

Posicionamiento visual con resolución subpixel de objetos marcados que se desplazan en un plano: conceptos básicos y aplicaciones

Vision is a convenient tool for position measurements. In this paper, we present several applications in which a reference pattern can be defined on the target for a priori knowledge of image features and further optimization by software. Selecting pseudoperiodic patterns leads to high resolution in absolute phase measurements. This method is adapted to position encoding of live cell culture boxes. Our goal is to capture each biological image along with its absolute highly accurate position regarding the culture box itself. Thus, it becomes straightforward to find again an already observed region of interest when a culture box is brought back to the microscope stage from the cell incubator where it was temporarily placed for cell culture. In order to evaluate the performance of this method, we tested it during a wound healing assay of human liver tumor-derived cells. In this case, the procedure enabled more accurate measurements of the wound healing rate than the usual method. It was also applied to the characterization of the in-plane vibration amplitude from a tapered probe of a shear force microscope. The amplitude was interpolated by a quartz tuning fork with an attached pseudo-periodic pattern. Nanometer vibration amplitude resolution is achieved by processing the pattern images. Such pictures were recorded by using a common 20x magnification lens.La visión es una herramienta conveniente para mediciones de posición. En este artículo, presentamos aplicaciones en las que un patrón de referencia puede ser adherido al objeto de interés. Ésto permite tener un conocimiento a priori de las características de la imagen y así poder optimizar el software. Como patrón de referencia se usan patrones pseudo-periódicos, los cuales permiten una alta resolución en las mediciones de fase absoluta. El método es adaptado para codificar la posición de soportes de cultivos celulares, con el fin de documentar cada imagen biológica registrada con su posición absoluta. Por lo tanto, resulta sencillo encontrar de nuevo una región de interés, observada previamente, cuando una caja de cultivo es traída de nuevo al microscopio luego de estar en una incubadora. Para evaluar el método, éste se utilizó durante un ensayo de “cicatrización de herida” de un cultivo celular derivado de tumores hepáticos. En este caso, el método permite obtener mediciones más precisas de la tasa de “cicatrización”, comparado a los resultados obtenidos con el método usual. El método propuesto también se aplica a la caracterización de la amplitud de vibración en el plano de una sonda de un microscopio de fuerza atómica. La amplitud fue interpolada por medio de un diapasón de cuarzo al cual se la adhirió un patrón pseudo-periódico. A partir del procesamiento de las imágenes del patrón, se logra obtener resolución nanométrica en la medida de la amplitud de la vibración. Estas imágenes fueron obtenidas con un microscopio óptico con magnificación 20x

Microfabrication of position reference patterns onto glass microscope slides for high-accurate analysis of dynamic cellular events

Los portaobjetos de microscopio se utilizan ampliamente como sustratos base in situ para la realización de diversos sistemas o elementos microfabricados. Para estos fines, el proceso de microfabricación consiste en transferir un diseño predefinido sobre el sustrato correspondiente a una lámina de vidrio utilizada como portaobjetos de microscopio. Este proceso se conoce como “patterning”, que es una técnica que también se puede utilizar en la transferencia de diseños específicos que permite la recuperación de una región de interés (ROI) bajo el microscopio. En estos casos, aparecen dos desafíos principales: 1) Las perturbaciones en la transmisión de la luz deben permanecer mínimas para mantener la alta calidad de observación del objeto de interés bajo el microscopio. 2) El tamaño del patrón debe ser entonces suficientemente pequeño, pero, sin embargo, mayor que el límite de difracción para ser observable satisfactoriamente para propósitos de posicionamiento. En este artículo presentamos los procedimientos involucrados en la microfabricación de Patrones Pseudo-Periódicos (PPP) los cuales encriptan la posición absoluta de un área extendida. Esos patrones están embebidos en placas de Pétri para permitir la recuperación absoluta y de alta precisión de una ROI, al igual que su orientación. La microfabricación presentada se basa en una técnica conocida como “liftoff” que, tras el ajuste de parámetros, permite la obtención de PPP cumpliendo los dos requisitos anteriormente mencionados. Los resultados corresponden a la realización de PPP en portaobjetos de vidrio y compuesto por puntos laterales de 2μm hechos de aluminio con un grosor de 30nm.Glass microscopes slides are widely used as in situ base-substrates carrying diverse micro-fabricated systems or elements. For such purposes, the micro-fabrication process consists in transferring a pre-defined design onto the substrate made of a glass microscope slide. This is known as patterning, which is a technique that can also be used in transferring specific designs that allows region of interest (ROI) recovery under the microscope. In those cases, two main challenges appear: 1) Disturbances in light transmission should remain minimum to keep the high quality of observation of the object of interest under the microscope. 2) The pattern-size should then be small enough but, however, larger than the diffraction limit to be observable satisfactorily for positioning purposes. In this article, we present the procedures involved in the microfabrication of Pseudo-Periodic Patterns (PPP) encrypting the absolute position of an extended area. Those patterns are embedded in Pétri dishes in order to allow the highaccurate retrieval of absolute position and orientation. The presented microfabrication is based in a technique known as lift-off, which after parameter adjustment, allows the obtaining of PPP fulfilling the two previously mentioned requirements. The results report on PPP realized on glass microscope slides and composed by 2µm side dots made of aluminum with a thickness of 30nm

Position-referenced microscopy for live cell culture monitoring

Position-referenced microscopy (PRM) is based on smart sample holders that integrate a position reference pattern (PRP) in their depth, allowing the determination of the lateral coordinates with respect to the sample-holder itself. Regions of interest can thus be retrieved easily after culture dish transfers from a cell incubator to the microscope stage. Images recorded at different instants in time are superimposed in a common coordinate system with subpixel accuracy. This paper presents such smart Petri culture dishes and their use for live cell culture monitoring. The impact of the PRP on the light budget is discussed and performances are demonstrated. First results on the application of PRM to the observation of apoptotic body internalization are reported

Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux.

Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell's refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells' division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images

Inertial microfluidic programming of microparticle-laden flows for solution transfer around cells and particles

Control of particles/cells and the surrounding fluid is enabling toward the purification of complex cellular samples, which still remains a bottleneck for point-of-care diagnostic devices. We explore a newly developed approach to engineer fluid stream motion while simultaneously controlling particles using inertial lift force. We use inertial flow deformations induced by sequences of simple pillar microstructures to control the fluid stream. Instead of iterative experimental procedures to identify optimal sequences of structures, we use software that numerically predicts the total deformation function for any pillar sequence. Using this program, we engineer the cross-stream translation of a fluid stream to achieve solution exchange around particles, where both the particles and fluid stream remain focused and can be extracted at high purity. An extraction device, called a pillar separation device, is then designed and validated with suspensions of rigid particles to identify optimal operating parameters. At a flow rate of 250 A mu L/min, up to 96 % beads and 70.5 % of an initial buffer stream inputted into the system can be collected downstream in separate outlets, respectively, with 10.9 % buffer and 0.3 % bead contamination. This device was further applied to a functionalized bead bioassay, achieving high-yield and continuous separation of 98 % of biotin-coated beads from 72.2 % of extra FITC-biotin. In a last study, we performed the extraction of 80 % of leukocytes from lysed blood, which validates our platform can be applied on living cells and used for various functions of cellular sample preparation

Recognition of synthetic polyanionic ligands underlies “spontaneous” reactivity of Vγ1γδTCRs

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1‐containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self‐ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate‐like αβ T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long‐standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation‐independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.ISSN:0741-5400ISSN:1938-367